THE THAI JOURNAL OF VETERINARY MEDICINE Office:

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under the Royal Patronage Miss Pringsri Ingkaninun Assoc Prof. Dr. Wimon Pothiwong President of Chulalongkorn University Veterinary

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TJVM would like to thank the followings for their expertise contribution to the Journal in 2010-2013

Animal Husbandry: Boonrit Thongsong, Duangsmorn Suwattana, Somchai Chanpongsang, Thanathip Suwannasopa Animal Nutrition: Jowaman Khajarern, Karen L. Keller, Pongtorn Sungpuag, Suwanna Kijparkorn, Uttra Jamikorn Aquatic Animal Medicine: Aranya Ponpornpisit, Kanit Chukanhom, Weena Koeypudsa Biochemistry: Gunnaporn Suriyaphol, Meena Sarikaputi, Prapruddee Piyaviriyakul Companion Animal Medicine: Achara Tawatsin, Rosama Pusoonthornthum, Sirilak Disatian, Siriporn Khumsub Complementary and Alternative Medicine: Chalaiporn Iamsirithaworn, Krongthong Oraveerakul, Namtip Iamsoongnoen Pharmaceutical Sciences: Kanokrat Siripanichgon, Nijsiri Ruangrungsi, Somboon Tanasupawat, Sunan Pongsamart Physiology: Anusak Kijtawornrat, Chollada Buranakarl, Chutamas Benjanirut, Kittipong Tachampa, Kris Angkanaporn, Narongsak Chaiyabutr, Sumpun Thammacharoen, Varodom Charoensawan, Weera Supornsilpchai Livestock Animal Medicine: Anucha Sirimalaisuwan, Jaruwan Kumpa, Kittisak Ajariyakhajorn, Nalin Upragarin, Niwat Chansiripornchai, Pornchalit Asawacheap, Supol Luengyosleuchakul, Wisanu Wanasawaeng, Witaya Suriyastaporn Laboratory Animal Medicine: Piengchai Kupradinun Medicine: Kanokrat Siripanichgon, Lallida Pariyakanok, Padet Siriyasatien, Pinnita Tanthuvanit, Pongsak Yuktanandana Theriogenology: Annop Kunavongkrit, Chanchao Lorthongpanich, Chatchote Thitaram, Kaywalee Chatdarong, Kumpon Kaeoket, Mongkol Techakumphu, Nikorn Thongtip, Padet Thummarak, Rangsan Parnpai, Stig Einarsson, Sukanya Leethongdee, Sukanya Manee-in, Suneerat Aiumlamai, Theerawat Tarasanit Veterinary Anatomy: Paisan Tienthai, Peerapol Sukon, Prasarn Tangkawattana, Sayamon Srisuwattanasakul, Weerapong Koykul Veterinary Microbiology: Channarong Rodkhum, Nuvee Prapasarakul, Varaporn Vuddhakul Veterinary Parasitology: Manop Muangyai, Oliver Sparagano, Sonthaya Tiewsirisup, Woraporn Sukhumavasi

Veterinary Pathology: Chaleow Salakij, Eric D. Lombardini, Nopadon Pirarat, Roongroje Thanawongnuwech, Sirikachon Tangkawattana, Somporn Techangamsuwan, Taweesak Songserm, Thanongsak Mamom, Theerayuth Kaewamatawong, Wijit Banlunara Veterinary Pharmacology: Kanjana Imsilp, Nipattra Suanpairintr, Sukanya Phalitkul, Supatra Srichirat Veterinary Public Health: Benjamas Patamalai, Byeong Hwa Jeon, Nipa Chokesajjawatee, Piyawat Saipun, Rungtip Chuanchuen, Sumalee Boonmar, Suphachai Nuanualsuwan, Suthep Ruangwises, Thanit Damrongwattanpokin, Waraporn Phimpraphai Veterinary Surgery: Marissak Kalpravidh, Sumit Durongpongtorn, Suwicha Chuthateph, Voraphan Na Songhla, Wanna Suriyastaporn Veterinary Virology: Juthathip Kaewcharoen, Kanisak Oraveerakul, Pravina Kitikoon

The Thai Journal of Veterinary Medicine

Vol. 43 No. 2 June 2013

Contents

Review Article Indices of Myocardial Contractility Anusak Kijtawornrat

167

Original Article

Glycerol Concentration Effects on Quality and Longevity of Post-thaw Goat Semen Nitira Anakkul, Junpen Suwimonteerabutr, Theerawat Tharasanit, Nawapen Phutikanit, Jinda Singlor, Mongkol Techakumphu

179

Expression of Oestrogen Receptor Alpha and Progesterone Receptor in Sow Oviducts after Different Artificial Insemination

Sayamon Srisuwatanasagul, Padet Tummaruk, Annop Kunavongkrit

187

Probable Pig to Duck Transmission of the Pandemic H1N1 2009 (pH1N1) and Its Reassortant in Commingling Experimental Condition

Nataya Charoenvisal, Juthatip Keawcharoen, Donruethai Sreta, Siriporn Tantawet, Suphattra Jittimanee, Jirapat Arunorat, Korakrit Poonsuk, Alongkorn Amonsin, Roongroje Thanawongnuwech

197

Preference Testing as Environmental Enrichment Assessment for Laboratory Mice Navakanit Sachanonta, Waridtha Sa-ngeunreung, Somchai Sa-ing-kaew, Raywadee Butraporn

205

Effects of Myogenic Factor 5 (MYF5) Gene on Carcass and Meat Quality of Mong Cai Pigs Nguyen Thi Kim Khang Nguyen Trong Ngu

213

The mRNA and protein expression of prostaglandin E2 receptors (EP2 and EP4), cyclooxygenase-2 and prostaglandin E synthase in the cervix of cyclic bitches and those with pyometra

Pichanun Linharattanaruksa, Theerawat Swangchan-Uthai, Sayamon Srisuwatanasagul, Muhammad Khalid, Kaywalee Chatdarong

219

Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for Feline Coronavirus Detection Somporn Techangamsuwan, Araya Radtanakatikanon, Roongroje Thanawongnuwech

229

Welfare Assessment of Flight-restrained Captive Birds: Effects of Inhibition of Locomotion Shawn Jen-Lung Peng, Fang-Chia Chang, Judy Sheng-Ting I, Andrew Chang-Young Fei

235

Mutations in Topoisomerase Genes and Expression of the AcrAB Multidrug Efflux System in Fluoroquinolone-Resistant Salmonella enterica from Pork and Patients Wechsiri Wannaprasat, Rungtip Chuanchuen

243

Effect of Polysaccharide Gel Extracted from Durio zibethinus Rind on Immune Responses, Bacteria Counts and Cholesterol Quantities in Chickens Nustha Kitprathaung, Nattaya Ngamrojanavanich, Piyarat Chansiripornchai, Sununta Pongsamart, Niwat Chansiripornchai

251

Production of Black Goat Using Laparoscopic Artificial Insemination and Embryo Transfer Nitira Anakkul, Junpen Suwimonteerabutr, Theerawat Tharasanit, Saritvich Panyaboriban, Sarawanee Khunmanee, Natthakarn Thanomsuksinchai, Mongkol Techakumphu

259

Short Communication

Prevalence of Salmonella enterica, Escherichia coli and Staphylococcus aureus in Raw Meat in Thai Self-Service Style Restaurants in Khon Kaen Municipality Sunpetch Angkititrakul, Arunee Polpakdee, Rungtip Chuanchuen

265

Coccidiosis in a Greater Slow loris (Nycticebus coucang) Wijit Banlunara, Sawang Kesdangsakonwut, Piyaporn Kongmekee, Angkana Sommanustweechai

269

Histological Characteristics of the Auditory Tube Diverticulum of the Domestic Donkey (Equus asinus) Seyed Morteza Ghazi, Behzad Mobini

273

Antiparasitic Efficacy of 10% w/v Fipronil Spot on (Fiproline Spot on) against Experimental Tick (Rhipicephalus sanguineus) Infestations on Dogs Sonthaya Tiawsirisup, Kanokwan Thiansirikhun, Kanoksak Thanadumkerng, Nuttika Pastarapatee, Nunaree Trirattananuwong, Wilai Rattanatayarom

279

Differential Influence of Intracellular Glutamine Depletion on Glutamate Uptake Mediated by Amino Acid Transport Systems; EAATs and X-c, in Placental (BeWo) Cells Boonrit Thongsong

285

Genetic Diversity of Spirometra erinaceieuropaei from Dogs in Hunan province, China Based on Analyses of Two Mitochondrial Sequences Liang Liang, Jian Liang, Hui-Shen, Xiao-Lei Yao

291

Surgical Removal of Foreign Bodies in the Gastrointestinal Tract of Monocellate Cobra, Naja kaouthia Taksa Vasaruchapong, Lawan Chanhome

297

Implications of Cat Ownership Statistics and Social Changes: A Longitudinal Study in Taiwan from 2001 to 2009 Li-Huan Chen, Ting-Wei Chang, Meng-Chih Tung, Chang-Young Fei, I-Tsun Chiang

301

Comparison of repetitive sequence-based polymerase chain reaction (rep-PCR) and pulsed-field gel electrophoresis (PFGE) for genetic characterization of Arcobacter spp. Panvipa Phasipol, Nipa Chokesajjawatee, Taradon Luangtongkum

307

Susceptibility of Clostridium difficile isolated from healthy captive Asian elephants to metronidazole and vancomycin Panvipa Phasipol, Nipa Chokesajjawatee, Taradon Luangtongkum

313

Diagnostic Forum

ECG Quiz Chollada Buranakarl, Suwanakiet Sawangkoon, Chutamas Benjanirut, Monkon Trisiriroj, Thanusorn Phakhawambodee

317

Ophthalmology Snapshot Nalinee Tuntivanich

319

Ultrasound Diagnosis Phiwipha Kamonrat

321

165

INSTRUCTIONS TO AUTHORS

The Thai Journal of Veterinary Medicine publishes articles reporting interdisciplinary investigations concerning veterinary and animal sciences, at all levels of resolution, from basic to clinical, molecular to behavioral, and opinions that are of general interest to the broad community of veterinarians and biological scientists. Clinical or pathological investigations, protocols and reviews will also be considered for publication if they provide significant insight into the structure or function, the pathophysiology of a disease, or its treatment.

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166

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Thai J Vet Med. 2013. 43(2): 167-178.

Indices of Myocardial Contractility

Anusak Kijtawornrat

Abstract

Myocardial contractility is defined as the velocity of cycling of heavy meromyosin cross-bridges.

Contractility may be defined also as the velocity of myocardial fiber shortening or change in ventricular dimension during contraction when the fiber shortens or the ventricle contracts against no load. In the clinic; however, myocardial contractility may be estimated by some parameters of systolic function adjusted for the degree of stretch on the fibers (the preload) just before contraction begins and the hindrance to ejection of blood (the afterload). Excitation-contraction coupling is a physicochemical process during which electrophysiological changes (initiated by depolarization of the cell) translate to mechanical changes (manifested as a combination of contraction and generation of tension). The result of this process is a combination of the ejection of blood out of the ventricle into and through the arterial tree and of the generation of pressure. Many indices have been used in both humans and experimental animals to assess cardiovascular function: (1) Vmax (the maximal velocity of fiber shortening estimated from the imaginary conditions of the fiber shortening against no load), (2) parameters (ESPVR, PRSW, Ees) obtained from the left ventricular pressure-volume relationship, (3) dLVP/dtmax (maximal rate of rise of left ventricular pressure), (4) QA interval (time between onset of electrical activity in the ventricle to initial elevation of arterial pressure), (5) ejection fraction (ratio of stroke volume to preload). However, some (3, 4 and 5) of these indices must be used with caution for estimating contractility since they are also influenced by other factors (e.g. preload, afterload, stiffness of the arterial tree). Parameters of the pressure-volume loop are considered the gold standard for measuring contractility, lusitrope, myocardial energetics and ventricular-vascular coupling.

Keywords: dLVP/dtmax, ejection fraction, myocardial contractility, pressure-volume relationship, QA interval, Vmax

Department of Veterinary Physiology, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, 10330 Thailand Corresponding author E-mail: kanusak@hotmail.com

Review Article

168 Kijtawornrat A. / Thai J Vet Med. 2013. 43(2): 167-178.

บทคดยอ

ดชนตรวจความสามารถในการหดตวของกลามเนอหวใจ

อนศกด กจถาวรรตน

ความสามารถในการหดตวของกลามเนอหวใจ คอ ความเรวในการหมนของเมอโรไมโอซนหนกในกระบวนการ cross-bridges หรอความเรวในการหดตวของใยกลามเนอหวใจ หรอการเปลยนแปลงขนาดของหวใจหองลางขณะทหวใจหดตวในภาวะทไมมนาหนก ในทางคลนกความสามารถในการหดตวของกลามเนอหวใจอาจประมาณคาไดจากดชนของการหดตวของหวใจทปรบมาตรฐานกบระดบการยดของใยกลามเนอหวใจกอนการหดตว (พรโหลด) และอปสรรคในการสบฉดเลอด (อาฟเตอรโหลด) เอกไซเตชน-คอนแทรกชน คบปลง เปนกระบวนการทางฟสกสและเคมรวมกนซงการเปลยนแปลงทางไฟฟาสรรวทยาเปลยนเปนการหดตวของกลามเนอ ผลของกระบวนการดงกลาวทาใหเกดการการสบฉดเลอดจากหวใจหองลางสหลอดเลอดแดงและเปนตวกาหนดความดนเลอด ในสตวทดลองและในคนมการใชดชนสาหรบตรวจการทางานของหวใจหลายชนด เชน 1) วแมกซ เปนการบนทกความเรวในการหดตวของใยกลามเนอในภาวะทไมมนาหนก 2) ดชน (ESPVR, PRSW, Ees) ทไดจากการการหาความสมพนธระหวางความดนและปรมาตรของเลอดในหวใจหองลางซาย 3) dLVP/dtmax เปนอตราเรวสงสดของการเพมความดนเลอดในหวใจหองลางซาย 4) ระยะควเอ เปนชวงเวลาจากจดเรมตนของ Q wave บนภาพคลนไฟฟาหวใจจนถงจดเรมตนของความดนเลอดแดง และ 5) อเจคชนแฟรคชน เปนสดสวนของปรมาณเลอดทถกบบออกจากหวใจในแตละครงตอพรโหลด อยางไรกตามในการใชดชนท 3, 4 และ 5 ตองมความระมดระวงในการใชเนองจากมปจจยอนๆ ทไมใชแคความสามารถในการหดตวของหวใจทมผลตอดชนดงกลาว (เชน พรโหลด อาฟเตอรโหลด และความแขงของหลอดเลอดแดง) ดชนทไดจากการการหาความสมพนธระหวางความดนและปรมาตรของเลอดในหวใจหองลางซายถอวาเปนดชนมาตรฐานในการตรวจความสามารถในการหดตวของหวใจ ความสามารถในการคลายตว พลงงานของกลามเนอหวใจ และการคบปลงระหวางหวใจหองลางและหลอดเลอด

คาสาคญ: dLVP/dtmax อเจคชนแฟรคชน ความสามารถในการหดตวของหวใจ ความสมพนธระหวางความดนและปรมาตรของเลอด ระยะควเอ วแมกซ

ภาควชาสรรวทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 Corresponding author E-mail: kanusak@hotmail.com

Introduction

Overview of the cardiovascular system and its properties

Cardiovascular system is essential for multicellular organisms to sustain their intracellular environment (milieu interior) with optimal (i.e. suitable for good health) amounts of oxygen, carbon dioxide, pH, substrates for oxidation, waste products from metabolism. Since all of these factors either come from (e.g. oxygen, substrates) or are discharged to (e.g. carbon dioxide, waste products of metabolism) the external environment, and since only a very few cells of the body have direct (e.g. skin, sclera, tympanic membrane, mucous membranes) or semi-direct (e.g. GI tract, lung, kidney) contact with the external environment, the multicellular organism has evolved with a cardiovascular system that brings each of the approximately one quadrillion cells into intimate contact with the external environment. This system consists of the heart, arteries, veins, capillaries, and the blood within.

The principal task of the heart is to generate the pressure difference that drives blood from arteries to veins through the microcirculation (capillaries), and from the left and right ventricles into the initial portion of the aorta and pulmonary trunk. The heart has five so-called physiological properties that allow it to function:

1. Chronotrope is the ability of a region of the heart—the SA node—to discharge a wave of depolarization at a certain and variable rate to sustain the heart beat.

2. Dromotrope is the ability of various structures (e.g. atrial myocardium, internodal pathways, AV conduction system, ventricular myocardium) in the heart to conduct waves of depolarization.

3. Bathmotrope refers to excitability or the ability to respond to a stimulus.

4. Inotrope is the ability of the heart muscle, once depolarized, to contract and to generate a combination of tension and shortening.

5. Lusitrope is the ability of the heart, once contracted and having ejected blood, to refill.

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The following will be a review of the myocardial contractility—the inotropic state. The inotropic state is an important physiological property of the heart, and when it is either decreased or increased, significant morbidity and/or mortality may be expected. Therefore, it is important to measure contractility and, when it is altered, to attempt to return it to levels that might reduce morbidity and/or mortality. It is enigmatric that although everyone knows of the importance of inotrope—and the purpose of this missive is to define and discuss how to measure it—there is virtually no consensus on what change in inotrope is meaningful for patients.

When contractility is too low, the heart may not be able pump enough blood at a sufficient pressure to serve the needs of the organism. When contractility is too high, the heart may consume so much oxygen in which an oxygen debt leads to an energetic imbalance.

A failing heart is a heart with reduced contractility. Heart failure is a clinical entity—syndrome—in which an organism manifests signs and symptoms because of a failing heart and a pathological interaction with the blood vessels (Katz and Konstam, 2008). However, many organisms with a failing heart are not in heart failure but may be at risk for heart failure.

Definition of contractility

Myocardial contractility may be defined simply and most precisely as the velocity of cycling of heavy meromyosin heads (cross-bridges). It is cycling of those cross-bridges, emanating from the thick myosin filament and attaching to the thin actin filament, that actually generates tension and or shortening (Hamlin and del Rio, 2010). This cycling is the most fundamental unit of contraction. Rate of hydrolysis of ATP and the amount of Ca++ available to bind to troponin-C are often used as surrogates for contractility. However, the energy from hydrolysis of ATP fuel contraction and binding of Ca++ to troponin-C displaces tropomyosin to allow cycling, but not actually, directly measures contractility (Hamlin and del Rio, 2010).

Excitation-Contraction (EC) coupling

A completed review of cardiac excitation-contraction coupling is presented by several authors (Hamlin and del Rio, 2010; Bers, 2002; Jafri, 2012). Contraction of the myocardium resides in the cycling of heavy meromyosin heads, but the process is fueled by energy from the hydrolysis of ATP and initiated when the cell is stimulated allowing Na+ to enter and depolarize it. The total process, from stimulation to contraction, is termed excitation-contraction (EC) coupling. The following is a sequence of the steps constituting EC coupling:

Step 1: The stimulus to initiate the process is anything that will drastically increase the permeability of the cell membrane to Na+. Although a cell may be stimulated mechanically (as by pricking with a pin) or pharmacologically (as with a drop of aconitine), the usual/normal stimulus is the passage of current

across the cell membrane (Negulyaev et al., 1990). The current flux is produced by voltage differences between neighboring regions that possess differing concentrations of ions.

Step 2: Once the permeability of the cell membrane to Na+ has increased, Na+ rushes into the cell down its concentration gradient (~140 mEq/L outside to ~4 mEq/L inside) and its electrical gradient (because the inside of the resting cell is electrically negative (~-80 mV). This entry of Na+ depolarizes the cell to ~+15 mV.

Step 3: Depolarization of the cell opens up the voltage-gated, L-type, Ca++ channel; Ca++ enters the cell, and a small portion of Ca++ binds to troponin-C (Tc) but the major portion binds to the ryanodine channel (RyR) in the sarcoplasmic reticulum (SR).

Step 4: When Ca++ binds to the RyR channel, the channel opens, and Ca++ exits the SR and binds to Tc.

Step 5: When Ca++ binds to Tc, the Tc tugs on tropomyosin (TM) and removes TM from a position between the heavy meromyosin crossbridge and a site on actin to which the heavy meromyosin head may attach. When the heavy meromyosin heads (that contains ATPase) attach to the binding sites on actin, ATP is hydrolyzed and energy is released which fuels cycling of the heavy meromyosin.

Step 6: Heavy meromyosin heads, and the necks connecting the heads to myosin, constitute the molecular motor which, when fueled with energy from ATP, produces the cycling (Precisely why cycling occurs is not known, but it probably involves changes in molecular geometry of the heavy meromyosin head and the neck.).

Step 7: Swinging of heavy meromyosin heads and tugging on the actin filament is translated to tugging on the Z band (to which the actin filament is attached), and that tugging are translated to a combination of sarcomere shortening or generation of active tension.

If shortening is prohibited by loading conditions, then all cycling is translated to tension only. Under impossible (impossible since the muscle or sarcomere must contract against at least its own mass) conditions of no load, then no tension will be developed and all tugging is translated to shortening. The physiological status of the muscle can be described by the velocity tension curve in which the maximal velocity of shortening is defined as contractility.

Step 8: The cell then repolarizes (i.e. returns towards the resting potential of ~-80 mV) when K+ exits through a number of specific ion channels (e.g. ITO, IKr, IKS) whose conductances increase. Simultaneous to the repolarization, Ca++ is released from Tc and a small amount (i.e. ~30%) exits the cell via the Na+/Ca++ exchanger, but the greatest amount (i.e. ~70%) is resequestered into the SR through the sarcoendoplasmic reticulum Ca++(SERCa) channel. The removal of Ca++ from Tc allows TM to reorient itself to prevent interaction between the heavy meromyosin heads and actin binding sites (Bers,

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2002).

Step 9: After repolarization and return to normal Ca++ distribution, (1) the cell relaxes and returns to normal, resting geometry generating neither motion nor active tension, (2) there is an ionic exchange of Na+ and K+ across the sarcolemma to reestablish ionic balance, and (3) ATP is replenished by mitochondrial metabolism.

Methods of measuring contractility

The primary purpose of this paper was to review several indices of myocardial contractility by defining the indices, discussing how they are measured, discussing limitations of their uses, and discussing how they are used in experimental animals and humans.

1. Vmax: the maximal velocity of fiber shortening estimated from the imaginary conditions of the fiber shortening against no load

A single myocardial fiber, a group of fibers forming a muscle, or a group of muscles forming a cardiac chamber of the heart generate tension or be shorten due to cycling of heavy meromyosin heads. Myocardial contractility is defined as the rate of cycling of those heavy meromyosin heads. Is it possible to establish the rate of cycling from the rate of shortening of a muscle fiber, muscle, or even the entire ventricle? Clearly the rate of shortening depends upon both the rate of cycling of heavy meroymosin heads and the load hindering the shortening. If it was possible to measure rate of shortening without any hindrance to shortening, then the rate of shortening would be determined only by the velocity of cycling of heavy meromyosin heads, and would measure myocardial contractility (Hamlin and del Rio, 2010). The problem is that it is impossible to have a fiber shorten against no hindrance because, even if there appeared to be no hindrance, the fiber would still have to shorten against itself. The method for estimating the maximal velocity of fiber shortening that could occur only against no load is shown (Fig 1) and explained as follows.

A load, too heavy for the muscle to shorten against, is attached to the fiber or muscle. When the fiber or muscle is stimulated with a maximal electric current, it contracts and tries to shorten but it cannot move the heavy load, therefore the velocity of shortening is zero. But it does generate the maximal tension it can. This produces a point that intersects the abscissa showing great tension but intersects the ordinate showing zero velocity. Now as the load is lightened and lightened further, the tension generated is reduced, but the velocity of shortening increases and increases further proportional with the reduction in load. This produces the points (obtained by experimentation) shown as asterisks on the hyperbolic curve. Since these points identify a hyperbola, it then becomes simple extrapolation to extend the curve to the Y-axis intercept (at which the X-axis intercept is zero), which gives the velocity of fiber shortening against no load. This intercept is Vmax, the maximal velocity that can occur only at zero load, which is also

Figure 1 shows the relationship between the load against

which the ventricle contracts and the velocity of fiber shortening. Vmax is the maximal velocity of fiber shortening at zero load.

the maximal velocity of cycling of heavy meromyosin or myocardial contractility.

This concept originated from studies on skeletal muscle that was not allowed to shorten because of a very high load, and was then allowed to shorten against lower and lower loads (Sonnenblick, 1962). With the assumption that Vmax of muscle fibers is equivalent to Vmax of the contractile element, Vmax has been used, in both physiology laboratories and the clinic, as an index of the inotropic state of cardiac muscle (Hefner and Bowen, 1967; Parmley et al., 1969; Falsetti et al., 1971). Based on the work of Pollack (1970), Vmax of the contractile element is not independent of fiber length but increases at least 50% for a 25% increase of fiber length. Therefore, it is unlike that of the muscle fibers. His work indicated that Vmax is invalid for estimating cardiac inotrope since a shift of Vmax can be attributable to either changing contractility or changing fiber length (Pollack, 1970).

The mean rate of ventricular circumferential fiber shortening (Vcf), as may be approximated by angiography or echocardiography (Ruschhaupt et al., 1983; Karliner et al., 1971), appears to estimate accurately ventricular inotrope for both normal humans and humans with heart diseases. Vcf can be estimated from changes in the minor axis of the left ventricle divided by left ventricular ejection time (the time required for those changes to occur). Mahler and colleagues (1975) performed an experiment in conscious dogs and found that the Vcf was insensitive to volume overload but was sensitive to pressure overload, whereas the opposite effect was found with the dP/dtmax. Data indicate, however, that although Vcf derived from M-mode echocardiography is a useful index of inotrope, it is not truly reflective of the precise change of circumference during systole (Ruschhaupt et al., 1983).

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Active tension

PreloadEnd‐diastolic pressure/volumeZ‐Z (sarcomere) band length# cross‐bridges cyclingBinding of Ca++ to troponin‐C

Cyon‐Frank‐Starling curvesforce of contraction;active tension

resting tension curves

normal

+inotrope

‐inotrope

stiffnormal

Cardiac ou

tput, Stroke volume,

Aortic pressure, dP/dt

max

Figure 2 shows Cyon-Frank-Starling curves expressing the “law-of-the-heart”. The parameters of systolic function are plotted against the degree of preload just before the ventricle contracts as shown by the different size of valentine heart.

2. Cyon-Frank-Starling curves:

Myocardial contractility may be measured/inferred/estimated in either the clinic or physiology laboratory from Cyon-Frank-Starling curves expressing the “law-of-the-heart” (Fig 2).

In this plot, parameters (e.g. CO = cardiac output, AoP = aortic pressure, dP/dtmax = maximal rate of rise of left ventricular pressure) of systolic function are graphed on the ordinate against the degree of stretch on the myocardial fibers just before they contract graphed on the abscissa. The degree of stretch on fibers forming the wall of the left ventricle is, of course, intimately related to the EDV, and (in the clinic) it is termed the preload (PL). Actually, Cyon, Frank, and Starling stated that systolic function generated by the left ventricle depended upon the level of resting stretch on the fibers if all other factors—in particular hindrance to ejection—remained constant (Cyon, 1866; Frank, 1899; Starling, 1918). The molecular mechanism serving as the basis for this relationship is that the degree of stretch on the fibers just before they contract determines the number of heavy meromyosin cross-bridges that may contact binding sites on Tc, and on the avidity with which Ca++ binds to Tc. However, increase in force generation by increasing stretch depends principally on improved binding of Ca++ to Tc.

In Fig 2, the vertical, dotted line, originates from a point on the abscissa that represents a degree of stretch (i.e, PL), the number of heavy meromyosin cross-bridges that cycle, and the avidity with which Ca++ binds to Tc. The horizontal dotted lines from the curves terminate at points on the ordinate that represent systolic function. The curved lines on the graph represent 3 different states of contractility (inotrope). For each inotropic state, the systolic force increases for increases in preload. For each inotropic state, the systolic force decreases for decreases in preload. Increased force can be generated by either increasing PL or from the same preload but increasing contractility. Similarly, decreased force can be generated by decreasing preload or from the same

preload by decreasing contractility.

Thus, systolic function depends upon both the inotropic state and PL, and attempting to infer the inotropic state by examining systolic events without considering if PL is incorrect (Hamlin and del Rio, 2010).

Contractility may be assessed using Cyon-Frank-Starling curves that require assessment of systolic function and knowledge of preload. Of course heart rate, stiffness of the aorta, compliance of the left ventricle, and many other parameters of cardiovascular function must be held constant to comply with the Cyon-Frank-Starling restriction. Systolic function is then monitored by SV, CO, developed left ventricular pressure (i.e. peak systolic minus end-diastolic), aortic pressure, dLVP/dtmax, or many other indices, and preload is altered by increasing or decreasing venous return. The plot of systolic function versus preload (degree of stretch on the fibers just before contraction) produces a baseline Cyon-Frank-Starling curve. Often left ventricular end-diastolic pressure (LVEDP) is used as an assessment of preload (PL). However, PL is determined not only by LVEDP, but also by intrapleural pressure (Ppl) and stiffness of the ventricle (εLV). Therefore, if EDP is substituted for PL, it must be confirmed—or assumed—that both Ppl and εLV were not affected by the intervention. Next, an intervention (drug or disease) that may change contractility is imposed, and another curve of systolic functions versus preloads is generated. The points at equal preloads for the 2 curves are compared. If the points have identical coordinates, there has been no change in contractility; if the points for the second curve are above those for the baseline curve, the intervention was a positive inotrope; if the points for the second curve are below those for the initial curve, the intervention was a negative inotrope.

3. Pressure-Volume (PV) loop, end-systolic pressure-volume relation (ESPVR), and preload recruitable stroke work (PRSW)—time-varying elastance (Ees)

The concept of time-varying elastance (elastance is the reciprocal of compliance or ease of filling) model was established in 1973 by Suga and colleagues (Suga et al., 1973) and has been used as a contractility index in man since 1976 (Weisfeldt, 1976). A small catheter with a high-fidelity micromanometer (Millar instruments, Texas, USA or Scisense, Ontario, Canada) at the tip for measuring the left ventricular pressure and with 4-10 metal rings used for measuring volume (Baan et al., 1984) by electrical impedance is advanced into the ventricle via a carotid artery. A plot (pressure-volume loop) of ventricular pressure (ordinate) versus ventricular volume (abscissa) is generated. In addition, a family of loops is made as the ventricle becomes temporarily smaller due to partial occlusion of venous return. From analysis of this family of loops, maximal information can be gleaned about (1) systolic and diastolic function of the ventricle, (2) how the heart interacts with the arterial tree, and (3) the energetic balance of the heart (i.e. the ratio between oxygen demand and work).

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The single pressure-volume loop:

The left ventricle generates a pressure-volume loop (A, B, C, D) for each cycle of contraction (Fig 3). BD occurs during systole; DB occurs during diastole. Isovolumetric phases, during which times pressures change but volumes do not, are BC for systole and DA for diastole; isotonic phase, during which volumes change but pressure changes only slightly, are CD for systole and AB for diastole. BC is produced when the ventricular pressure rises quickly but the volume does not change. This isovolumetric period occurs because the mitral valve is closed (ventricular pressure exceeds atrial pressure) and the aortic valve has not yet open (ventricular pressure has not yet exceeded the pressure in the aorta resulting from the elastic recoil of the aorta). CD is produced when ventricular pressure exceeds aortic pressure and the ventricle pumps a stroke volume (SV) into the proximal portion of the aorta. In fact, stroke volume is equal to the horizontal distance between C and D. During this phase ventricular pressure elevates only slightly, since energy of contraction is transferred to motion rather than tension. DA is the period of relaxation when ventricular pressure falls rapidly, but there is no change in volume. The rapidity with which this pressure falls is related to the rapidity with which Ca++ is driven off of Tc and resequestered into the SR through the SERCa channel in the SR membrane (Bers, 2002). Energy for this event comes from further hydrolysis of ATP. During this phase of diastole, the ventricle is closed because the aortic valve is closed (aortic pressure exceeds left ventricular pressure) and the mitral valve has not yet opened (left ventricular pressure still exceeds left atrial pressure). It is important to note that, despite the ventricle relaxing, there is no ventricular filling. However, during AB the left ventricle fills even though there is little change in pressure. The aortic valve remains closed but the mitral valve is open. The slope of AB reflects the property of dynamic stiffness. The steeper the slope (i.e., then greater the pressure required to achieve a given volume), the stiffer the ventricle, since a steep slope indicates that a greater pressure is required to achieve each volume. The end-diastolic pressure volume point (B) reflects static stiffness, since it is stiffness at only a single time. Stiffness is the opposite of lusitrope, therefore analysis of the PV loop is useful for estimating both the dynamic and static components of lusitrope. Since time is not expressed in the PV loop, the loop provides no information about isovolumetric events such as rate of decrease in pressure, the maximal value of which is determined by the rate of resequestration of Ca++ from Tc to the SR via the SERCa channel. It is estimated that approximately 50% of the morbidity and mortality from heart disease arise from reduction in lusitrope; therefore, lusitrope is as important a parameter as inotrope (Aurigemma, 2006; Owan et al., 2006).

With respect to valves opening and closing, the mitral valve opens at A and closes at B; the aortic valve opens at C and closes at D. With respect to

Figure 3 shows Pressure-volume loop of the left ventricle.

The mitral valve closes after the onset of systole. This phase is called isovolumic contraction because the mitral valve closed and the aortic valve is not yet opened; therefore, the volume is constant. When the aortic valve opens and the pressure in the ventricle meets the aortic pressure (afterload), the ejection phase begins. Aortic valve closes at the point of end-systolic pressure-volume relationship (ESPVR). The next phase is called isolumic relaxation since the aortic valve closed but the mitral valves are not open yet; therefore, the volume is constant again. The mitral valve opens when the left ventricular pressure falls below the pressure in the left atrium. The blood flows from left atrium to the left ventricle, ventricular filling. The filling phase ends at the point of end-diastolic pressure-volume relationship (EDPVR).

ventricular volume, end-diastolic volume occurs at B but remains until C; end-systolic volume occurs at D but remains until A.

The pressure-volume loops proceeds with counterclockwise rotation. The area within the counterclockwise component of the pressure-volume loop measures total work performed by the ventricle. Two very important instants are B (the end-diastolic pressure-volume point) that estimates end-diastolic stiffness, and D (the end-systolic pressure-volume point) that measures end-systolic stiffness (elastance) which is used for calculating contractility.

Pressure-volume loops are enormously useful for identifying and understanding the pathophysiology of aortic and mitral regurgitation and of an interventricular septal defect. With all 3 of those lesions, the left ventricle changes volume during what should be isovolumetric phases (when no changes ion volume should occur), and the changes and the degree to which they change identifies both the lesion and the magnitude of the abnormal blood flow.

A family of pressure-volume loops:

From the family of PV loops (Fig 4), myocardial contractility may be inferred from either the end-systolic pressure-volume relationship (ESPVR) or from preload recruitable stroke work (PRSW). Both methods require obtaining a family of curves generated by recording individual loops during a brief period of decreased venous return leading to reduction in preload. While recording a number of pressure-volume loops, they are normally

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Figure 4 shows a family of pressure-volume loops. A series

of loops was obtained by inflation of a balloon in the caudal vena cava to reduce preload. Myocardial contractility is measured as the slope of the lines connecting the end-systolic points on the family of loops.

superimposed except for slight variations in their positions produced, when during inspiration, end-diastolic volume and pressure, peak systolic pressure, and stroke volume decrease. During expiration just the opposite occurs. However, if 6 to 10 heart beats, venous return is diminished by occluding the caudal vena cava, a family of pressure-volume loops is generated in which end-diastolic pressure and volume, stroke volume, and end-systolic pressure and volume decrease according to the Cyon-Frank-Starling law-of-the-heart. During diminished venous return, the centroid of the area of the pressure-volume loop drifts leftward and the area diminishes. Three enormously valuable bits of information about the myocardium can be gleaned from this family of curves: (1) contractility, (2) lusitrope, and (3) energetics.

End systolic pressure volume relationship (ESPVR)

ESPVR identify the end-systolic points of pressure-volume for all loops as the heart becomes smaller due to occlusion of the vena cava. Each point refers to end-systolic elastance because it occurs at end-systole, and the ratio of pressure to volume is elastance. Connect the end-systolic points, and construct a straight line (Ees), named the end-systolic pressure volume relationship (ESPVR) that connects the points (Suga et al., 1973; Sunagawa et al., 1983). This line represents time-varying elastance. During some times the points may not fall on the line (i.e. the line may be curvilinear), so it is important to select those points for which the r2 of the line is > 0.9. The slope of the line, Ees, represents myocardial contractility because it expresses the maximal pressure that can be developed from any/all preloads.

The line that represents time-varying elastance also possesses enormous importance in estimating the energetic balance of the heart (Asanoi

et al., 1996). Energetic balance is the ratio between myocardial oxygen demand to oxygen delivery. Myocardial oxygen demand (MVO2) is determined principally by heart rate (HR), myocardial contractility (Vmax), and peak (p) myocardial tension. Whereas peak myocardial tension (Tp) equals aortic diastolic arterial blood pressure (PAodiast.) times end-diastolic radius (EDr) or volume (EDV) of the ventricle divided by end-diastolic wall thickness (EDWT) of the ventricle. Delivery of oxygen depends upon lung function, amount and health of hemoglobin (Hb), and coronary blood flow [Qcor≈(PAo-PRA)/HR]. Thus, energetic balance depends upon HR, Vmax, PAodias., PRA, Hb, EDV, ESV, SV; Vmax, EDV, SV, PAodiast which may be measured from the PV loop and the slope of ESPVR, and other determinates of energetics (e.g. HR, Hb, EDWT) can be measured by other means (e.g. echocardiography).

Though slope of the P-V relation is used as a gold standard for contractility index, there are some potential limitations. Firstly, the end-systolic P-V relation is not always linear. The work of Burkhoff and colleagues (1987) demonstrated that the ESPVR tended to be concave downward at a high Emax and upward at a low Emax. Secondly, Emax must be normalized for different heart size (Suga, 1984). Finally, Emax is slightly dependent upon loading conditions (Sugiura et al., 1989). Despite these limitations, the Emax concept is still considered the gold standard to characterize the myocardial contractility.

Preload recruitable stroke work (PRSW):

Instead of estimating contractility as the slope of the line produced by graphing end-systolic pressure (ESP) versus end-systolic volume (ESV) during reductions in preload and representing ESPVR, contractility may be estimated even better as the slope of the line produced by graphing stroke work (SW) versus left ventricular end-diastolic volume (EDV) during reductions in preload and representing preload recruitable stroke work (Takeuchi et al., 1992; Takaoka et al., 1995; McClain et al., 1998). From each PV loop, SW is taken as SV (EDV-ESV) times ESP, and preload is taken as EDV. Then, a line is produced by graphing SW versus EDV for all PV loops at differing preloads. The line connecting points of PRSW is superior for estimating contractility than the line connecting points of ESPVR for 2 major reasons. First, the points forming PRSW produce a near straight line. Second, the slope of the PRSW line is independent of the size of the animal, i.e. it is nearly the same for all animals. ESP for all animals is approximately 100 mmHg, whereas ESV varies from 0.25 ml for a mouse with an EDV 0.5 ml to 50 ml for a large dog with an EDV of 100 ml. This produces ratios of ESP: ESV of 100: 0.25 (400) for a mouse and 100: 50 (2) for a dog, and of producing highly variable slopes from the highly variable ESP: ESV ratios. On the other hand, for PRSW based upon ratios of SW: EDV, SW is SV x ESP, EDV is EDV, so the SW: EDV ratio is (SV x ESP): EDV, or SV/EDV x ESP. SV/EDV (approximately 0.5) and ESP

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(approximately 100) are fairly constant (approximately 0.5) among species; therefore, the slopes from PRSW have larger r2 and have similar values among species than for ESPVR.

4. dLVP/dtmax

The maximal rate of rise of left ventricular pressure (dP/dtmax or dLVP/dtmax) is obtained by differentiation of the left ventricular pressure pulse. dP/dtmax usually occurs at the end of the period of isovolumetric contraction, just before the ventricle begins to eject. In both experimental and clinical settings, dP/dtmax can be obtained by placing a micro-tip manometer catheter within the left ventricular cavity and recording both left ventricular pressure and its derivative (Fig 5). When left ventricular pressure is constant (i.e. there is no change in pressure), the derivative is 0; when left ventricular pressure increases, dP/dt is positive; when left ventricular pressure decreases, dP/dt is negative; and the dP/dtmax or dP/dtmin are measured, the rates of change of pressure are the greatest. dP/dtmax is species and heart rate dependent, and varies from 400 mmHg/sec for a guinea pig (Hamlin et al., 2004; Mooney et al., 2012), to 7,000 mmHg/sec for a mouse (Reddy et al., 2007), and is between 1000 and 5000 mmHg/s for dog (Markert et al., 2012). The cost for obtaining dP/dt is relatively low, and the technique is quite simple when compared to the gold standard obtained from pressure-volume loops. Although used frequently, dP/dtmax , in fact, may or may not be an index of cardiac contractility, since it is influenced by afterload and preload, and afterload (peak myocardial tension) is influenced by left ventricular radius, pressure and wall thickness that occur at end-diastole just before ejection begins (Katz, 2006). Therefore, the change of dP/dtmax reflects the change of contractility only when other factors that might influence it are unchanged. Recently, Hamlin and del Rio (2012) proposed the new term of dP/dtmax as “baroinometry”. Since it is well-known that dP/dtmax is affected by both afterload (i.e. aortic diastolic pressure) and preload (left ventricular end diastolic pressure). dP/dtmax may decrease in response to arteriolar dilator drugs even though myocardial contractility has not changed or may have even increased (Hamlin and del Rio, 2012). On the other hand, dP/dtmax may increase without an increase in contractility in response to increase venous return via the Cyon-Frank-Starling mechanism (Cyon, 1866; Frank, 1895; Starling, 1918). Schmidt and Hoppe (1978a) demonstrated that the dLVP/dtmax was strongly dependent on preload in the canine heart and lung preparation. The authors also showed that the relation between the dLVP/dtmax and EDP demonstrated the steepest slope right after isolation and continued to decline once the heart deteriorated. The same authors showed that dLVP/dtmax, when compared with other indices of cardiac contractility (e.g. Vmax, VCEmax), was more affected by changes in preload than Vmax. Of course if preload was held constant, then dP/dtmax was a sensitive indicator of contractility (Schmidt and Hoppe, 1978b).

Figure 5 shows left ventricular pressure (LVP; top panel) and

its maximum rate of rise of the LVP (dP/dtmax; bottom panel) of the anesthetized dog.

Figure 6 shows a simultaneous ECG (top trace) and arterial

pressure pulse (bottom trace) from a guinea pig, demonstrating how QA interval is measured. The QA interval represents time from the beginning of Q wave in the electrocardiogram (ECG) tracing to the beginning of the aortic pressure. It is an indirect measure of the duration of isovolumetric contraction without need for left ventricular pressure measurement.

According to the Bowditch effect (Lakatta, 2004), it is also recognized that dP/dtmax, and all other indices of contractility, is influenced by heart rate. That is dP/dtmax will increase when the heart rate (frequency) increases and vice versa when heart rate decreases. In the canine heart and lung preparation, increased heart rate by 30 bpm resulted in a 20% rise of dLVP/dtmax (Schmidt et al., 1979). Although much work has been done to determine the equation for adjusting dP/dtmax to heart rate, Pedraza-Toscano (2006) found that dP/dtmax (mmHg/sec) = 1394 (mmHg/sec) + 12.42HR (beats/minute) in awake, freely-moving dogs. Recently, a complicated equation that has been used for correcting dP/dtmax for heart rate in conscious dogs, minipigs, and cynomolgus macaques has been proposed by Markert and colleagues (2012). The proposed formula was LVdP/dtmaxc = a*(HR)b*exp(c*HR), whereas the parameters (a,b, and c) were estimated using the glm

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function in R 2.13.0, which could be downloaded from http://www.r-project.org/. By using both positive and negative inotropic agents, the formula demonstrated the inotropic effects of drugs independent from the change of heart rate.

5. QA Interval

The QA interval was first purposed as an indirect index of myocardial contractility in 1986 as a result of studies on anesthetized dogs (Cambridge and Whiting, 1986). The shorter the QA interval, the greater the contractility. The QA interval is defined as the duration from the onset of the Q wave on the electrocardiogram (ECG) to the initial upstroke on the aortic pressure wave recorded, most often, from a catheter-tip or transducer located in the aorta between the renal and iliac arteries (Fig 6). It is thought that QA interval measures the duration of isovolumetric contraction without need to catheterize the left ventricle, and that this interval relates inversely to contractility.

However, there are numerous factors—other than myocardial contractility—that contribute to the duration of QA: (1) electropressor latency (time between depolarization and completion of excitation-contraction coupling when contraction begins), (2) isovolumetric contraction (time between onset of contraction and beginning of ejection), (3) the amount of pressure the ventricle must generate from the onset of contraction until the onset of ejection, (4) velocity of propagation of the pulse wave down the aorta (determined principally by the stiffness of the aorta), and (5) distance between the ventricle and the point in the aorta where the pulse is being measured. Therefore, it seems that QA interval depends on many factors other than myocardial contractility (Hamlin and del Rio, 2010) and, if it changes in response to a drug or disease, the change may not be attributed to altered contractility, alone, unless potential effects on other determinants are known.

The QA interval has been demonstrated to correlate inversely with other indices of myocardial contractility (i.e. dP/dtmax) in rats, guinea pigs, dogs, and monkeys (Gras et al., 2000; Takahara et al., 2001; Tontodonati et al., 2007; Priori et al., 2008; Adeyemi et al., 2009; Norton et al., 2009; Mooney et al., 2012; Saengklub et al., 2012). Mostly, the animals were instrumented with a radiotelemetry device for simultaneous recording of electrocardiograms and pulsatile arterial pressure. In both conscious rat and dog, inverse linear correlation between LVdP/dtmax and log10 QA interval was reported (Adeyemi et al., 2009; Norton et al., 2009).

6. Ejection Fraction (EF) and Fractional shortening (FS)

It is well-known that echocardiography is now standard diagnostic non-invasive technique in human cardiologic examinations. The M-mode echocardiography was first described in 1953 by Inge Edler and Hellmuth Hertz (Singh and Goyal, 2007). After that the technique was carried forward and the article about the left ventricular wall thickness,

Figure 7 shows an echocardiogram from a dog. In the short-

axis view of the left ventricle, an M-mode cursor is placed through a left ventricle, perpendicular to the interventricular septum and left ventricular wall just below the tips of the mitral valves at the largest ventricular chamber size (top panel). Bottom panel shows the M-mode image of left ventricular chamber at the middle of the picture and the electrocardiogram located at the bottom below the left ventricular free wall. Dash lines represent the measurement of the left ventricular dimension and the values are demonstrated in the table on the left side of the picture. (Picture by courtesy of Dr. Pedro Vargas Pinto).

internal dimensions, stroke volume, ejection fraction, and valvular regurgitation was first published in 1968 by Feigenbaum and colleagues (1968). Ejection fraction is defined as the fraction of the end-diastolic volume that is ejected as the stroke volume (EF = SV/EDV). It measures how well the heart pumps the blood out which reflects the myocardial contractility. In fact, EF is a mathematic equation and may be obtained from cardiac imaging techniques other than echocardiography (i.e. magnetic resonance imaging, computered tomography). The EF in both experimental animals and patients is usually obtained from M-mode of left ventricular image of echocardiogram (Fig 7). The proper image is obtained by placing M-mode cursor through the septal and posterior LV wall just above the tip of the mitral leaflets in a parasternal long axis view or placed the M-mode cursor directed between the papillary muscles in a parasternal short axis view at the level just beneath the mitral valve (Schiller et al., 1989). By placing a marker of the LV wall dimension during systole and diastole, the echocardiogram machine will generate the number of EF. In several clinically settings, the EF can be used as a predictor of early and late mortality in patients with coronary bypass and patients with heart failure (Soliman Hamad et al., 2010; Campbell et al., 2012).

As with other myocardial contractility indices, the EF is load-dependent with afterload more influential than preload. Moreover, measurement of EF by m-mode evaluates information of myocardial contractility only along a single line. If the regional wall motion of the LV is abnormal (i.e. subject with coronary artery disease), the M-mode beam may not transit through that region which will interpret EF higher than what actually happen. In addition, the M-mode beam usually transverses the LV in a tangential manner which may not reflect the true minor axis

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dimension in some situations (i.e. subject with emphysema or elderly patients) and thus overestimates the true internal dimension (Suga et al., 2000).

Summary/Conclusions

At the present time, data derived from a single PV loop or from a family of PV loops are the closest to ideal index of cardiac contractility due to its less sensitivity to load and frequency. The technique is challenging and will be improved to use in clinical setting in the near future. Other indices (i.e. dP/dtmax, ejection fraction, QA interval, and Vmax) are also usable for the measurement of myocardial contractility with caution of their limitations and assumptions.

Acknowledgement

The author would like to thank Professor Robert L. Hamlin from The Ohio State University and QTest Labs, LLC, USA for his kindness and advice to this manuscript. The author also would like to thank Thailand Research Fund (MRG5480108).

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Thai J Vet Med. 2013. 43(2): 179-186.

Glycerol Concentration Effects on Quality and Longevity of

Post-thaw Goat Semen

Nitira Anakkul Junpen Suwimonteerabutr Theerawat Tharasanit

Nawapen Phutikanit Jinda Singlor Mongkol Techakumphu*

Abstract

This study determined the effects of glycerol concentration (7, 10 and 14% (v/v)) supplemented into freezing

medium on post-thaw sperm qualities. Semen was collected from 3 Black Bengal bucks and then pooled to reduce individual variation. Motility, viability, morphology and acrosome integrity were evaluated before and after cryopreservation. Computer-assisted semen analysis was used to assess sperm motion patterns at 0, 1, 2 and 3 hours post-thawing. The laparoscopic artificial insemination was performed to test the fertilizing ability of frozen-thawed semen. The spermatozoa cryopreserved with 10% glycerol tended to have a higher percentage of motility, progressive motility, spermatozoa with intact acrosome membrane and live spermatozoa especially when examined at 1 hour after thawing. Motion characteristics in all groups were similar except that the beat cross-frequency of spermatozoa frozen with 14% glycerol was significantly lower than the 10% glycerol group (p = 0.0211). The laparoscopic AI using frozen-thawed semen obtained from 10% glycerol group yielded 60% (3/5) conception rate and three live offspring. In conclusion, glycerol concentration affects the qualities and longevity of post-thaw goat semen. The freezing medium containing 10% glycerol sufficiently protected goat spermatozoa against cryoinjury during freezing and thawing, and provided an acceptable pregnancy rate.

Keywords: cryopreservation, glycerol, goat, semen Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330. Thailand *Corresponding author: E-mail: tmongkol@chula.ac.th

Original Article

180 Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 179-186.

บทคดยอ

ความเขมขนของกลเซอรอลมผลตอคณภาพและความยาวนานของการมชวตของอสจแพะภายหลงแชแขงและทาละลาย นธรา อนคกล จนทรเพญ สวมลธระบตร ธรวฒน ธาราศานต นวเพญ ภตกนษฐ จนดา สงหลอ มงคล เตชะกาพ *

การศกษาครงนเพอพจารณาผลของกลเซอรอลทระดบความเขมขนรอยละ 7 10 และ 14 ในสารละลายเจอจางนาเชอสาหรบแชแขงตอคณภาพนาเชอแพะแชแขงหลงการละลาย ทาการรดเกบนาเชอจากพอแพะพนธแบลค เบงกอล จานวน 3 ตว และทาการประเมนอตราการเคลอนทของตวอสจ อตราสวนตวอสจมชวต ความผดปกตของรปรางอสจ และความผดปกตของอะโครโซมในนาเชอสดและภายหลงการละลาย การตรวจประเมนรปแบบการเคลอนทของอสจโดยการใชเครองตรวจวเคราะหอสจ ทาภายหลงการละลายและเกบรกษาท 37 องศาเซลเซยส ท 0 1 2 และ 3 ชวโมง รวมทงการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปเพอทดสอบความสามารถในการปฏสนธของนาเชอภายหลงการแชแขง การศกษาพบวากลมทแชแขงดวยสารละลายทมกลเซอรอลรอยละ 10 มแนวโนมของอตราการเคลอนทของตวอสจ รอยละของอสจทมอะโครโซมปกต และอตราสวนตวอสจมชวต สงกวากลมอนๆ โดยเฉพาะท 1 ชวโมงหลงการละลาย รปแบบการเคลอนทของอสจมลกษณะคลายคลงกนในทกกลมการทดลอง ยกเวนคาความถในการสายหวของอสจในการเคลอนทของกลมทแชแขงดวยสารละลายทมกลเซอ รอลรอยละ 14 มคาตากวา กลมกลเซอรอลรอยละ 10 อยางมนยสาคญ (p = 0.0211) อตราการผสมตดจากการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปโดยใชนาเชอทแชแขงดวยสารละลายทมกลเซอรอลรอยละ 10 คดเปนรอยละ 60 (แพะตงทอง 3 ตว จาก 5 ตว) ไดลกแพะ 3 ตว สรปไดวาความเขมขนของกลเซอรอลในสารละลายเจอจางนาเชอสาหรบแชแขงสงผลตอคณภาพ และความยาวนานของการมชวตของอสจแพะภายหลงการทาละลาย สารละลายเจอจางนาเชอสาหรบแชแขงทมกลเซอรอลรอยละ 10 สามารถปองกนตวอสจจากความเยนทเกดขนระหวางกระบวนการแชแขงและการละลาย รวมทงใหอตราการผสมตดในเกณฑทยอมรบได

คาสาคญ: การแชแขง กลเซอรอล แพะ นาเชอ ภาควชา สตศาสตร เธนเวชวทยา และ วทยาการสบพนธ คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย กรงเทพฯ 10330 *ผรบผดชอบบทความ E-mail: tmongkol@chula.ac.th

Introduction

The application of artificial insemination (AI) is important for genetic improvement programs in goat. However, pregnancy rate following AI with frozen-thawed semen is generally poorer than that obtained from fresh semen (Sohnrey and Holtz, 2005). This indicates that freezing and thawing inevitably induce cryodamage to spermatozoa, thus modification of freezing media to improve the goat sperm viability is required. To date, various freezing media and protocols have been developed aiming specifically at reducing cryoinjury and, in turn, improving the quality of post-thawed spermatozoa. Among factors affecting the freezing ability of spermatozoa, supplementation of freezing medium with various types of cryoprotectant plays a central role in minimizing the physical and chemical stresses occurring during cryopreservation procedure (Purdy, 2006; Peterson et al., 2007). Although it is generally accepted that high concentration of cryoprotectant reduces lethal intracellular ice formation (Mazur and Kleinhans, 2008), this is frequently consequent with

probable toxicity of the particular cryoprotectant (Fahy, 2010). Furthermore, other chemical components added into the freezing medium, in particular non-penetrating cryoprotectant such as lactose (Singh et al., 1995; Chang et al., 2006), trehalose (Aboagla and Terada, 2003; Peterson et al., 2007), and surfactants either sodium dodecyl sulfate (SDS) (Aboagla and Terada, 2004a,b) or Equex STM paste (Anakkul et al., 2011) have also been reported to affect the freezing ability of goat semen.

Glycerol has three hydroxyl (OH) groups which their hydrogen atoms are likely to form H-bonding with the oxygen atoms of the phosphate groups of membrane phospholipids (Kundu et al., 2000). It acts as a membrane-permeable cryoprotectant, resulting in dehydration of spermatozoa due principally to the osmotically driven efflux of intracellular water (Watson, 1995; Amann, 1999). This prevents the sperm from supercooling and also decreases intracellular ice formation. It is most widely used as a membrane-permeable cryoprotectant for preserving mammalian spermatozoa (Tuli and Holtz, 1994). This cryoprotectant is superior for

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protecting buck spermatozoa during cryopreservation to other chemicals (Leboeuf et al., 2000; Purdy, 2006; Peterson et al., 2007). However, suitable amount of glycerol added into the freezing medium remains controversial.

High concentrations (10-14%, v/v) were applied in an early study of buck semen cryopreservation (Ritar et al., 1990) but, at the present time, the glycerol was reduced to 3-9% (v/v) (Leboeuf et al., 2000). Moreover, the amounts of glycerol added into the freezing extender are likely to differ between breeds and individual males (Holt, 2000b), for example, in pigs (Waterhouse et al., 2006), marsupials (Taggart et al., 1996) and African antelopes (Loskutoff et al., 1996). The excess amount of glycerol induces osmotic damage to spermatozoa, hence reducing post-thaw semen quality (Holt, 2000a). On the other hand, too low glycerol concentration poorly protects the spermatozoa against cryoinjury (Silva et al., 2002). Therefore, appropriate glycerol concentration in the freezing medium, as well as a long-term consequence such as frozen-thawed semen longevity and conception rate following AI should be cautiously determined. In this study, we used semen from Black Bengal bucks as a model since they have high climate-adaptability, fertility and superior skin quality (Husain et al., 1996). However, spermatozoa from Black Bengal buck are quite sensitive to cold stress, and large variability of post-thaw sperm motility (5 to 50%) have been reported (Biswas et al., 2002; Afroz et al., 2008).

This study was carried out to investigate the effects of different glycerol concentrations on the quality and longevity of frozen-thawed Black Bengal buck spermatozoa. The pregnancy rate after laparoscopic artificial insemination with frozen-thawed semen was additionally examined.

Materials and Methods Chemical reagents: All chemicals used in this study were purchased from Sigma St Louis, USA, unless otherwise specified.

Experimental animals: This study was approved by the Institutional Animal Care and Use Committee (IACUC), Chulalongkorn University (Approval No. 11310030). Three Black Bengal bucks, aged between 2 to 4 years old, were used in this experiment. The animal facility was located at the Veterinary Student Training Center, Nakhon Pathom Province (latitude 13oN and longitude 100oE). The animals were fed concentrates containing 14% (w/w) protein, ad libitum of grass with free access to mineral salt blocks and water.

Semen processing and cryopreservation: Semen from three bucks was collected twice a week using an artificial vagina during May to August 2011 (summer to rainy season). The ejaculate volume was evaluated from a graduated collection tube. Semen pH was evaluated by a pH-indicator paper (Neutralit®, Merck, Darmstadt, Germany). Mass movement and progressive motility of fresh and equilibrated semen were subjectively evaluated under a phase contrast

microscope (CX41RF; Olympus, Japan). Sperm concentration was calculated using a hemocytometer (Neubauer, Boeco, Germany) after a 1: 400 dilution with formal saline solution. The ejaculates with an acceptable progressive motility (> 65%) and concentration (> 1,500x106 spermatozoa/ml) were selected and pooled for further processes.

Tris-citric acid-fructose (TCF) solution (pH 7.0 to 7.2), consisting of 250 mM Tris (hydroxymethyl animomethane), 90 mM citric acid, 70 mM fructose (BDH, Poole, UK), 100 IU/ml penicillin G and 100 µg/ml streptomycin, was supplemented with 10% (v/v) fresh hen egg yolk and 1% (v/v) Equex STM paste (Nova Chemical Sales Inc, Scituate, USA). The extender containing different glycerol concentrations (7, 10 and 14% (v/v) glycerol) was then finally prepared. To prepare each 10 ml of three different glycerol concentration in extender in brief, the TCF solution (7.5x3 ml) was diluted with egg yolk (1x3 ml) and Equex STM paste (0.1x3 ml). After mixing well, the medium was separated into 3 aliquots (7.5 ml each). The three amounts of glycerol (0.7, 1 and 1.4 ml) and TCF (0.7, 0.4 and 0 ml) were then added to each aliquot, respectively.

Pooled samples were diluted with TCF (1 : 9 at 370C) and then centrifuged at 940 x g for 10 min at room temperature in order to remove the seminal plasma. The supernatant was discarded, and the sediment containing spermatozoa was divided into three equal aliquots, each was diluted with extender containing 7, 10 or 14% glycerol. The sperm concentration was finally adjusted to 400x106 spermatozoa/ml. The diluted semen was then equilibrated at 40C for 4 hours. After equilibration, the semen was loaded into a 0.25 ml French mini straw (Minitüb, Landshut, Germany), and the open-end was sealed with polyvinyl powder. The straws were placed horizontally in liquid nitrogen vapor at 4 cm above the liquid nitrogen level in a styrofoam box for 10 min and then plunged directly into liquid nitrogen. Semen thawing was performed at 370C in a water bath for 30 sec (Deka and Rao, 1987; Leboeuf et al., 2000).

Assessment of semen quality: The fresh, equilibrated and post-thaw samples were assessed for motility, morphology, viability and acrosome integrity. Motion analysis of frozen-thawed semen was additionally performed using the computer-assisted semen analysis (CASA, Hamilton-Thorne Biosciences IVOS, Version 12.3, Bevery, MA, USA). To evaluate the motility of fresh semen, samples were blind-assessed under a phase contrast microscope (100x) by the same technician throughout the experiment. The CASA procedure/setting was performed as the manufacturer's recommendation. The frame rate was set up at 60 Hz (Sundararaman and Edwin, 2008; Bucak et al., 2010). The frozen semen samples to be analyzed with CASA were thawed and diluted (1: 9) with phosphate buffered saline and incubated at 370C throughout the evaluation process. The motility assessment was carried out at four time points: within 5 min post-thawing (T0), 1 hour (T1), 2 hours (T2) and 3 hours (T3) post-thawing. For CASA, 10 µl

182 Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 179-186.

of the diluted sample was placed in a 2X-CEL chamber slide (Hamilton-Thorne, Inc, MA, USA) pre-warmed at 370C prior to analysis. Three different fields were selected and scanned for motility (MOT, %), progressive motility (PMOT, %), average path velocity (VAP, µm/s), straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/sec), amplitude of lateral head displacement (ALH, µm), beat cross-frequency (BCF, Hertz), straightness (a ratio of VSL/VAP; STR, %) and linearity (a ratio VCL/VAP; LIN, %). The mean value calculated from three analyzed fields was used for statistical analysis.

The viability of the spermatozoa was evaluated following eosin–aniline blue staining (Peterson et al., 2007). The morphology of 500 sperm heads was evaluated after William’s staining. The viability and head morphology of spermatozoa were examined under a bright-field microscope (1000x) (Eclipse E200; Nikon, Japan). For tail morphology, a total of 200 spermatozoa fixed in formal saline solution were examined under a phase contrast microscope (400x) (CX41RF; Olympus, Japan). A fluorescein isothiocyanated peanut agglutinin (FITC-PNA) staining was used to evaluate the acrosome integrity as previously described by Axnér et al. (2004), with minor modifications (Anakkul et al., 2011). In brief, 2 µl of sperm suspension was smeared onto a glass slide, and then the sperm membrane was permeabilized with 95% ethanol for 30 seconds. The sperm smear was spread over with the mixture of FITC-PNA (100 µg/ml in PBS) and propidium iodide (PI, final concentration 18 µM) and incubated in a moist chamber at 40C for 30 min then rinsed with 40C distilled water before air drying. At least 200 spermatozoa per sample were evaluated using an epifluorescent microscope (BX51; Olympus, Japan). Spermatozoa with intensive bright fluorescent acrosomal cap indicated the acrosome-intact spermatozoa.

Laparoscopic artificial insemination and pregnancy diagnosis: To examine the fertilizing ability of frozen-thawed semen providing the best freezing protocol from the experiment, which was 10% glycerol, five female 75% Saanen does, aged between 2-3 years old, were used for laparoscopic artificial insemination. The estrus was synchronized by an intravaginal progesterone sponge containing 65 mg of Medroxyprogesterone (Sincro-gest sponges®; Laboratorios Ovejero, Leon, Spain) for 13 days. An injection of 300 IU pregnant mare serum gonadotropin (Sincro-gest PMSG®; Laboratorios Ovejero, Leon, Spain) was done two days before the sponge removal. The estrus detection was carried out using a teaser buck and the insemination was done at 24 hours (Martinez-Rojero et al., 2007) after the standing estrus. The does were sedated with xylazine hydrochloride (0.1 mg/kg) and ketamine (4 mg/kg), and were inseminated using laparoscopic technique as described by Mobini et al. (2002) with some modification. In brief, the does were sedated and placed in dorsal recumbency, the animal’s head downwards. The carbon dioxide (CO2) was inflated in the abdomen via verres needle. Two incisions were made at approximately 5 cm cranial to the udder and

4 cm to either side of the midline to facilitate the penetration of trocar and cannula. The 5 mm laparoscope (Schölly, Denzlingen, Germany) was inserted through the first incision, and then a post-thawed spermatozoon was inseminated through the other incision. A total of 120x106 frozen-thawed spermatozoa were deposited into the lumen of both uterine horns (60x106 spermatozoa per horn). Pregnancy was diagnosed at 45 days post insemination by a real-time B mode ultrasound (HS-2000, Honda Electronics Co, Ltd, Aichi, Japan). The numbers of live birth were also recorded.

Statistical analysis: The results were presented as means with pooled SEM. Semen characteristics of pooled samples were descriptively analyzed. The comparisons among semen parameters were analyzed by general linear model procedure (GLM) using the Statistical Analysis Systems software package (Version 9.0, SAS Institute Inc, 1996, Cary, NC, USA). The values with p < 0.05 were considered statistically significant.

Results The semen characteristic data of pooled samples were summarized in Table 1. The mean volume per ejaculate of fresh semen from all bucks was 0.59±0.5 ml. Following the addition of freezing extender, the spermatozoa were slowly cooled at 40C for 4 hours prior to spermatozoa evaluation. It was found that different concentrations of glycerol supplemented in the freezing extender did not affect the spermatozoa quality, in terms of viability, acrosome integrity, percentage of normal morphology (Table 2, p > 0.05). There were also no significant differences among glycerol concentrations for post-thaw viability and percentages of normal sperm morphology in all incubation times (Table 2). Nevertheless, spermatozoa cryopreserved with 10% glycerol tended to have a higher percentage of live spermatozoa when compared to other concentrations. This finding coincided with the results that the 10% glycerol preserved integrity of acrosomal membrane better than other concentrations especially when examined at T1. Although MOT and PMOT gradually decreased along the incubation time, the buck spermatozoa frozen with 10% glycerol showed superior results of MOT and PMOT compared to the 7% and 14% glycerol groups in all evaluation times (Table 3). The significant differences were shown at T0 for PMOT and at T0 and T1 for MOT (p < 0.05). Motion characteristics in all glycerol groups were similar except that the beat cross frequency of the

Table 1 Semen characteristics of pooled samples obtained from Black Bengal goat bucks

Semen parameter Mean±SEM pH Mass movement (0-4 score) Motility (%) Concentration (x 109/ml) Viability (%) Normal head morphology (%) Normal tail morphology (%) Acrosome intact spermatozoa (%)

7.6±0.5 2.6±0.9 68.1±1.8 3.6±1.3 77.3±2.4 98.5±1.2 90.4±2.3 88.2±2.3

Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 179-186. 183

spermatozoa frozen with 14% glycerol was significantly lower than the 10% glycerol group (p = 0.0211, Table 3). The spermatozoa frozen with the 10% glycerol protocol retained the fertilizing ability since the ultrasound scanners diagnosed 60% of pregnancy (three of five does) with 100% accuracy. This resulted in a total of three live offspring.

Discussion

Goat sperm are sensitive to cryopreservation as sperm quality and pregnancy are usually poorer than those obtained from non-cryopreserved sperm (Medeiros et al., 2002; Apu et al., 2012). In this study, we found that the amount of glycerol supplemented

Table 2 Mean percentages of viability, acrosome intact, normal head and normal tail morphology of equilibrated and post-thaw spermatozoa frozen with different glycerol concentrations (7, 10 and 14%)

Parameter Viability Intact acrosome Normal head Normal tail (%) (%) morphology (%) morphology (%) Post-equilibration 7% glycerol 65.4 80.5 98.9 86.9 10% glycerol 66.5 79.4 99.3 85.1 14% glycerol 61.09 80.4 99.4 84.1 Post-thawing (T0) 7% glycerol 33.3 83.7 99.2 91.4 10% glycerol 40.4 87.8 99.8 90.3 14% glycerol 31.4 86.8 98.9 91.1 Post-thawing (T1) 7% glycerol 14.1 74.7ab 99.2 91.6 10% glycerol 21.7 80.1a 98.8 91.9 14% glycerol 21.5 70.7b 99.2 91.4 Post-thawing (T2) 7% glycerol 12.3 66.9 99.1 91.4 10% glycerol 14.8 69.5 98.9 92.0 14% glycerol 14.3 67.2 99.0 93.2 Post-thawing (T3) 7% glycerol 9.7 64.7 99.3 91.9 10% glycerol 10.4 66.6 98.7 91.9 14% glycerol 10.3 57.3 99.0 93.2

SEM 1.44 1.40 1.07 0.48

Statistical analysis was used to compare the values obtained from different glycerol concentrations. Within the same sperm parameter, different superscripts denote values that differ statistically significant (p < 0.05). SEM indicates standard error of the mean.

Table 3 Motility characteristics of post-thaw spermatozoa frozen in freezing extender supplemented with 7, 10 and 14% glycerol

Parameter MOT (%) PMOT (%) VAP ( / )

VSL ( / )

VCL ( / )

ALH ( )

BCF ( )

STR (%)

LIN (%)Post-thawing (T0)

7% glycerol 30.9a 7.6a 84.1 56.1 161.7 7.5 32.7 64.6 35.7

10% glycerol 42.3b 12.0b 90.8 62.4 173.7 7.4 32.9 66.0 36.0

14% glycerol 37.4ab 8.3a 79.5 51.1 157.7 7.2 30.6 64.4 35.9

Post-thawing (T1)

7% glycerol 18.7a 6.2 70.3 53.0 133.0 6.9 32.4 72.9 41.9

10% glycerol 28.3b 9.7 77.5 55.9 149.4 7.3 32.1 69.3 38.0

14% glycerol 18.9a 6.2 69.2 50.1 133.5 6.9 32.7 69.8 38.6

Post-thawing (T2)

7% glycerol 11.4 4.0 64.7 48.5 120.2 6.2 31.2 71.8 40.1

10% glycerol 17.7 6.2 62.9 46.4 122.6 5.9 34.4 68.8 37.7

14% glycerol 14.3 4.8 58.8 43.3 113.3 6.3 32.7 69.6 38.1

Post-thawing (T3)

7% glycerol 7.1 2.1 51.8 38.7 102.4 5.6 31.0ab 72.7 41.3

10% glycerol 11.6 4.0 57.1 41.7 103.2 5.7 32.3a 68.8 38.6

14% glycerol 10.0 3.3 52.3 38.5 102.6 5.8 28.4b 71.5 41.7

SEM 1.29 0.66 2.10 1.83 3.80 0.24 0.67 1.22 1.28

MOT: motility, PMOT: progressive motility, VAP: average path velocity, VSL: straight line velocity, VCL: curvilinear velocity, ALH: amplitude of the lateral head displacement, BCF: beat cross-frequency, STR: straightness, LIN: linearity. Within a column and time point, different superscripts denote values that differ statistically significant (p < 0.05). SEM indicates standard error of the mean.

184 Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 179-186.

into the freezing extender affected the post-thaw sperm quality. Following semen collection, some semen parameters were slightly different compared with previous reports such as lower in motility but higher in semen volume and concentration (Biswas et al., 2002; Apu et al., 2008). This may be a consequence from the variation of age, semen collection method, living condition and individual effect (Apu et al., 2008). Overall, the observed semen characteristics in this study were typical of sexually mature Black Bengal bucks as previously reported (Khan, 1999; Apu et al., 2012). This present study indicated that the concentration of glycerol in freezing extender affected the quality and longevity of cryopreserved semen. The addition of 10% glycerol in the TCF extender supplemented with 10% hen egg yolk and 1% Equex STM paste significantly improved the percentage of PMOT and MOT of the spermatozoa after thawing (PMOT and MOT) or until 1 hour of incubation (MOT), whereas the lowest sperm quality was observed in the extender containing 7% glycerol. The appropriate amount of glycerol increases the membrane fluidity by rearranging the membrane lipid and protein, thus increasing the survival ability of spermatozoa during cryopreservation (Holt, 2000a). The range of 3 to 14% glycerol is generally used in freezing extender for several goat breeds (Leboeuf et al., 2000) while 5 and 7% glycerol are frequently reported for Black Bengal breed (Biswas et al., 2002; Afroz et al., 2008; Apu et al., 2012). However, we found that the 7% glycerol protocol poorly protected the spermatozoa against cryoinjury during freezing and thawing procedure. This finding was in agreement to other reports indicating that the addition of 7% glycerol was not suitable for proper dehydration of the goat sperm cells (Afroz, 2008). By contrast, Biswas et al. (2002) reported that 7% glycerol protocol produced higher post-thaw Black Bengal sperm motility than the 10% glycerol supplementation (51±1% and 7±1%, respectively). The different results may be due to different freezing extender compositions, for example, amount of egg yolk, kind of sugar and absence of Equex STM Paste.

Moreover, the latter study used subjective sperm evaluation which variations of 30 to 60% have been reported, while this study used CASA, an objective method (Verstegent et al., 2002) which provides more accurate estimates and has demonstrated to be a useful tool to evaluate kinematics properties of individual spermatozoa (Tuli et al., 1992; Mortimer, 2000; Verstegent et al., 2002). On the other hand, poor sperm quality obtained from a high glycerol concentration (14%) probably causes an extreme osmotic stress that can damage the cell structures and impair the sperm motility (Deka and Rao, 1986; Gil et al., 2003; Sönmez and Demirci, 2004; Sundararaman and Edwin, 2008).

In this study, glycerol concentration did not significantly affect velocity or speed characteristics (VAP, VSL and VCL) as well as STR and LIN in frozen-thawed semen. Only BCF at T3 showed a significant difference between the 10 and 14% glycerol

groups (p = 0.0211). BCF is a frequency of sperm head crossing the sperm average path in Hertz. It gives an indication of the flagella beat frequency and assesses the time that flagella beat changes its pattern, which low BCF values may impair the penetrating ability of spermatozoa (Mortimer, 1997). We found that BCF values were different, despite the similar head and tail morphology between groups. It is possible that optimal glycerol and Equex STM paste concentration used in the freezing extender synergistically protected the spermatozoa plasma membrane (Peña et al., 2003). Although the numbers of inseminated does would have been increased, the pregnancy and kidding rate indicated conclusively that the spermatozoa frozen with the 10% glycerol protocol retained the fertilizing ability. The pregnancy result obtained was in a normal range (20% to 90%) reported in laparoscopic insemination (Mobini et al., 2002).

In conclusion, the glycerol concentration in semen freezing extender affected the quality and longevity of frozen-thawed Black Bengal buck semen. The freezing medium containing 10% glycerol sufficiently protected goat spermatozoa against cryoinjury during freezing and thawing, and provided an acceptable pregnancy rate. Further examination of other factors that interact and influence the spermatozoa quality following freezing and thawing remains to be studied.

Acknowledgements

Nitira Anakkul is a Ph.D. student under the

Royal Golden Jubilee (RGJ) Ph.D. Program, Thailand Research Fund. The authors are grateful to Dr. Em-on Olanratmanee, Faculty of Veterinary Science, Chulalongkorn University for advice in statistical analysis. This work was financially supported by the Chulalongkorn University Centenary Academic Development Project. The CASA evaluation was kindly provided by the Zoological Park Organization, Thailand.

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Thai J Vet Med. 2013. 43(2): 187-195.

Expression of Oestrogen Receptor Alpha and Progesterone

Receptor in Sow Oviducts after Different Artificial

Insemination

Sayamon Srisuwatanasagul1* Padet Tummaruk2 Annop Kunavongkrit3

Abstract

In pig production, different insemination methods which allow deposition of sperm in the uterine body

(intrauterine insemination, IUI) and uterine horn (deep intrauterine insemination) have been applied in order to reduce number of spermatozoa. Earlier studies reported that steroid hormones influenced the transportation of spermatozoa, ovum and embryos in the sow reproductive tract which was related to the presence of their specific receptors; oestrogen receptor alpha (ERα) and progesterone receptor (PR). Therefore, the present study aims to evaluate the immunolocalization of ERα and PR in different parts of sow oviducts, after different artificial insemination methods. Twelve sows were divided into 3 groups according to the different insemination methods, which were AI (n = 3), IUI (n = 4) and DIUI (n = 4). Percentage of ERα and PR immunostaining was evaluated by manual scoring and image analysis system. Results showed significantly higher percentage of positive staining in AI group compared with IUI and DIUI groups in the oviduct for ERα and PR. It has been demonstrated that oestrogen (E2) in boar semen can up-regulate steroid receptors in the pig reproductive tract. The small volume of semen used for IUI and DIUI groups might also influence the lower expression of these steroid receptors due to the lower amount of E2. In conclusion, the different insemination methods regarding the volume of semen can have the effects on the expression of steroid receptors in the sow oviduct.

Keywords: artificial insemination, deep intrauterine insemination, intrauterine insemination, oestrogen receptor

alpha, progesterone receptor 1 Department of Veterinary Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330 Thailand 2 Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330 Thailand 3 Office of the Commission on Agricultural Resource Education (OCARE), Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand *Corresponding author: E-mail: ssayamon@chula.ac.th

Original Article

188 Srisuwatanasagul S. et al. / Thai J Vet Med. 2013. 43(2): 187-195.

บทคดยอ

การแสดงออกของตวรบฮอรโมนเอสโตรเจนชนดแอลฟาและตวรบฮอรโมนโปรเจสเตอโรนในทอนาไขของสกรนางหลงจากการผสมเทยมดวยวธตางกน ศยามณ ศรสวฒนาสกล 1* เผดจ ธรรมรกษ 2 อรรณพ คณาวงษกฤต 2

ในอตสาหกรรมการผลตสกรไดมการนาเทคนคตาง ๆ ของการผสมเทยมมาประยกตใช โดยทาการปลอยนาเชอทตาแหนงมดลก (การผสมเทยมแบบปลอยนาเชอทมดลก, IUI) และทปกมดลก (การผสมเทยมแบบปลอยนาเชอทปกมดลก, DIUI) เพอลดความเขมขนของจานวนอสจในนาเชอลง จากการศกษากอนหนานพบวาสเตยรอยดฮอรโมนมความเกยวของกบการขนสงตวอสจ เซลลไข และตวออนในทอทางเดนสบพนธของสกรเพศเมยโดยมความสมพนธกบการแสดงออกของตวรบสเตยรอยดฮอรโมนทจาเพาะคอ ตวรบฮอรโมนเอสโตรเจนชนดแอลฟา และตวรบฮอรโมนโปรเจสเตอโรน การทดลองในครงนมจดประสงคเพอศกษาการแสดงออกของตวรบฮอรโมนเอสโตรเจนชนดแอลฟาและตวรบฮอรโมนโปรเจสเตอโรนในสวนตาง ๆ ของทอนาไขของสกรนาง โดยใชสกรนางจานวนทงสน 12 ตวแบงเปน 3 กลมตามวธการผสมเทยมไดแก การผสมเทยมแบบดงเดม การผสมเทยมแบบปลอยนาเชอทมดลก และการผสมเทยมแบบปลอยนาเชอทปกมดลก โดยผลการศกษาจะถกวเคราะหสองวธ คอ วธการใหคะแนนจากภาพ และการใชโปรแกรมวเคราะหภาพ ผลการศกษาพบวาการแสดงออกของตวรบฮอรโมนเอสโตรเจนชนดแอลฟาและตวรบฮอรโมนโปรเจสเตอโรนในทอนาไขสกรนางมระดบสงในกลมทผสมเทยมแบบดงเดมเปรยบเทยบกบกลมทผสมเทยมแบบปลอยนาเชอทมดลกและปกมดลก จากการศกษากอนหนานพบวาในนาเชอสกรนนมระดบของฮอรโมนเอสโตรเจนในระดบสงทาใหสามารถกระตนใหเกดการแสดงออกของตวรบฮอรโมนทเพมขนไดในอวยวะสบพนธของสกร ดงนนการผสมเทยมดวยวธปลอยนาเชอทตาแหนงมดลกและปกมดลกซงใชปรมาณของนาเชอและตวอสจลดลงอยางมากอาจสงผลใหการแสดงออกของตวรบฮอรโมนนอยกวาในกลมทผสมเทยมแบบดงเดม จากผลการศกษาครงนสรปไดวาการผสมเทยมดวยวธตาง ๆ มอทธพลตอการแสดงออกของตวรบสเตยรอยดฮอรโมนในระบบทางเดนสบพนธของสกรนาง

คาสาคญ: การผสมเทยมแบบดงเดม การผสมเทยมแบบปลอยนาเชอทปกมดลก การผสมเทยมแบบปลอยนาเชอทมดลก ตวรบฮอรโมนเอสโตรเจนชนดแอลฟา ตวรบฮอรโมนโปรเจสเตอโรน

1 ภาควชากายวภาคศาสตร คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 2 ภาควชาสตศาสตร เธนเวชวทยา และ วทยาการสบพนธ คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 3 สานกงานคณะกรรมการการศกษาวจยทรพยากรการเกษตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 *ผรบผดชอบบทความ E-mail: ssayamon@chula.ac.th

Introduction

In Thailand, artificial insemination (AI) has an important role in swine industry in order to increase the efficiency in swine production. Regarding conventional artificial insemination, diluted fresh semen was used and released at the cervix of female pigs. This can reduce the numbers of spermatozoa to 5-10 folds compared to natural mating. However, Sumransap et al. (2007) found that this technique caused a loss of more than 90% of spermatozoa before they could reach the fertilization site which was caused by a drawback of the semen (Steverink et al., 1998). In order to overcome this problem, a newer technique of artificial insemination was introduced. It is the insertion of insemination cohet into the female reproductive tracts and the release of semen in the uterus, which is called “intrauterine insemination” (IUI), and deeper into the uterine horn, which is called

“deep intrauterine insemination” (DIUI) without any surgical treatment. An early study on the IUI technique by Watson and Behan (2002) showed that the number of spermatozoa used was only 1x109 per dose which did not affect the number of fertilization rate. In addition, a study by Martinez et al. (2001; 2002) revealed that the DIUI technique, which released the semen at the uterine horn, could reduce the concentration of the semen to 60 folds compared to convention AI. In Thailand, the number of 150 million spermatozoa was used for DIUI technique which resulted in the number of fertilization rate in 5 from 8 gilts. Moreover, the embryos were also found in both sides of uterine horn with the average of 11.4 embryos per gilts (Tummaruk et al., 2007). This certifies that these new methods of artificial insemination may be able to use with freezing semen, sex determination semen and may also be able to develop to use for embryonic transfer in the future.

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The mechanism of sperm transportation in the female reproductive tracts is complicated and regulated by several biological factors both from the female as well as the quality and concentration of semen (Rodriguez-Martinez et al., 2005). After insemination, spermatozoa will be transported through the reproductive tract to sperm reservoir. Studies revealed that ovulation could affect sperm distribution and transportation, i.e. sperm distribution from sperm reservoir, by the changes in hormonal levels during ovulation (Hunter, 2008). In addition, the contraction of female reproductive tract was also needed in order to transport sperm to sperm reservoir (Langendijk et al., 2002a). Moreover, the contraction of the oviduct took part in the transportation of ovum to the fertilization site (Orihuela et al., 2001).

The ovarian steroid hormones, mainly oestrogen and progesterone, interplay the roles of controlling the morphological and functions of female reproductive organs of all mammals, e.g. control of reproductive cycle, ovulation as well as pregnancy (Cooke et al., 1998; Spencer and Bazer, 2002; Lessey, 2003; Drummond, 2006). These steroid hormones elicit their functions by binding through specific receptor proteins in target tissues (Jensen, 1991; O'Malley et al., 1991; Yamashita, 1998), therefore the presence of steroid receptors is as important as the levels of steroid hormones as they involve in the effective functions of reproductive control. Several studies reported the different localization of steroid receptor proteins in various reproductive organs and it was shown that steroid receptors such as oestrogen receptors and progesterone receptors could be found mainly in the uterus, cervix, oviduct and ovary (Mowa and Iwanaga, 2000; Pelletier and El-Alfy, 2000; Pelletier et al., 2000; Wang et al., 2000). However, the study of steroid receptors in newly wean anoestrous sows demonstrated the high presence of steroid receptors in the uteri, although the level of steroid hormones, oestradiol 17-β and progesterone, were low (Sukjumlong et al., 2004). Moreover, the presence of ER in the gene level was involved in the reproductive performance of the pigs (van Rens et al., 2000; Isler et al., 2002). Though the studies of steroid receptors in normal reproductive tracts are widely documented, there are no data of these receptors localization in the different parts of the sow oviduct after different artificial insemination methods. Therefore, the present study aims to investigate the presence of steroid receptors, oestrogen receptor subtype alpha (ERα) and progesterone receptor (PR) in the sow oviduct after different artificial insemination methods.

Materials and Methods Animals: Twelve crossbred Landrace x Yorkshire multiparous sows were purchased from a commercial swine herd and were brought to the department of Obstetrics, Gynaecology and Reproduction, Nakhon Pathom province, Thailand on the day of weaning. The sows were kept in individual pen and were fed twice a day (approximately 4.0-5.0 kg per day) on a commercial feed (Starfeed176® BP Feed Co Ltd,

Saraburi, Thailand) containing 15.0% protein, 2.0% fat and 10.0% fiber. Water was provided ad libitum via water nipples. The sows were carefully detected for the onset of standing estrus twice a day (am/pm) after weaning. The proposal of the present study was approved by the Institutional Animal Care and Use Committee (IACUC) (Approval No. 1031018), Chulalongkorn University.

Detection of estrus and ovulation: Estrus detection was initially performed on the day after weaning (Day 1), by allowing the sows to have nose-to-nose contact with a mature boar and applying the back pressure test. Sows with a standing reflex were considered in estrus. The onset of estrus was defined as the first time the sow showed standing reflex minus 6 hours. At standing estrus, hCG (Chorulon®, Intervet Ltd, Boxmeer, The Netherlands) 750 IU was administrated intramuscularly to the sows in order to induce ovulation (Wongkaweewit et al., 2012). The time of ovulation was determined by monitoring an appearance of the follicles every 8 h using transrectal realtime B-mode ultrasonography adjusted to a 5-MHz linear transducer (Honda Electronics Co, Ltd, Tokyo, Japan). The ovulation time was defined as 4 hours before the first time when no follicle was visible.

Artificial insemination: All sows were divided into 3 groups according to the insemination methods, which were conventional artificial insemination (AI, n = 4), Intrauterine insemination (IUI, n = 4) and Deep intrauterine insemination (DIUI, n = 4). The sows were inseminated with a single dose of diluted semen during the second oestrus after weaning. The time of ovulation during the first oestrus was used to determine the timing of insemination, which was carried out at 6-8 hours prior to the expected time of ovulation. Semen with a motility of > 70%, a concentration of > 150x106 spermatozoa/ml and with normal sperm > 85% was extended with Beltsville thawing solution (Pursel and Johnson, 1976). The sperm dose contained 3000x106 spermatozoa in 100 ml for AI, 1000x106 spermatozoa in 50 ml for IUI and 150x106 spermatozoa in 5 ml for DIUI. Both the IUI and the DIUI methods were adapted from Sumransap et al. (2007). Briefly, after cleaning the perineal area of the sows, a commercial AI catheter (Goldenpig®, Minitube, Tiefenbach, Germany) was inserted through the vagina into the cervix, where the diluted semen was deposited in AI group. In IUI group, the IUI device (Magaplus®, Magapor, Ejea de los Caballeros, Spain) was inserted through the vagina into the cervix. Thereafter, the inner tube extended about 20 cm beyond the tip of the outer catheter and resided in the uterine body or the posterior uterine horn in order to deposit the diluted semen. In DIUI group, the long flexible catheter (1.8 m) was inserted through the conventional AI catheter. This long catheter was moved forward and deposited diluted semen in the uterine body along one uterine horn (unknown side) for its full length. The diluted fresh semen with 150x106 motile sperm in 5.0 ml was deposited in the proximal third of one side of the uterine horn. Subsequently, a warm BTS, 2.5 ml in volume, was used to flush the semen into the uterine

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horn after insemination.

Tissue collection: Approximately 12 hours after insemination, all sows were slaughtered and the reproductive tracts were removed. Post-mortem examination was performed on each part of the reproductive organs. Three different parts of the oviduct, which were isthmus, ampulla and infundibulum, were collected and preserved separately in 4% paraformaldehyde for 24-36 hours. Thereafter, they were routinely histological processed and 4 µm thick sections were cut from each block and mounted on Polysine™ slides (Menzel-Glazer, Germany). One section from each block was stained with Hematoxylin-Eosin for histological investigation and the other sections were used for immunohistochemistry.

Immunohistochemistry: Before immunohisto-chemistry, sections were deparaffinized in xylene and rehydrated in graded alcohol. The immunohistochemical protocol was described previously by Sukjumlong et al. (2003). Briefly, antigen unmasking technique by mean of heating in the microwave at 750 watt (in 0.01M citrate buffer, pH 6.0) was performed, followed by endogenous peroxidase blocking in 3% hydrogen peroxide in methanol as well as a background blocking with normal horse serum. A standard avidin-biotin immunoperoxidase technique (Vectastain® ABC kit, Vector Laboratories, Inc, USA) was applied to detect ERα and PR. The primary antibodies used were mouse monoclonal antibody to oestrogen receptor alpha, ERα (clone 1D5, Dako Denmark, dilution of 1: 25) and mouse monoclonal antibody to PR (clone 10A9, Immunotech, dilution of 1: 200). The PR primary antibody can recognize both PR-A and PR-B, so the results shown in the present study was the accumulation of PR-A and PR-B. The incubation time for both primary antibodies was 1 hour at room temperature. The sow uterus at oestrus which was known to contain both ERα and PR was served as positive controls, while the negative controls were obtained by replacing the primary antibodies to ER� or PR with normal mouse IgG (sc-2025, Santa Cruz Biotechnology Inc, USA) in a dilution of 1: 200. In the final step, a chromogen which was 3,3’-diaminobenzidine (DAB, Dakopatts AB, Älvsjö, Sweden) was added to visualize the bound enzyme (brown color). All sections were counterstained with Mayer’s hematoxylin followed by mounting in glycerine-gelatin before investigation.

Evaluation of the results and statistical analysis: The oviduct was divided into 3 parts which were isthmus, ampulla and infundibulum. The results of the immunostaining were evaluated semi-quantitatively by a manual scoring method as well as image analysis by computer software (Image-pro plus version 6.0, Media Cybernetics, Inc, MD, USA).

The manual scoring of ERα and PR positive cells was done by classification into three different levels of intensity: weak, 1; moderate, 2 and strong, 3. Since not all cells stained positively in some

compartments of the tissues, the proportion of positive to negative cells was also included for these tissues. The proportions were estimated into four different levels (marked 1-4): low proportion (< 30% of positive cells, 1); moderate proportion (30-60% of positive cells, 2); high proportion (> 60-90% of positive cells, 3) and almost all cells positive (more than 90%, 4) (Sukjumlong et al., 2005). The total scores were calculated by the summary of intensity and proportional scores of each compartment of the oviductal tissues which were surface epithelium (SE), stromal layer (STR) and muscular layer (M). Since the muscular layer of the infundibulum was not accessible, therefore the manual scoring was done only in the SE and STR in this part of the oviduct.

Quantification of the immunostaining was performed on five randomly selected fields which comprised all compartments of the oviduct. The results from image analysis are presented as mean percentage of total area of positive staining per total area of cell nuclei.

The data obtained from both manual scoring and image analysis were analysed using SAS (Statistical Analysis System, SAS Inst V 9.1, Cary, NC, USA). Descriptive statistics including the mean and the standard deviations (SD) of all parameters were calculated. The total score or percentage of positive staining from each compartment of the oviduct were compared between groups using Kruskal-Wallis’s test and Wilcoxon rank sum test (NPAR1WAY procedure of SAS), and p < 0.05 were regarded to have statistical significance.

Results Immunohistochemistry

In general, positive immunostaining of both ERα and PR were observed in the nuclei of oviductal epithelium, stroma and muscular layer, but with different proportions and intensities (Figs 1 and 2). Furthermore, cytoplasmic staining for ERα was observed periodically whereas it was not found for PR immunostaining. The immunostaining results were summarized according to the different artificial insemination methods in Tables 1.1 and 2.1 for image analysis and in Tables 1.2 and 2.2 for manual scoring.

By using image analysis, higher percentage of both ERα and PR was found in the AI groups compared to the other insemination methods. Regarding different oviductal parts, significantly higher percentage of ERα was observed in the AI group for the ampulla and the infundibulum part

Table 1.1 Percentage of ERα positive staining from image analysis in different parts of the oviduct (mean±SD)

Insemination AI IUI DIUI Isthmus 37.78 ± 9.4 31.08 ± 16.54 33.89 ± 18.5 Ampulla 31.95 ± 13.82 a 37.66 ± 23.58 a 15.36 ± 10.63 b Infundibulum 45.33 ± 26.01a 36.15 ± 21.89 a 25.19 ± 19.26 b

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while higher percentage of PR was found in the isthmus and the infundibulum part of AI group (p < 0.05) (Tables 1.1 and 2.1.). For the manual scoring results, similar pattern was observed that high immunostaining score was found in the AI groups compared to the other insemination groups for both ER� and PR (Tables 1.2 and 2.2). In addition, significant difference was observed mainly in the SE of the oviductal tissue from the manual scoring method. Moreover, for PR immunostaining, significant difference was found only in the SE of the infundibulum part by manual scoring (Tables 2.2).

Table 1.2 ERα positive staining score by manual scoring in different parts of the oviduct (mean±SD)

ERα immunostaining AI IUI DIUI Isthmus-SE Isthmus-STR Isthmus-M Ampulla-SE Ampulla-STR Ampulla-M Infundibulum-SE

4.0 ± 2.0 3.16 ± 1.04 5.5 ± 0.4 4.16 ± 2.0a 3.33 ± 0.76 3.33 ± 0.5 3.6 ± 2.08a

2.16 ± 0.28 2.16 ± 0.28 3.83 ± 1.04 2.16 ± 0.28 b 1.83 ± 0.28 3.5 ± 1.3 1.34 ±1.15 b

2.83 ± 0.76 2.6 ± 1.15 4.16 ± 0.28 2.16 ± 0.28 b 2.67 ± 1.15 4.67 ± 0.5 0.67 ± 1.15 b

Infundibulum-STR 3.00 ± 1.0 1.83 ± 1.15 2.33 ± 0.5 Infundibulum-M NA NA NA

AI: conventional artificial insemination, IUI: intrauterine insemination, DIUI: deep intrauterine insemination, SE: surface epithelium of the oviduct, STR: stromal layer of the oviduct, M: muscular layer of the oviduct Different letters within the same row represent significant difference, NA: not accessible.

Figure 1 ERα immunostaining in different parts of the sow oviduct after different insemination methods. IST-AI: isthmus after conventional artificial insemination, IST-IUI: isthmus after intrauterine insemination, IST-DIUI: isthmus after deep intrauterine insemination, AM-AI: ampulla after conventional artificial insemination, AM-IUI: ampulla after intrauterine insemination, AM-DIUI: ampulla after deep intrauterine insemination, INF-AI: infundibulum after conventional artificial insemination, INF- IUI: infundibulum after intrauterine insemination, INF-DIUI: infundibulum after deep intrauterine insemination

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Figure 2 PR immunostaining in different parts of the sow oviduct after different insemination methods. IST-AI: isthmus after conventional artificial insemination, IST-IUI: isthmus after intrauterine insemination, IST-DIUI: isthmus after deep intrauterine insemination, AM-AI: ampulla after conventional artificial insemination, AM-IUI: ampulla after intrauterine insemination, AM-DIUI: ampulla after deep intrauterine insemination, INF-AI: infundibulum after conventional artificial insemination, INF- IUI: infundibulum after intrauterine insemination and INF-DIUI: infundibulum after deep intrauterine insemination

Table 2.1 Percentage of PR positive staining from image

analysis software in different parts of the oviduct (mean±SD)

Insemination AI IUI DIUI Isthmus 51.97 ± 11.95a 27.89 ± 26.07b 30.27 ±16.42b Ampulla 41.72 ± 8.6 33.07 ± 25.01 46.63 ± 15.23 Infundibulum 43.59 ± 24.12a 23.45 ± 18.65b 26.61 ± 24.08b

Table 2.2 PR positive staining score by manual scoring in different parts of the oviduct (mean±SD)

PR immunostaining AI IUI DIUI Isthmus-SE Isthmus-STR Isthmus-M Ampulla-SE Ampulla-STR Ampulla-M Infundibulum-SE

6.67 ± 0.28 5.5 ± 0.5 5.8 ± 0.75 5.33 ± 0.28 4.33 ±0.29 5.3 ± 0.57 4.83 ± 0.57a

4.5 ± 0.86 3.83 ± 0.76 3.1 ± 1.32 4.67 ± 0.76 3.5 ± 0.86 3.5 ± 1.29 2.0 ±0b

6.3 ± 0.29 5.83 ± 0.58 4.16 ± 0.2 6.16 ± 0.28 5.16 ± 0.28 4.6 ± 0.50 2.5 ± 1.0b

Infundibulum-STR 3.83 ± 0.76 2.33 ± 0.57 4.66 ± 0.28 Infundibulum-M NA NA NA AI: conventional artificial insemination, IUI: intrauterine insemination, DIUI: deep intrauterine insemination, SE: surface epithelium of the oviduct, STR: stromal layer of the oviduct, M: muscular layer of the oviduct. Different letters within the same row represent significant difference, NA: not accessible.

Discussion

Regarding the different insemination methods, the present results showed higher immunostaining in the AI group compared to the others. This demonstrates that different insemination methods may have the influence on the expression of these steroid receptors. It has been demonstrated that oestrogen (E2) in boar semen can up-regulate steroid receptors in the pig reproductive organs (Langendijk et al., 2002a; Langendijk et al., 2002b). The small volume of semen used for IUI and DIUI groups might also influence the lower expression of these steroid receptors due to the lower amount of E2.

In the oviducts, the present study demonstrated the higher PR staining in the oviductal part of uterotubule junction (UTJ) of the AI sows comparing to the IUI and DIUI sows (Tummaruk et al., 2010). DIUI may influence the lower expression of PR in the UTJ from the lower number of spermatozoa in the UTJ which serves as sperm reservoir (Tummaruk and Tienthai, 2008). Similar to our present results, the lower PR was found in the isthmus part of the oviduct, which is located near the UTJ, as well as in the infundibulum part. As progesterone can influence the transportation of spermatozoa both before and after fertilization

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(Mburu et al., 1996), this mechanism may mediate through the expression of PR which showed lower expression when the number of sperm was lower by IUI and DIUI techniques.

In addition to sperm transportation, oviductal cilia are believed to have a critical role in ovum transport from the oviduct to the uterus in cyclic and pregnant rats (Halbert et al., 1989). Oestrogen may have roles in ovum transport by regulating oviductal ciliogenesis in rats indirectly via ERα in the epithelium (Okada et al., 2003). Moreover, a single injection of 17β-estradiol (E2) on day 1 of the reproductive cycle or pregnancy can shorten oviductal transportation of eggs (Croxatto, 2002) and this mechanism was believed to mediate via ERα in the oviductal epithelium as well (Croxatto, 2002; Orihuela et al., 2003). From the results of the present study, we found positive ERα in all parts of the oviducts (isthmus, ampulla and infundibulum) which may involve in the mechanism of ciliogenesis regulation via ERα. However, the oviductal epithelium consisted of two cell types which were ciliated cells and secretory cells, thus further study should be considered regarding the differentiation between these two cell types. Comparing different insemination methods in regards to gamete transportation, the higher ERα staining found in the AI group may indicate that more successful fertilization after conventional artificial insemination could result from the better transportation of ovum which may be regulated by ERα in the oviductal epithelial cells as well.

In general, there are two isoforms of PR; PR-A and PR-B which arise from single gene. It was well documented that the levels of PR-A and PR-B are differently regulated during the reproductive cycle and, therefore, may mediate different physiological responses to progesterone. In the ovary and uterus, the studies in mice revealed that ablation of PR-A resulted in severe abnormalities in ovarian and uterine function leading to female infertility but not for PR-B (Conneely et al., 2003). Furthermore, a recent study showed that PR-A was absent in all compartments of the uterus in anoestrous sows (Karveliene et al., 2007). As the results of the present study were the accumulation of PR-A and PR-B, therefore the difference between these two isoforms of PR could not be demonstrated. On the other hand, there may be some differences in the expression of PR-A in these inseminated sows, but it may also be balanced by the level of PR-B in the tissue compartments and, therefore, causes the similar expression of PR in some of the oviductal tissues. However, the different localization of PR-A should be further studied as it may reveal or explain the possible effects on the localization of PR in different inseminated sows.

From the results of the present study, it was also demonstrated that ERα immunostaining was found in the cytoplasm of the oviductal epithelium. This is in agreement with previous studies reporting localization of ERα in non-nuclear sites of reproductive cells (Welshons et al., 1984; Marquez

and Pietras, 2001; Monje and Boland, 2001; Monje et al., 2001). Moreover, the study in rats demonstrated that mating could increase the number of ERα in non-nuclear compartments. The increase in ERα in non-nuclear compartment involved the changes from non-genomic pathway in cyclic rat to genomic pathway in mating rat (Okada et al., 2003). The changes in these pathways have been designated "intracellular path shifting, or IPS" (Parada-Bustamante et al., 2007). Therefore, the marked ERα cytoplasmic staining could be involved in the changes of IPS induced by mating or insemination in the present study since no cytoplasmic staining was observed in cyclic sows (Sukjumlong et al., 2005) nor in the epithelial cells for PR immunostaining.

In the present study, immunostaining results of both ERα and PR were evaluated by two different methods, the manual scoring and the image analysis. The results from these two methods were in agreement with each other, although minor differences could be observed in some parts of the oviduct. The explanation of the difference was that the manual scoring showed variations in patterns of immunostaining with regard to both proportion and intensity while the image analysis quantified the total amount of positive staining in randomly selected area. Moreover, cytoplasmic staining could be excluded from manual scoring method but still be detected by image analysis system.

In conclusion, the present study showed the differences in steroid receptor expression among different insemination methods and in varied parts of the sow oviduct. The methods of insemination regarding the volume of semen can have the effects on the expression of steroid receptors in the sow oviduct and this may also influence the successful fertilization in sows.

Acknowledgements

The present study was granted by Thailand

Research Fund (MRG5380173). We would like to thank Dr. Panida Chananpiwat and Dr. Sukanya Manee-in for assistance with sample collection, Mr. Sinchai Pianchop for his assistance with histological technique and Miss Jongkol Sangvirun for assistance with immunohistochemical procedure.

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Thai J Vet Med. 2013. 43(2): 197-203.

Probable Pig to Duck Transmission of the Pandemic H1N1 2009

(pH1N1) and Its Reassortant in Commingling Experimental

Condition

Nataya Charoenvisal1 Juthatip Keawcharoen2 Donruethai Sreta3 Siriporn Tantawet4

Suphattra Jittimanee1 Jirapat Arunorat1 Korakrit Poonsuk1 Alongkorn Amonsin5,6

Roongroje Thanawongnuwech1,6*

Abstract

Pandemic H1N1 2009 (pH1N1) virus is considered as a low pathogenic influenza virus, however, it rapidly

spread among humans and was finally found in the swine population of 6 continents. Interspecies transmissions among different animal species are of interest. In this study, sentinel ducks were commingled with pH1N1 or pandemic H1N1 reassortment virus (rH1N1) inoculated pigs in separate groups. According to the results, both studied viruses were able to cross-species transmit to a few sentinel ducks with mild or no clinical signs. Viral shedding measured by a modified real time RT-PCR detection from the oropharyngeal and cloacal swabs were also observed in both studied viruses. Interestingly, ducks commingled with pH1N1-infected pigs showed higher number of infected ducks detected by viral shedding in cloacal swabs. This present study suggested that pH1N1 and rH1N1 were able to transmit from pigs to ducks but viral replication in ducks were limited. As a result clinical signs were not obvious and low levels of viral shedding were detected in both sentinel duck groups.

Keywords: ducks, interspecies transmission, pandemic H1N1 2009, pigs, reassortant 1 Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd., Bangkok, 10330, Thailand 2 Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd., Bangkok, 10330, Thailand 3 Faculty of Veterinary Medicine, Rajamangala University of Technology Tawan-ok, Bangpra, Chonburi, 20110, Thailand 4 Department of Clinical Science and Public Health, Faculty of Veterinary Science, Mahidol University, Salaya, NakhonPathom, 73170, Thailand 5 Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd., Bangkok, 10330, Thailand 6 Center of Emerging and Re-emerging Infectious Diseases in Animals, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd., Bangkok, 10330, Thailand *Corresponding author: E-mail: Roongroje.T@chula.ac.th

Original Article

198 Charoenvisal N. et al. / Thai J Vet Med. 2013. 43(2): 197-203.

บทคดยอ

ความเปนไปไดของการตดเชอขามชนดสตวจากสกรไปเปดของเชอไวรสไขหวดใหญสายพนธใหม H1N1 2009 (pH1N1) และไวรสลกผสมในสภาวะการทดลองเลยงรวมกน ณ ทยา เจรญวศาล 1 จฑาทพย เขยวเจรญ 2 ดลฤทย ศรทะ 3 สรพร ตณฑเวส 4 สภทตรา จตตมณ 1 จรภทธ อรโณรตน 1 กรกฤต พนสข 1 อลงกร อมรศลป 5,6 รงโรจน ธนาวงษนเวช 1,6*

แมวาเชอไวรสไขหวดใหญสายพนธใหม H1N1 2009 (pH1N1) ไมไดกอโรครนแรงในมนษย แตเชอไวรสสามารถแพรระบาดไปไดอยางรวดเรว ถง 6 ทวปทวโลกในระยะเวลาเพยง 2 เดอน และหลงจากการแพรระบาดในมนษย ไดมรายงานการพบเชอไวรสชนดนในสกรในหลายๆประเทศ รวมทงประเทศไทยดวย ดงนน การตดเชอไวรสขามชนดสตวจงเปนประเดนทนาสนใจในขณะน และเปนทมาของการศกษาในครงน ซงทาการศกษาโดยนาเปดมาเลยงรวมกบสกรทใหเชอไวรส โดยสกรในกลมท 1 ใหเชอ pH1N1 และสกรในกลมท 2 ใหเชอไวรสลกผสมของเชอ pH1N1 (rH1N1) ผลการศกษาพบวาไวรสทง 2 ชนดสามารถตดตอไปยงเปดได โดยทเปดไมแสดงอาการ หรอมอาการทางคลนกเลกนอย แตสามารถตรวจพบสารพนธกรรมของไวรสโดยวธ real-time RT-PCR ไดในระดบตาๆ ทงจากตวอยางทเกบจากชองปากและทวารหนกของเปด ซงพบวาเปดทเลยงรวมกบสกรในกลมทใหเชอ pH1N1 มจานวนตวทใหผลบวกมากกวากลม rH1N1 และพบเชอจากตวอยางทเกบจากทวารหนกมากกวาตวอยางจากชองปาก การศกษาในครงนบงชวา เชอไวรส pH1N1 และ rH1N1 สามารถตดจากสกรไปยงเปดได แตไวรสอาจมขอจากดในการแบงตวในเปด ดงนนเปดจงไมแสดงอาการ แตเปดสามารถขบเชอออกสสงแวดลอมได

คาสาคญ: เปด การตดเชอไวรสขามชนดสตว เชอไวรสไขหวดใหญสายพนธใหม H1N1 2009 สกร เชอไวรสลกผสม 1 ภาควชาพยาธวทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 2 หนวยไวรสวทยา ภาควชาจลชววทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 3 คณะสตวแพทยศาสตร มหาวทยาลยเทคโนโลยราชมงคลตะวนออก อ. บางพระ จ. ชลบร 20110 4 ภาควชาเวชศาสตรคลนกและการสาธารณสข คณะสตวแพทยศาสตร มหาวทยาลยมหดล ต. ศาลายา อ. พทธมณฑล จ. นครปฐม 73170 5 ภาควชาสตวแพทยสาธารณสข คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 6 ศนยเชยวชาญเฉพาะทางโรคอบตใหมและโรคอบตซาในสตว คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 *ผรบผดชอบบทความ E-mail: Roongroje.T@chula.ac.th

Introduction

In April 2009, a pandemic H1N1 influenza A virus (pH1N1) emerged and spread worldwide. The pH1N1 is a reassortant virus of the North American triple reassortant (TRIG) swine virus and the avian-like Eurasian swine lineage (Garten et al., 2009). The pH1N1 virus efficiently transmitted back to pigs observed in many countries (Ducatez et al., 2011), including Thailand (Sreta et al., 2010) and the pH1N1 reassortant virus (rH1N1) was evidently found in pigs in 2010 in Thailand. The rH1N1 contains Neuraminidase (NA) gene of a Thai endemic swine influenza virus (SIV) and the other 7 genes were closely related to the pH1N1 virus (Kitikoon et al., 2011). Influenza A virus is a highly contagious pathogen and able to cause disease in mammalian and avian species. Influenza viruses normally require 2 specific receptors; Sialic acid (SA) α2, 3 receptors commonly found in the intestinal tract of avian species, and SA α2, 6 receptors commonly found in the respiratory tract of humans (Forrest and Webster,

2010). Wild aquatic ducks are known as the natural reservoir of the influenza A virus. Influenza A viruses in avian species are commonly divided into highly pathogenic (HPAI) and low pathogenic (LPAI) avian influenza viruses (Kim et al., 2009) and the pH1N1 and rH1N1 viruses were classified as LPAI viruses (Babiuk et al., 2010). LPAI viruses normally cause limited lesions within 2 days in the epithelial cells of respiratory and digestive tracts showing mild pneumonia and infiltration of lymphocytes and macrophages. In addition, the replication site is mainly in the large intestine since high concentration of viral shedding is found in feces up to 7 days (Van Reeth, 2007; Kim et al., 2009). Interestingly, mallard ducks are able to act as the viral reservoir transmitting the virus to domestic poultry (Keawcharoen at al., 2008). Hence, the avian-like swine virus such as Eurasian H1N1 lineage containing PB2 and PA genes of the first isolated TRIG virus was closely related to the virus isolated from duck (Brown, 2000) indicating that duck’s viruses can effectively transmit either the whole virus or a few gene reassortment to pigs. It should be noted that Bao et al. (2010) challenged

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pH1N1 (human isolate) in Peking ducks and found no viral RNA detection in the lung tissue nor in the epithelial cells of the digestive tract, the main replication site of LPAI. Moreover, avian viruses such as the 1918 H1N1 and pH1N1 can cross species transmission resulting in fatal diseases in humans but no or mild clinical signs in poultry, particularly in ducks (Babiuk et al., 2010, Kalthoff et al., 2010). Commingling among domestic species is commonly seen in pig ecosystems particularly in backyard farming in most Asian countries. Interspecies transmission of influenza viruses is possible when pH1N1 is widespread in the domestic animals, particularly in pigs. The viruses used in this study were isolated from naturally infected pigs. The infectivity and pathogenesis of these viruses in sentinel ducks were elucidated.

Materials and Methods Viruses: A/swine/Thailand/CU-RA29/2009(H1N1) (Sreta et al., 2010), a pandemic H1N1 of pig origin (pH1N1) and A/swine/Thailand/CU-SA43/2010 (H1N1) (Kitikoon et al., 2011), a novel reassortant virus of pig origin (rH1N1) were individually propagated 3 times in 9 day old embryonated chicken eggs. Allantoic fluids were collected 72 hours later and virus concentrations were calculated using 50% tissue culture infectious dose (TCID50) in Madin-Darby canine kidney (MDCK) cells by Reed and Muench method. The virus concentration of both isolates was adjusted to104 TCID50/ml and kept at -800C until used.

Animals: Twenty one, 6-week-old ducks (Anas platyrhynchos domestica) were placed in the animal facility biosafety level 2 with adequate appropriate nutritional food and clean water. Before starting the experiment, oropharyngeal and cloacal swabs and sera were collected from each duck. All samples were tested negative using routine RT-PCR detection for influenza A virus (M gene).

At the same time, 21 nursery pigs were divided into 3 groups. Group 1 containing 9 pigs received intratracheally inoculation with pH1N1 virus (A/swine/Thailand/CU-RA29/2009(H1N1)). Group 2 also had 9 pigs receiving intratracheally inoculation with rH1N1 ((A/swine/Thailand/CU-SA43/2010 (H1N1)). Group 3 was a negative control group containing 3 pigs receiving mock inoculation with cell culture media.

One day post inoculation (1 DPI) in pigs, sentinel ducks were divided into 3 groups similar to the pig experiment and placed into each pig room. As a result, pigs per ducks ratio are 1:1 and those animals were commingling in each separate group throughout the experiment. At 3, 5 and 13 DPI, 3 ducks from group 1 and group 2 and one duck from the control group were randomly selected for necropsy. Adequate appropriate foods and water for pigs and ducks were provided in each room. The animal usage and procedures were approved by Chulalongkorn University-Faculty of Veterinary Science Animal Care and Use committee (protocol No. 11310052).

Clinical measurement and sampling: Clinical signs of ducks were observed at 2-8, 11 and 13 DPI. Oropharyngeal and cloacal swabs from all remaining ducks were collected at 2-8, 11 and 13 DPI. Nasal swabs were also collected from all remaining pigs on 1-7, 10 and 12 DPI. All collected swabs were tested for the presence of the influenza virus by a modified real time Reverse Transcriptase-Polymerase Chain Reaction (real time RT-PCR) and virus isolation (described below). Sera from ducks and pigs collected at necropsy were tested for influenza A virus antibody. Lung, pancreas and caecum of ducks and lung and bronchial lymph node of pigs were collected during necropsy for quantitative detection of viral RNA by the real time RT-PCR and viral isolation. Selected organs including brain, pancreas, liver, jejunum, caecum, spleen and kidney were fixed in 10% buffer formalin for histopathology study.

Viral detection by modified real time Reverse Transcriptase-Polymerase Chain Reaction (real time RT-PCR): Total RNA was extracted from collected swabs, lungs, pancreas and caecum using a commercial kit (NucleoSpin Extract Viral RNA Kit, Macherey-Nagel, Germany). A modified real time reverse transcriptase-polymerase chain reaction (real time RT-PCR) was performed using Superscript III platinum one-step quantitative RT-PCR system (Invitrogen, USA). Primers were specific to the Matrix (M) gene. Forward primer (MF3; 5’ TGATCTTCTTGAAAATTTGCAG 3’), reward primer (MR1+; 5’ CCGTAGMAGGCCCTCTTTTCA 3’) and M-probe (FAM-TTGTGGATTCTTGATCG-MGB) were used in this study as previously described (Payungporn et al., 2006). Briefly, the cycling conditions started at 480C for 45 min, then 950C for 10 min and followed by 40 cycles including denaturation (940C for 15 sec), annealing (550C for 30 sec) and extension (720C for 40 sec). Positive samples showed CT value over 40.

Meanwhile, homogenated tissue samples were filtrated and inoculated onto the monolayer of MDCK cells using a ten-fold serial dilution manner. The inoculated cell cultures were incubated for 72 hours. The virus was identified using anti-influenza A nucleoprotein monoclonal antibody as a primary antibody and rabbit anti-mouse IgG conjugated horseradish peroxidase as a secondary antibody (Dako Cytomation, Carpinteria, California). Then, the color was developed using a chromogen aminoethyl carbazole substrate (Sigma, St. Louis, Missouri) to identify the virus antigen in the nuclease of the infected cells (Sreta et al., 2009).

Pathological examination: Formalin-fixed tissues were embedded in paraffin and processed routinely. Briefly, sections were cut approximately 4-6 µm thick for histopathological and immunohistochemistry (IHC) staining for the influenza A virus antigen detection. The IHC staining was performed using a labeled streptavidin-biotin (LSAB) method. Primary antibody using anti-influenza A (H5N1) nucleoprotein monoclonal mouse antibodies (EVS238, B.V. European Veterinary Laboratory, the Netherlands) and secondary antibody using

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biotinylated rabbit anti-mouse IgG antibody and envision polymer (Envision Polymer DAKO®, Denmark) were concurrently performed with a negative control slide. A positive control slide was also included using the SIV-infected lung section from the previous experiment (Sreta et al., 2009). The sections were developed with 3, 3’-diaminobenzidine tetrahydrochloride (DAB) and counterstained with Mayer’s hematoxylin.

Serological test: All sera were tested for influenza A virus antibody by commercial ELISA (Avian Influenza virus antibody test kit, IDEXX Laboratories, USA). Concurrently, sera were randomly selected for hemagglutination inhibition (HI) test (1 sample/ group/necropsy day). Since the hemagglutinin (HA) genes of pH1N1 and rH1N1 are closely related, pH1N1 virus (A/swine/Thailand/CU-RA29/2009 (H1N1) was used as the representative antigen in the assay. Samples with HI titers ≥ 40 were considered as previously exposed to the specific tested antigen.

Results Clinical examination: Sentinel ducks in the pH1N1 group did not show any clinical signs during 2-4 DPI, only ocular and nasal discharges were observed at 5 DPI. All 3 remaining ducks in the pH1N1 group showed conjunctivitis between 6-13 DPI. Ducks in the control and the rH1N1 groups did not show any obvious clinical signs throughout the experiment. It should be noted that the inoculated pigs in both groups showed obvious clinical signs of SIV infection including nasal discharge, coughing and sneezing.

Virus detection: Both groups of inoculated pigs showed viral detection in the nasal swabs as early as 1 DPI and could be detected in low levels until 12 DPI (data not shown). In addition, the rH1N1-inoculated group showed higher number of pigs shedding the

virus based on the nasal swab results (data not shown). In the experimental ducks, virus in the pH1N1 group was detected by the real time RT-PCR from the oropharyngeal swabs at 3 DPI (1 of 9) and cloacal swabs at 2 (1 of 9), 3 (2 of 9), 11 (1 of 3) and 13 (1 of 3) DPI. Based on the viral isolation results in the pH1N1 group, only one oropharyngeal swab and one cloacal swab showed positive results with low levels of virus concentration at 4 DPI (Table 1). In the rH1N1 group, 1 oropharyngeal and 1 cloacal swab at 2 DPI were tested positive and the virus isolation of the rH1N1 group yielded negative results (Table 2). No viral detection was found in the negative control ducks throughout the experiment. Low levels of viral genetic material was detected only in the lung of one necropsied duck in the pH1N1 group by the real time RT-PCR at 3 DPI (data not shown).

Pathological examination: In both infected groups of pigs, typical lesions of SIV infection characterized by multifocal consolidated lung lesions with a checker-board lung pattern at the cranioventral area were observed. The viral antigens were detected by IHC only in the lung and bronchial lymph node of the rH1N1 infected pigs (data not shown). However, sentinel ducks in both inoculated pig groups as well as in the control group showed only mild interstitial pneumonia and mild airsacculitis. None of the duck tissues were tested positive by IHC technique.

Serological examination: At the beginning of the experiment, all duck sera were tested negative for influenza A virus antibody using commercial ELISA. Sera collected from necropsied ducks at 3, 5 and 13 DPI were also tested negative for influenza A antibody using a commercial ELISA. Similarly, all duck sera were tested negative for the pH1N1 virus using HI test.

Table 1 Viral detection measured from oropharyngeal and cloacal swabs of sentinel ducks in the pH1N1 group using modified real-time RT-PCR and viral isolation

Duck No. Virus detection 1 DPI 2 DPI 3 DPI 4 DPI 5 DPI 6 DPI 7 DPI 8 DPI 11 DPI 13 DPI

Oropharyngeal swab rt VI rt VI rt VI rt VI rt VI rt VI rt VI rt VI rt VI rt VI 1 - - - - - - - - - + N 2 - - - - - - - - - - - - - - - - - - - - 3 - - - - - - - - - - N 4 - - - - - - N 5 - - - - + - N 6 - - - - - - N 7 - - - - - - - - - - N 8 9

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

Cloacal swab 1 - - - - - - - + - + N 2 - - - - - - - - - - - - - - - - + - - - 3 - - - - - - - - - - N 4 - - - - + - N 5 - - - - - - N 6 - - + - - - N 7 - - - - - - - - - - N 8 9

- -

- -

- -

- -

- +

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

- +

- -

DPI: day post inoculation, rt: real time RT-PCR (+: Ct values < 40, - : Ct values ≥ 40), VI: viral isolation using MDCK , N: necropsy.

Charoenvisal N. et al. / Thai J Vet Med. 2013. 43(2): 197-203. 201

Table 2 Viral detection measured from oropharyngeal and cloacal swabs of sentinel ducks in rH1N1 group using modified real time

RT-PCR and viral isolation

Duck No. Virus detection 1 DPI 2 DPI 3 DPI 4 DPI 5 DPI 6 DPI 7 DPI 8 DPI 11 DPI 13 DPI

Oropharyngeal swab

rt VI rt VI rt VI rt VI rt VI rt VI rt VI rt VI rt VI rt VI

1 - - + - - - - - - - - - - - - - - - - - 2 - - - - - - - - - - N 3 - - - - - - - - - - - - - - - - - - - - 4 - - - - - - N 5 - - - - - - N 6 - - - - - - N 7 - - - - - - - - - - - - - - - - - - - - 8 9

- -

- -

- -

- -

- -

- -

- -

- -

- -

- -

N N

Cloacal swab 1 - - - - - - - - - - - - - - - - - - - - 2 - - - - - - - - - - N 3 - - - - - - - - - - - - - - - - - - - - 4 - - - - - - N 5 - - - - - - N 6 - - + - - - N 7 - - - - - - - - - - - - - - - - - - - - 8 - - - - - - - - - - N 9 - - - - - - - - - - N

DPI: day post inoculation, rt: real time RT-PCR (+: Ct values < 40, - : Ct values ≥ 40), VI: viral isolation using MDCK, N: necropsy.

Discussion

In this experiment, pigs and ducks were commingling in close contact and might share the same water basins. Although appropriate foods for ducks and pigs were provided in separate bowls, the animals randomly consumed foods in all bowls. Mimicking the field situation in backyard farming, ducks, chickens and birds freely share the same environment and might have direct contact among those animals. The poultry not only consume leftover feed but might also contaminate the pigs from their excretions or vice versa. As a result, cross-transmission of influenza virus from pigs to ducks or vice versa could happen in the mimicking pigs to ducks interfacing environment.

As expected, the sentinel ducks did not show obvious clinical signs or significant lesions. However, viral RNA was detected in the oropharyngeal and cloacal swabs of ducks in both inoculated pig groups. In addition, pH1N1 virus could be isolated from sentinel ducks in MDCK cells with low titers implying that both studied viruses could infect, replicate, and probably transmit from pigs to ducks when commingling. Previously, ducks were demonstrated to be insusceptible to the human pH1N1 infection and no viral RNA was detected from swabs or lungs (Swayne et al., 2009; Bao et al., 2010). It should be noted that the 1918 H1N1 influenza virus could replicate in ducks with low levels of virus titers (Babiuk et al., 2010).

Based on the present study, the pH1N1 virus obtained from pigs was able to infect and replicate in the duck intestine better than the respiratory tract. This result is well correlated with the evidence that the LPAI viruses shed via fecal-oral route and persist in duck population. As a result, all subtypes of the

viruses, especially LPAI, can be isolated from duck feces (Kim et al., 2009). It should be noted that the viral RNA was also found in the duck lung tissue of the pH1N1 group in this study. However, viral antigen could not be detected in all collected tissues of both inoculated groups. The results suggested that both pH1N1 and rH1N1 viruses had limited replication in sentinel ducks possibly due to the lack of SA α2, 6 receptors in ducks (Matrosovich et al., 2008). The HA genes of both pH1N1 and rH1N1 are similarly compatible with the SA α2, 6 receptors and able to infect pigs. In addition, the differences of body temperature in avian and mammals may affect the replication process (Forrest and Webster, 2010). As a result, both studied viruses had possibly limited replication in the sentinel ducks.

In the sentinel ducks, viral detection in the pH1N1 group yielded higher numbers of positive ducks than those of the rH1N1 group. In contrast, viral detection in the parallel infected pigs was observed more in the rH1N1-infected pigs. Since the NA gene of pH1N1 and rH1N1 were 89% homology and the other 7 genes had at least 99% homology, the difference of the NA gene might cause the variation in infectivity and virus shedding in the sentinel ducks. It should be noted that NA gene is responsible for releasing progeny viral particles from the infected cells (Suzuki, 2005). The NA gene of the studied rH1N1 obtained from the local swine influenza virus must be well adapted in the Thai pig population but might have the limitation on the release of new progeny virus in ducks.

Unfortunately, all collected sera showed negative results on influenza A antibody suggesting the limited infection and replication of the studied viruses. In contrast, antibody against HPAI (H5N1) could be detected as early as 4 DPI and gradually

202 Charoenvisal N. et al. / Thai J Vet Med. 2013. 43(2): 197-203.

increased by 14 DPI in experimental ducks (Jeong et al., 2009).

In conclusion, the results suggested that pH1N1 and rH1N1 isolated from pigs could probably be transmitted to the sentinel ducks when commingling in close contact with the infected pigs. However, the studied viruses had limited replication in ducks. The pH1N1 isolated from pigs appeared to have the potential for pig to duck transmission. Interspecies transmission may influence the antigenic drift and shift resulting in the emergence of a novel influenza virus. Each novel virus may adapt in the specific hosts before emerging and causing severe diseases and may transmit to another host or vice versa and so on. Since backyard farming, commonly seen in most Asian countries, creates a perfect interspecies transmission together with closely human-animal interface scenario, influenza A virus surveillance, monitoring, and pathogenesis studies are necessary.

Acknowledgements

This study was supported by the Higher

Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (HR1160A-55) and “Integrated Innovation Academic Center: IIAC” Chulalongkorn University Centenary Academic Development Project and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). In addition, the authors would like to express our sincere thanks to the Charoen Pokphand Food public company limited and AHTSO Lab, Thailand for providing the experimental animals and animal facilities.

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Thai J Vet Med. 2013. 43(2): 205-211.

Preference Testing as Environmental Enrichment Assessment

for Laboratory Mice

Navakanit Sachanonta* Waridtha Sa-ngeunreung Somchai Sa-ing-kaew

Raywadee Butraporn

Abstract

In the field of biomedical research, a wide variety of environmental enrichment items are available for

laboratory mice to fulfill its physiological and behavioral needs which influence the outcome of animal experiments. However, to take a decision on the most suitable enrichment, researchers are likely to consider the cost of enrichment item more than animal preference and animal well-being. To develop a suitable environmental enrichment program for mice in Laboratory Animal Center, National Institute of Health (LAC-Thai NIH), we evaluated mouse preference for cages containing different types of nesting material and enrichment device. We carried out a simple preference test in groups of mice. Mice were allowed to choose three nesting materials; woodchips, paper towel and paper strip; and three enrichment devices; commercial plastic mouse house, standard plastic mouse house and cassava-based cup for 48 hours, then we evaluate where the mice positioned their nests and the amount of time they spent in the cage. The mice showed a statistical significant preference of nesting materials for paper towels than paper strip and of enrichment devices for standard plastic mouse house than commercial plastic mouse house. They also showed preference of cassava-based cup comparing with standard plastic mouse house. These data will be supporting information for environmental enrichment program of laboratory mice in LAC-Thai NIH.

Keywords: environmental enrichment, mice, preference, welfare Laboratory Animal Center, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi 11000, Thailand *Corresponding author: E-mail: navakanit.s@dmsc.mail.go.th

Original Article

206 Sachanonta N. et al. / Thai J Vet Med. 2013. 43(2): 205-211.

บทคดยอ

การประเมนความพงพอใจของหนไมซตอชนดวสดรองนอนและอปกรณเพอการเพมพนสภาพแวดลอม นวขนษฐ สจจานนท * วรฏฐา สงวนเรอง สมชาย สองแกว เรวด บตราภรณ

สาหรบงานทดสอบและวจยดานวทยาศาสตรการแพทยนน สงเพมพนสภาพแวดลอมสาหรบสตวทดลองชนดหนไมซจะชวยเตมเตมความตองการดานสรรวทยาและพฤตกรรมซงมผลตอขอมลทไดจากสตวทดลองและสงผลกระทบตองานวจยโดยรวม การตดสนใจเลอกชนดของสงเพมพนสภาพแวดลอมมกถกพจารณาจากปจจยดานราคามากกวาปจจยดานความพงพอใจของสตวหรอเพอการเสรมสรางสวสดภาพสตวการพฒนาโครงการสงเพมพนสภาพแวดลอมสาหรบหนไมซของศนยสตวทดลอง สถาบนวจยวทยาศาสตรสาธารณสข กรมวทยาศาสตรการแพทย กระทรวงสาธารณสขนนไดดาเนนการประเมนความพงพอใจของหนไมซทงตอวสดทารงและอปกรณเพมพนสภาพแวดลอมโดยใชวธการทดสอบความพงพอใจแบบเบองตนโดยใหสตวเลอกระหวางกรงทมวสดทารงจานวน 3 ชนด ไดแก ขกบ กระดาษและกระดาษเสน และกรงทมอปกรณเพมพนสภาพแวดลอมจานวน 3 ชนด ไดแก บานพลาสตกเพอการคา บานพลาสตกและถวยมนสาปะหลง เปนเวลา 48 ชวโมง จากการสงเกตกรงทสตวเลอกทารงรวมทงเวลาทสตวใชในกรงชนดตางๆ พบวา หนไมซแสดงความพงพอใจอยางมนยสาคญทางสถตตอวสดทารงชนดกระดาษมากกวากระดาษเสน และพงพอใจอยางมนยสาคญทางสถตตออปกรณเพมพนสภาพแวดลอมชนดบานพลาสตกมากกวาบานพลาสตกเพอการคา นอกจากน หนไมซยงแสดงความพงพอใจตอถวยมนสาปะหลงมากกวาบานพลาสตก จากขอมลดงกลาว ศนยสตวทดลอง สถาบนวจยวทยาศาสตรสาธารณสขสามารถใชเปนขอมลสนบสนนประกอบการพจารณาคดเลอกวสดทารงและอปกรณเพมพนสภาพแวดลอมเพอพฒนาโครงการสงเพมพนสภาพแวดลอมใหเหมาะสมสาหรบหนไมซเพองานทดสอบและวจยทางวทยาศาสตรการแพทยตอไป

คาสาคญ: สงเพมพนสภาพแวดลอม หนไมซ ความพงพอใจ สวสดภาพสตว กลมสตวทดลอง สถาบนวจยวทยาศาสตรสาธารณสข กรมวทยาศาสตรการแพทย กระทรวงสาธารณสข จงหวดนนทบร 11000 *ผรบผดชอบบทความ E-mail: navakanit.s@dmsc.mail.go.th

Introduction

Environmental conditions such as housing and husbandry have a major impact on laboratory animal throughout its life and will thereby influence the outcome of animal experiments (Van de Weerd et al., 2002). Standard laboratory cages are designed to fulfill the most essential needs in a laboratory animal's life such as provision of food, water and bedding. However, animals also have physiological and behavioral needs. Physiological needs include eating, drinking, sleeping and should logically include some provision of shelter. Behavioral needs include performing behavior necessary for the maintenance of a normal physiological and psychological state (Poole, 1998), most of which cannot be met in these cages. The animals are able to perform only a part of their complete species specific behavioral repertoire (Van de Weerd et al., 1994), which can result in abnormal behavior. Furthermore, animals maintained in unresponsive environments and highly unnatural groupings are less adequate models for extrapolating experimental results to humans (Markowitz and Gavazzi, 1995). Enrichment provides a more

structured environment. Enrichment can focus on several aspects of the environment such as housing animals in pairs or groups are a form of social enrichment. Foraging by scattering food particles in the bedding is a part of nutritional environment. The cage and its contents are part of the physical environment (Van de Weerd and Baumans, 1999). It is generally agreed that environmental enrichment is beneficial for the well-being of laboratory animals and that it should be applied whenever appropriate which enables them to perform more of their species-specific behavior and which gives them more control over their environment (Kaliste and Mering, 2004). The introduced enrichment should be interesting for the animals by meeting their behavioral requirements but, from the human point of view, it should be easy to provide, remove and clean (Van de Weerd and Baumans, 1995). Nesting material is an easily applicable form of enrichment for laboratory mice. Both males and females will build a nest when offered nesting materials. Nesting material is also used as a source of protection e.g. against extreme environmental temperatures, when physiological systems alone are inadequate to maintain body temperature. Nests also offer an opportunity to hide

Sachanonta N. et al. / Thai J Vet Med. 2013. 43(2): 205-211. 207

from predators and in the laboratory to avoid aggressive con specifics, or to provide a shelter from overexposure to light. Mice in the enriched environment exhibited less anxiety-like behavior and more activity compared with mice in the standard cages (Benaroya-Milshtein et al., 2004). When applying enrichment, it is necessary to evaluate the suitability of the enrichment program, as various species or strains may respond differently to the methods of enrichment. To assess the preference of an animal for a certain feature, one can use well-designed choice tests. Specifically, this technique has been used to identify laboratory animal preferences and avoidances for specific housing conditions including cage size, bedding type, nesting type, etc. Individual animal can be offered different housing conditions in different test cages. During the test period, the movements of the animal between the test cages can be detected (Blom et al., 1992). The aim of the present study was to investigate possible differences in preference for nesting materials and enrichment devices as a supporting data for a better development of suitable enrichment program of LAC-Thai NIH, accredited by AAALAC International. For this purpose, major strain and sex of animal used in LAC-Thai NIH were selected in a preference test for different types of nesting materials and also enrichment devices added to otherwise standard environmental conditions were evaluated.

Materials and Methods Animals: 10 male ICR mice were used. The mice were 10-12 weeks of age. The use of animals was approved by Thai-NIH Animal Care and Use Committee (#53-013). Prior to this study, the mice were housed in groups of five animals in shoebox cages (9x14x5.5 inch) on corncob contact bedding that was changed twice a week. Cages were supplied with food pellets and RO water in bottle ad libitum. The animals were kept in conventional rooms with controlled photo period (12: 12 L: D, lights on at 06:00 hour, approx. 200 lux at 1 m above the floor), temperature (20-220C), relative humidity (50-60%) and ventilation (12-15 air changes per hour). Environmental conditions in the experimental rooms were similar. One day prior to a preference testing, each mouse was introduced into the test system for 3 hour in order to familiarize the mice with test system and they have a recess time, not less than 7 days, between each experiment.

Figure 1 The preference test system (top view).

Preference test system: Three nesting materials and three enrichment devices were evaluated with the use of two-cage choice housing system (Fig 1). It consisted of two test cages connected by transparent tubes (inner dimension 4.5 cm, 20 cm long) to a central cage (15x15x16 cm). The test system was gently rotated 180 degrees every 24 hours to prevent bias due to external influences in the experimental room which could interfere with the choice behavior of the mouse. The test cages were supplied with 150 g of corncob bedding, a food hopper with 50 g of food pellets and 250 g RO water in bottles. The central choice cage had no food, water or bedding. The movements of the

Figure 2 The three nesting materials tested and background

bedding material. A: Woodchips (left), scanning electron micrograph x 500 (right), B: Paper series; Paper towel and Paper strip (left), scanning electron micrograph x 500 (right), C: Corn cob (left), scanning electron micrograph x 500 (right).

Figure 3 The three enrichment devices tested. A: Commercial

plastic mouse house, B: Standard plastic mouse house, C: Cassava-based cup.

208 Sachanonta N. et al. / Thai J Vet Med. 2013. 43(2): 205-211.

mice between the test cages were detected automatically by means of photo-electrical devices in the passage tubes.

Preference test; nesting materials and enrichment devices: We used one system containing positions for two cages with a photo-electrical device for measuring dwelling time for one cage. Mouse was introduced into the test system by placing into the central choice cage between 10:00 and 12:00 and tested individually during 48 hours. To discourage the mice from remaining in the central choice cage, we did not place bedding, food or water. The behavior of one animal was observed via video camera for the first 2 hours, so that movements of the mouse could be followed in the test system. We also observed and monitored the mouse and test system 3 times daily. Food and water of each test cage were weighed before and after the experiment.

Two assessments were performed to test types of each nesting materials and enrichment devices. For the nesting material assessment; three nesting materials (woodchips and paper series; paper towel and paper strip) were tested by comparing woodchips with paper towel (experiment 1) and paper towel with paper strip (experiment 2). For the enrichment device assessment; three enrichment devices (cassava-based cup and plastic mouse house series; standard and commercial plastic mouse house) were tested by comparing standard with commercial plastic mouse house (experiment 3) and standard plastic mouse house with cassava-based cup (experiment 4). Table 1 describes the materials and gives the amounts provided per cage and Figs 2 and 3 show nesting materials and enrichment devices.

Scanning Electron Microscopy (SEM): The ultrastructure of bedding and nesting materials were studied using SEM. Specimens (corn cob, woodchips and paper) were attached to aluminum stubs with double-stick tape and coated with gold (Au) in a sputter-coating apparatus before being viewed with a JEOL JSM-5910LV scanning electron microscope (JEOL: Japan).

Statistical analysis: Dwelling time per cage data was collected from individual mouse at 24 and 48 hours. The dwelling time data were analyzed which of the cages were preferred or avoided using paired t-test. Statistical significance was preset at p < 0.05. Data on

food and water intake were not transformed because they were normally distributed.

Results For the nesting material assessment (Fig 4) at 24 hours, there were no significant differences in cage preferences (dwelling time) between paper towel and woodchips of experiment 1, paper towel and paper strip of experiment 2, (paired t-test, p < 0.05). At 48 hours, comparing between woodchips and paper towel in experiment 1, no significant difference in dwelling times was found but we inferred that mice preferred paper towels because 60% of the mice dragged paper towels from one cage to the other cage containing woodchips to combine two types of nesting material. Comparing between paper series (paper towel and paper strip) in experiment 2, the cage containing paper towel was found to significantly affect mouse preference with mice spending greater time than in the cage containing paper strip, p < 0.05. Anyway, all mice showed the same activity shredding sheet of paper towel and also paper strip to build nest. Also, mice showed nest building behavior with all nesting materials. No significant difference of all nesting materials from 24 to 48 hours.

Figure 4 The percentages of dwelling time of nesting

material assessment PT: Paper towel, WC: Woodchips, PS: Paper strip;

-*-p < 0.05 at 48 hours of paper towel compared with paper strip (paired t-test, values presented mean±SEM).

Table 1 Nesting materials and enrichment devices

Type Material Trade name Appearance (size) Amount Woodchips

Versele-Laga™, Belgium Wood (variable) 50 g

Paper towel

River pros™, Thailand Sheet (20 x 24.5 cm) 1 piece

Nes

ting

mat

eria

ls

Paper strip

River pros™, Thailand Strip (2 x 24.5 cm) 10 piece

Commercial plastic mouse house

Techniplast™, Italy Transparent, polycarbonate, triangular-shaped box

1 piece

Standard plastic mouse house

Bio-Serv™, USA Transparent, polycarbonate house

1 piece

Enri

chm

ent

devi

ces

Cassava-based cup KU-GREEN™, Thailand Opaque, biodegradable cup (83 mm D x 52 mm H)

1 piece

Sachanonta N. et al. / Thai J Vet Med. 2013. 43(2): 205-211. 209

Figure 5 Percentages of dwelling time of enrichment device

assessment SMH: Standard plastic mouse house, CMH:

Commercial plastic mouse house, CBC: Cassava-based cup

-*- p < 0.05 at 48 hours of standard plastic mouse house compared with commercial plastic mouse house (paired t-test) ** p < 0.05 from 24 to 48 hours of standard plastic mouse house and commercial plastic mouse house (paired t-test) (values presented mean±SEM)

For the enrichment device assessment (Fig 5) at 24 hours, there were no significant differences between standard and commercial plastic mouse house of experiment 3, cassava-based cup and standard plastic mouse house of experiment 4. At 48 hours, comparing between plastic mouse house series in experiment 3, it showed significant differences between commercial and standard plastic mouse house. The mice preferred standard than commercial plastic mouse house. Comparing between cassava-based cup and standard plastic mouse house in experiment 4, the result showed no statistical significant difference. Although the mean of data showed as it seemed to make a difference but the range of standard deviation is so wide that they showed no significant difference using paired t-test analysis. The standard plastic mouse house and cassava-based cup were clearly preferred by mice than commercial plastic mouse house. There were significant differences from 24 to 48 hours standard and commercial plastic mouse house in experiment 3. The mice showed more activities such as sleeping in and turning over with cassava-based cup than plastic mouse house series. In every experiment, the behavioral data on video tape of the first 2 hours of experiment showed an exploration of mice cages. Observing the mouse and test system 3 times daily, all mice showed nest building behavior within the first 24 hours using nesting materials and clearly decided to choose one cage within 48 hours. The mice used the preferred cage to sleep in and ate mostly in the cage they did not live in. Eating and drinking behavior of the mice was in normal range. Water intake did not differ between the cages.

Discussion

The results of this study indicate that mice preferred cages with paper towel as nesting material

than paper strip. The mice did not make a significant choice between the two nesting materials offered; paper towel and woodchips, but in most cases combined them. We exclude the data that mice dragged paper towel in the experiment because the mice showed their decision to combine both nesting materials. All mice dragging paper towel to cage containing woodchips because the structure of paper towel might be easier to move than woodchip. Behavioral observation showed that manipulation of the bedding and resting in nests was performed mostly on paper towel. The fact that some animals combined nesting materials might suggest that mice preferred to make more complicated nest or there is not a clear preference for the nature of nesting materials e.g. paper or wood but that other features of the nesting materials such as the structure e.g. sheet or strip also play a role. The results suggest that the nature of the nesting materials might be less important than the structure. On the other hand, the structure may be important because it determines the nesting ability of the material. In the present study, the characteristic feature which the preferred nesting material has in common is that the mice can manipulate them to build nests, and by doing this, they are able to construct their own environment. Another aspect of the nesting materials which could be an important criterion for selection by the mice is the degree of light absorption. Mice are nocturnal animals which often prefer to hide and sleep in dark places during daytime. However, the nesting materials in the present study allowed some penetration of light but the paper towel could provide a shelter for light, completely covered by the materials of their nests. Not only shelter for light, paper towel as nest can also control temperature of microenvironment for mice. Besides, a recessive animal can use shelter as a safe area for hiding that may increase its sense of security. When aggressive males are being used, individual housing is common practice. Single housing in order to avoid aggression between male mice is a solution with evident negative consequences for the animals, the presence of nesting material could partly compensate for the deprivation of social contact (Van Loo et al., 2004). Some study indicated that the male ICR mice showed preference in reusable cloth as nesting material than recycled-paper (Kawakami et al., 2007). In Blom's study, the mice showed a preference for shredded filter paper in comparison with smaller particle bedding material (Blom et al., 1996). The study by Blom (1992) showed that 65% of the time is spent on sleeping, grooming and digging behavior. In the present study, nest building behavior could be performed with the nesting materials and digging was less frequently observed. It is possible that female mice would demonstrate a different preference, however, results from previous preference tests (Van de Weerd et al., 1997) indicated that enrichment preferences of male mice did not differ drastically from those of female mice. This is able to use an active strategy to manipulate and control more aspects of environment for mice and be a simple method to contribute to the well-being of laboratory mice.

210 Sachanonta N. et al. / Thai J Vet Med. 2013. 43(2): 205-211.

In view of enrichment device results presented here, it presumes that the cassava-based cup and standard plastic mouse house have features which provide added attractive value to mice. The commercial plastic mouse house seems to lack these features. It is interesting that the mice showed significantly lower preference for commercial plastic mouse house comparing with standard type which has the same material. This may suggest that the shape of enrichment device is an important factor for animal needs. Anyway, the material of enrichment device is also important. Some study indicated a strong preference by mice for paper-based triangular-shaped enrichment device more than commercial plastic mouse house which has almost the same shape (Van Loo et al., 2005). Commercial plastic mouse house has more complex structure, triangular-shaped box with a small tunnel opening on the long side with an extra triangular opening in the top, than standard type. The top opening allowed light penetrating to the cage floor. Tunnel opening is only 3 cm in height which made it difficult for adult male mice to pass so they only used top opening to enter the box. These factors might affect the mice preference. Although the cassava-based cup and standard plastic house did not significantly show which were more preferred by the mice in statistical analysis but the mean of data clearly showed that the mice preferred cassava-based cup. Cassava-based cup and standard plastic mouse house differ with regard in not only construction material; rough, opaque and eatable vs smooth, transparent and uneatable but also weight; light vs heavy, respectively. These features make it possible to manipulate the cassava-based cup while the plastic mouse house is more rigid. The plastic mouse house always stayed in position once placed in the cage while the mice usually moved around and manipulated the cassava-based cup. We also observed that mice turned over the cassava-based cup to create their own rocking chair. These observations provide evidence that enrichment device increases environmental control for mice which is an important stress-reducing propensity (Sambrook and Buchanan-Smith, 1997).

It is upon individuals who use and care for laboratory animals to seek to improve the quality of the captive environment. To that end, well-designed and carefully communicated enrichment approaches are required. More data are needed to provide information related to the effects of specific enrichment programs on the animal, on specific animal species and on experimental results. The most important aim of environmental enrichment is to make the animals feel secure and give them a sense of control within a complex, challenging environment that meets essential species-typically behavioral needs of animals in order to improve their well-being in captivity. The selection for suitable enrichment items of laboratory animal center should be adapted from the originals, for example cassava-based cup was adapted from standard plastic mouse house and paper towel was adapted from commercial paper nesting material. The reason for enrichment item adaptation is harmonization between animal needs

with scientific evidence and human needs, economy and ergonomics. The advantage of cassava-based cup and paper towel is that they can produce in Thailand, therefore, they are inexpensive. They are also disposable, making them easier to be managed than other items. Anyway, both of them need to have analysis of contaminations; aromatic hydrocarbon, heavy metals, bleach, etc.; to make sure that it is safe for animals when using in long-term experiment. To comply with the animals’ needs while keeping in mind human requirements, we propose that the ultimate enrichment item should be tried on the basis of knowledge before use in laboratory animal center.

Acknowledgements

We would like to extend our gratitude to

Laboratory Animal Center, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand for supporting this project. We would also like to thank Dr. Maskiet Boonyareth for helps in the statistical analysis and Miss Kingkarn Tobngam for picture arrangement.

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Blom H, Van Tintelen G, Van Vorstenbosch CJ,Baumans V and Beynen AC 1996. Preferences of mice and rats for types of bedding material. Lab Anim. 30(3): 234-244.

Blom H, Van Vorstenbosch C, Baumans V, Hoogervorst M, Beynen A and Van Zutphen LF 1992. Description and validation of a preference test system to evaluate housing conditions for laboratory mice. Appl Anim Behav Sci. 35: 67-82.

Kaliste E and Mering S 2004. The welfare of laboratory rats. In: The Welfare of Laboratory Rats. Kluwer Academic Press. Dordrecht: 153-180.

Kawakami K, Shimosaki S, Tongu M, Kobayashi Y, Nabika T, Nomura M and Yamada T 2007. Evaluation of bedding and nesting materials for laboratory mice by preference tests. Exp Anim. 56(5): 363-368.

Markowitz H and Gavazzi A 1995. Eleven principles for improving the quality of captive animal life. Lab Anim. 24(4): 30-33.

Poole T 1998. Meeting a mammal's psychological needs: Basic principles. In: Meeting a Mammal's Psychological Needs: Basic Principles. Smithsonian Institution Press. Washington. 94 pp.

Sambrook T and Buchanan-Smith H 1997. Control and complexity in novel object enrichment. Anim Welf. 6: 207-216.

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Van de Weerd H and Baumans V 1995. Environmental enrichment in rodents. In: Environmental Enrichment in Rodents, 2. AWIC Resource Series: 145-149.

Van de Weerd H and Baumans V 1999. Evaluation of environmental enrichment for laboratory mice. AWIC Bulletin. 9(3-4): 18-19.

Van de Weerd H, Aarsen EL, Mulder A, Kruitwagen CL, Hendriksen CF and Baumans V 2002. Effects of environmental enrichment for mice: Variation in experimental results. J Appl Anim Welf Sci. 5(2): 87-109.

Van de Weerd H, Baumans V, Koolhaas JM and Van Zutphen LF 1994. Strain specific behavioural response to environmental enrichment in the mouse. J Exp Anim Sci. 36(4-5): 117-127.

Van de Weerd H, Van Loo PL, Van Zutphen LF, Koolhaas JM and Baumans V 1997. Nesting material as environmental enrichment has no adverse effects on behavior and physiology of laboratory mice. Physiol Behav. 62(5): 1019-1028.

Van Loo PL, Blom H, Meijer M and Bauman V 2005. Assessment of the use of two commercially available environmental enrichments by laboratory mice by preference testing. Lab Anim. 39 (1): 58-67.

Van Loo PL, Van de Weerd H, Van Zutphen LF and Baumans V 2004. Preference for social contact versus environmental enrichment in male laboratory mice. Lab Anim. 38 (2): 178-188.

212

Thai J Vet Med. 2013. 43(2): 213-218.

Effects of Myogenic Factor 5 (MYF5) Gene on Carcass and Meat

Quality of Mong Cai Pigs

Nguyen Thi Kim Khang Nguyen Trong Ngu*

Abstract

The aim of this study was to analyze the impact of Myogenic Factor 5 gene (MYF5) on carcass and meat

quality of Mong Cai pigs. PCR-RFLP was applied to genotype 100 animals at three loci defined as MYF5/Hin1II, MYF5/HaeIII and MYF5/MspI. Results showed that MC pigs had two genotypes at each locus namely CD and DD, EF and EE, and GH and HH for the MYF5/Hin1II, MYF5/HaeIII and MYF5/MspI loci, respectively. The frequencies of DD, EE and HH were predominant (0.78-0.94) in the population. In addition, association analysis of three loci revealed that animals carrying CD genotype provided higher dressing percentage (70.30 vs. 67.32%) and loin weight (841 vs. 787 g) than those of DD genotype; however, these DD pigs had lower compression force value (4.91 vs. 5.79 kg). The other two polymorphisms were completely linked and no significant association was found with carcass and meat quality traits. Furthermore, the constructed haplotypes were identified to associate with hot carcass and dressing weight, of which the highest values were in CDEEHH pigs. These results suggested that the MYF5/Hin1II polymorphism was valuable for some carcass traits and compression force, but on the basis of this polymorphism, a simultaneous selection for these traits in MC pig remained a difficult task.

Keywords: carcass, pork quality, Vietnamese pigs College of Agriculture and Applied Biology, Can Tho University, 3/2 Street, Can Tho City, Viet Nam *Corresponding author: E-mail: ntngu@ctu.edu.vn

Original Article

214 Nguyen TKK & Nguyen TN / Thai J Vet Med. 2013. 43(2): 213-218.

บทคดยอ

ผลของยน Myogenic Factor 5 (MYF5) ตอคณภาพซากและเนอของสกรพนธ Mong Cai Nguyen Thi Kim Khang Nguyen Trong Ngu *

จดมงหมายของการศกษานเพอวเคราะหผลกระทบของยน Myogenic Factor 5 (MYF5) ตอคณภาพซากและคณภาพเนอสกร Mong Cai โดยใชเทคนค PCR-RFLP ในวเคราะหสายพนธสตวจานวน 100 ตว โดยกาหนดตาแหนงทงหมดสามตาแหนง ไดแก MYF5/Hin1II MYF5/HaeIII และ MYF5/MspI ผลการศกษาพบวาสกร Mong Cai มสองยนในแตละทคอ CD และ DD, EF และ EE และ GH และ HH สาหรบตาแหนง MYF5/Hin1II, MYF5/HaeIII และ MYF5/MspI ตามลาดบ โดยพบความถของ DD, EE และ HH เปนหลก (0.78-0.94) จากกลมประชากร นอกจากนการวเคราะหความสมพนธของตาแหนงทงสามแสดงใหเหนวาสกรทมจโนไทปชนด CD มเปอรเซนตซากสกร (70.30 เทยบกบ 67.32%) และนาหนกเนอสนหลง (841 เทยบกบ 787 กรม) สงกวาของจโนไทป DD อยางไรกตามสกรทมจโนไทปชนด DD มคาแรงกด (compression force value) ทตากวา (4.91 เทยบกบ 5.79 กก.) สาหรบความหลากหลายอกสองประเภทนนมการเชอมโยงกนและไมมความสมพนธอยางมนยสาคญคณภาพซากและคณภาพเนอสกร นอกจากนพบ haplotype ทสมพนธกบนาหนกซากอนและเปอรเซนตซากสกร ซงมคาสงสดในสกรกลม CDEEHH ผลการศกษานชใหเหนวาความหลากหลายของ MYF5/Hin1II มความสาคญตอคณภาพซากบางประการและตอคาแรงกด แตเมอคานงถงความหลากหลายเหลานการคดเลอกเพอใหเกดลกษณะรวมกนในสกร Mong Cai นนยงคงเปนงานทยาก

คาสาคญ: คณภาพซาก คณภาพเนอหม สกรเวยดนาม College of Agriculture and Applied Biology, Can Tho University, 3/2 Street, Can Tho City, Viet Nam *ผรบผดชอบบทความ E-mail: ntngu@ctu.edu.vn

Introduction

Myogenic factor 5 (MYF5), a product of the MYF5 gene belonging to the MyoD family, plays an important role in the control of myogenesis and is responsible for the primary muscle fiber formation to their postnatal maturation and function (Hughes and Schiaffino, 1999). The MYF5 is related to the primary muscle cell migration and proliferation, especially for the satellite cell proliferation in the postnatal process of muscle regeneration (Pierzchała et al., 2008). This gene has been mapped to the porcine chromosome 5 (Soumillion et al., 1997), which contains three exons and two introns (te Pas et al., 1999). The expression and effects of MYF5 are different depending on breeds and it has been considered a candidate gene for meat production and meat quality (te Pas, 2004; Carmo et al., 2005). Because of its function involved in myoblasts proliferation, MYF5 gene was found to affect the proportion muscle fibers and the metabolic properties of muscle (Kłosowska et al., 2004). In cattle, MYF5 has been evidenced to influence the expression of muscle fiber types (Muroya et al., 2002) and thereby some meat quality traits (Ujan et al., 2011), while in pigs many studies have described the effect of MYF5 gene on carcass traits (te Pas et al., 1999; Cieślak et al., 2002; Liu et al., 2007a) but little is reported on its association with meat quality, especially in indigenous pig breeds. In Vietnam, Mong Cai (MC) is

a popular local pig breed in the Central coastline, the Red River delta and other Northern provinces. Although it has slow growth rate, it has favored characteristics of good adaptation to poor-quality feed and superior meat quality traits (Ngu, 2006). The aim of this study was to reveal the association of MYF5 polymorphisms with carcass characteristics and meat quality traits obtained in MC pigs.

Materials and Methods Animals and Sampling: This study was carried out on a hundred MC castrated male pigs, reared at a state farm in Quang Ninh province of Vietnam, at the body weight of 28.8±5.9 kg and at the age of 198±17.9 days. The pigs were fasted 12 hours before slaughtering following a commercial standard procedure and under the supervision of the veterinary service. Hot carcass weights were recorded and used for dressing percentage calculation. Longissimus dorsi (LD) muscle samples were collected for DNA extraction and meat quality evaluation. Meat color was determined using a Minolta Chromameter (CR310, Minolta, Japan) on a cut surface 24 hour post-mortem. Muscle pH was measured at 45 min (pH45 min) and 24 hour (pH24) with a Delta-320 portable pH meter (Richmond Scientific Ltd.). Drip loss was calculated as the weight loss of a meat sample in an inflated bag at 40C for 24 hours (DL24) and 48 hours (DL48) (Honikel,

Nguyen TKK & Nguyen TN / Thai J Vet Med. 2013. 43(2): 213-218. 215

1998). For compression force values, samples were thawed at 40C and cut into 10 x 10 mm cross-sections and six samples for each loin were measured using a TA–XT2i Texture Analyzer (Stable Micro Systems Ltd) (Florowski et al., 2006). Meat chemical compositions such as the percentage of dry matter (DM), crude protein (CP) and ether extract (EE) of LD muscle were analyzed using standard AOAC methods (AOAC, 1998).

Genotyping: Total DNA was extracted using phenol-chloroform method. Primer sequences, restriction enzymes and PCR condition of the MYF5 gene (GenBank Acc. No.Y17154) were detailed by Liu et al. (2008). In brief, fragments were amplified in 20 µl reaction volume containing 25 ng genomic DNA as template, 0.25 μM dNTP, 0.25 μM of each primer, and 1 U Taq polymerase and PCR buffer. The reaction started with 4 min of initial denaturation at 940C, followed by 35 cycles of amplification with denaturation at 940C for 45 sec, annealing at 570C (MYF5-p1) or 630C (MYF5-p2) for 45 sec, extension at 720C for 60 sec, and a final elongation at 720C for 10 min. The PCR products of the MYF5-p1 and MYF5-p2 primers were subsequently used for genotyping by PCR-RFLP (Restriction Fragment Length Polymorphism) method. The three polymorphic sites, namely A1205C (exon 1), G2368A and A2165G (intron 1, exon 2, intron 2), were recognized by Hin1II, HaeIII and MspI, respectively. A total volume of 10 µl including 8.5 µl PCR product, 0.5 µl restriction enzyme and 1 µl reaction buffer was incubated at 370C for 4 hours. The digested products were checked and genotypes were directly determined by electrophoresis on a 2% agarose gel at 80 volt for 30 min. From the detected SNPs, a construction of haplotype using Merlin software (Abecasis et al., 2002) was performed and these haplotypes together with the SNPs were analyzed in the association study.

Statistical analysis: Association analysis between MYF5 genotypes and traits of interest was performed using least squares method of the GLM procedure in Minitab version 13.20 with the following model: Yijk = µ + Gi + Cj + eijk, where Yijk was the observation of traits, µ was the overall mean of each trait, Gi was the genotype effect, Cj was the covariate (body weight at slaughter) and eijk was the random error. The difference between genotypes was tested using Turkey pair wise comparison at the 5% significance

level. Data were presented as Least square means±standard error.

Results Genotype and allele frequency: The PCR-RFLP data allowed identifying different genotypes at three polymorphic positions of the MYF5 gene. The electrophoresis results obtained from restriction enzyme fragments were: 243+210+33 bp for MYF5/Hin1II CD genotype; 210+33 bp for MYF5/Hin1II DD genotype; 1016+193 bp for MYF5/HaeIII EE genotype; 1016+777+239+193 bp for MYF5/HaeIII EF genotype; 575+461+173 bp for MYF5/MspI HH genotype and 1036+575+461+173 bp for MYF5/MspI GH genotype (Fig 1). The frequencies of genotypes and haplotypes of the SNPs (Single Nucleotide Polymorphism) are presented in Table 1. There was a similar trend of genotype distribution at all loci, in which the majority of animals carried DD, EE and HH genotypes (> 78%); however, there were only 2 genotypes found at each locus. The MYF5/HaeIII and MYF5/MspI were completely linked; their genotype frequencies for both mutations had the same values. Additionally, the most common haplotype was DDEEHH, followed by CDEEHH and DDEFGH.

Effects of polymorphisms on carcass and meat quality traits: Association analysis of the MYF5/Hin1II genotypes showed significant impacts of CD and DD genotypes on dressing percentage and loin weight (p < 0.05) (Table 2). Moreover, the pH45 min value was detected to be higher in DD compared to CD pigs (p < 0.05). The DD genotype had significantly lower compression force value than CD genotype (p < 0.05). For the other two SNPs, significant associations between MYF5/HaeIII and MYF5/MspI genotypes with carcass and meat quality traits were not detected (data not shown). In addition, MYF5 haplotypes were significantly associated with hot carcass and dressing percentage (p < 0.05) with highest values in animals carrying CDEEHH haplotype, whereas no differences were apparent for other meat quality traits (Table 3). There was also a trend of increased loin weight in CDEEHH pigs with the significance of p = 0.052. At all loci examined, statistical effects of either genotypes or haplotypes on meat chemical composition (Table 2 and Table 3) were not established (p > 0.05).

Figure 1 Agarose gel electrophoresis of PCR-RFLP at MYF5/Hin1II, MYF5/HaeIII and MYF5/MspI loci. M: 100 bp DNA ladder,

Fermentas.

216 Nguyen TKK & Nguyen TN / Thai J Vet Med. 2013. 43(2): 213-218.

Table 1 Genotype and haplotype frequencies of MYF5 gene in MC pigs

MYF5/Hin1II MYF5/HaeIII MYF5/MspI MYF5 haplotype Item CD DD EF EE GH HH DDEEHH CDEEHH DDEFGH

No. of pigs 22 78 6 94 6 94 73 21 5 Frequency 0.22 0.78 0.06 0.94 0.06 0.94 0.74 0.21 0.05

Table 2 Association of MYF5/Hin1II genotypes and meat quality traits in MC pigs

Genotype Traits

CD (n = 22) DD (n = 78) p-value

Carcass Dressing percentage (%) 70.30 ± 0.97 67.32 ± 0.53 0.009 Loin weight (g) 841.4 ± 22.23 786.9 ± 12.11 0.039

Meat quality pH45 min 6.53 ± 0.05 6.65 ± 0.02 0.018 pH24 6.09 ± 0.04 6.12 ± 0.02 0.530 Drip loss24 (%) 1.98 ± 0.16 1.79 ± 0.09 0.499 Drip loss48 (%) 2.87 ± 0.20 2.58 ± 0.11 0.237 Cooking loss (%) 23.35 ± 0.89 22.21 ± 0.49 0.264 Compression force (kg) 5.79 ± 0.37 4.91 ± 0.19 0.034

Meat color L* (Lightness) 48.94 ± 0.34 49.29 ± 0.19 0.353 a* (Redness) 4.97 ± 0.60 5.59 ± 0.32 0.369 b* (Yellowness) 8.79 ± 0.31 8.92 ± 0.17 0.997

Meat chemical composition (%) Dry matter 25.45 ± 0.30 25.63 ± 0.16 0.946 Crude protein 21.99 ± 0.22 22.09 ± 0.12 0.612 Ether extract 2.42 ± 0.11 2.58 ± 0.06 0.148 Ash 0.94 ± 0.08 1.01 ± 0.04 0.238

Table 3 Association of MYF5 haplotypes and carcass and meat quality traits in MC pigs

Haplotype Traits

DDEEHH (n = 74) CDEEHH (n = 21) DDEFGH (n = 5) p-value

Carcass Dressing percentage (%) 67.44 ± 0.53b 70.64 ± 0.97a 68.01 ± 2.02ab 0.016 Loin weight (g) 792.0 ± 12.17 847.9 ± 22.39 753.4 ± 46.78 0.052

Meat quality pH45 min 6.63 ± 0.03 6.55 ± 0.04 6.69 ± 0.10 0.261 pH24 6.09 ± 0.02 6.10 ± 0.04 6.15 ± 0.09 0.423 Drip loss24 (%) 1.83 ± 0.11 2.12 ± 0.21 1.73 ± 0.44 0.433 Drip loss48 (%) 2.57 ± 0.13 2.94 ± 0.23 2.55 ± 0.50 0.388 Cooking loss (%) 22.40 ± 0.51 22.41 ± 0.96 21.57 ± 1.99 0.920 Compression force (kg) 4.95 ± 0.20 5.17 ± 0.38 5.50 ± 0.79 0.715

Meat color L* (Lightness) 49.37 ± 0.19 48.87 ± 0.34 48.62 ± 0.71 0.287 a* (Redness) 5.72 ± 0.33 4.89 ± 0.60 7.76 ± 1.26 0.110 b* (Yellowness) 8.94 ± 0.17 8.71 ± 0.32 8.80 ± 0.66 0.804

Meat chemical composition (%) Dry matter 25.43 ± 0.17 25.49 ± 0.30 25.25 ± 0.63 0.937 Crude protein 21.94 ± 0.12 22.27 ± 0.22 21.92 ± 0.46 0.404 Ether extract 2.52 ± 0.06 2.37 ± 0.12 2.70 ± 0.24 0.367 Ash 1.04 ± 0.05 0.86 ± 0.08 1.01 ± 0.17 0.173

a, b Values in the same row with different superscripts are significantly different (p < 0.05)

Discussion

Our study confirms the findings in many pig breeds that the presence of homozygous genotypes CC, FF and GG was very rare and by investigating the association of three SNPs on carcass and meat quality traits, only the MYF5/Hin1II was observed to influence dressing percentage, loin weight, meat pH

and compression force.

In the current study, the MYF5/Hin1II polymorphism provided two genotypes with the majority being the DD animals. This partly supported the report of Liu et al. (2007b) that in a commercial population of Yorkshire, Landrace and their cross, this locus was polymorphic for the D allele whereas

Nguyen TKK & Nguyen TN / Thai J Vet Med. 2013. 43(2): 213-218. 217

three genotypes were detected in the Meishan, Yorkshire and Meishan x Yorkshire cross. At the MYF5/MspI locus, the GG genotype could not be found and the HH genotype was predominant in both Large White and Landrace breeds (Verner et al., 2007). In the LW x Meishan F2 population, a small number (5.1%) of GG animals was also described by Liu et al. (2007a). This was later validated by Liu et al. (2008), who found no GG genotype in the Large White and Landrace, whereas in the Meishan breed the GG and GH frequencies were 0.18 and 0.38, respectively, which was higher than those of MC pigs (0 and 0.06, respectively). Thus, irrespective of breed, the G allele was in low frequency across populations.

The first polymorphism in exon 1 of the MYF5 gene resulted in amino acid substitution (Met

Leu), which may be responsible for changing the functional properties of the protein and affecting muscle maturity (Liu et al., 2008). In the present study, the analysis of MYF5/Hin1II polymorphism with carcass traits indicated that pigs with genotype DD had significantly lower dressing percentage and loin weight as compared to those with genotype CD. In the Large White x Meishan F2 population, Liu et al. (2007b) observed significant associations between this locus with fat deposition traits and carcass length, in which the difference mainly between the CC and DD animals implied that allele “C” was closely related to higher back fat and buttock fat thickness content. Although CC animals were not available in this study, it could be that the impacts were breed-specific due to different genetic backgrounds and selection pressure or it might originate from the difference in muscle fiber type proportion, where higher percentages of IIx and IIa were found in MC pigs compared to IIb fibers (data not shown). It also suggested that this mutation could not be causal for the differences in carcass traits being investigated. The haplotype analysis, of which the lowest carcass values in pigs bearing DDEEHH variants, additionally confirmed the data.

The MYF5 gene was reported to have a relationship with the expression of fast-twitch oxidative fiber (Kłosowska et al., 2004) and thus it is likely to have further impact on metabolic activity of the muscle and meat quality. Regarding to this point, for the MYF5/Hin1II SNP, Liu et al. (2008) demonstrated changes of intramuscular fat and meat moisture content with the substitution of amino acid caused by this polymorphism. However, these outcomes were not confirmed in this study, showing only significant effects on pH45 min and compression force of the meat. In the present work, although there was a discrepancy of compression force values between two genotype groups, the intramuscular fat reflected by EE value was similar. According to the observations of Florowski et al. (2006) on a Polish indigenous pig breed, the relationship between compression force and intramuscular fat content was negative (-0.36), and thus higher fat deposition would be expected in homozygous DD pigs. The non-significant difference was probably because of the limited animals examined, especially the imbalance among the genotypes. In combination with the results of the association with carcass, it can be stated that a

simultaneous selection for both better carcass and meat quality in MC pig based on this polymorphism would be a difficult task to accomplish. As there were only a few animals bearing the EF and GH genotype, a wider study with larger numbers and in other native breeds would be of interest for analysis.

In conclusion, among the investigated MYF5 polymorphisms, the MYF5/Hin1II locus is of valuable SNP that has certain impacts on pH45 min, meat compression force in LD muscle, dressing percentage and loin weight. Further studies are needed to confirm the association in other native populations before applying this gene as a genetic marker for pig breeding selection.

Acknowledgements

This project was financially supported by the

Vietnam's National Foundation for Science and Technology Development (NAFOSTED), Grant No. 106.06.62.09.

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Thai J Vet Med. 2013. 43(2): 219-227.

mRNA and Protein Expressions of Prostaglandin E2 Receptors

(EP2 and EP4), Cyclooxygenase-2 and Prostaglandin E Synthase

in the Cervix of Cyclic Bitches and those with Pyometra

Pichanun Linharattanaruksa1 Theerawat Swangchan-Uthai1 Sayamon Srisuwatanasagul2

Muhammad Khalid3 Kaywalee Chatdarong1*

Abstract

Prostaglandins play a vital role in regulation of cervical patency. Prostaglandin E2 (PGE2) synthesis is

regulated by cyclooxygenase (COX) and prostaglandin E synthase (PGES). PGE2 acts through prostaglandin E receptors (EP), EP2 and EP4, to stimulate muscle relaxation. The aim of this study was to investigate mRNA expressions (EP2, EP4, COX-2, and PGES) and protein expressions (EP2, EP4, and COX-2) in the bitch cervix. Two groups of bitches, normal cyclic bitches and bitches with pyometra, were studied. Cyclic bitches were categorized into anestrus (n = 10), estrus (n = 7), and diestrus (n = 11), whereas bitches with pyometra were defined as open- (n = 18) or closed-cervix pyometra (n = 8) depending on the presence or absence of vaginal discharge, respectively. Cervices from the internal to external os were collected immediately after ovariohysterectomy. RNA extraction from cervical tissue was determined for levels of EP2, EP4, COX-2, and PGES mRNA using real-time qPCR. Western blot was performed to evaluate the protein expressions of EP2, EP4, and COX-2. There were no differences of mRNA and protein expressions in the bitch cervix among the stages of the estrous cycle. However, the expressions of PGES mRNA was higher in the cervix of bitches with open-cervix than closed-cervix pyometra (p < 0.05). The differences of protein expressions of EP2, EP4, and COX-2 were not observed. Our findings suggest that mRNA and protein expressions of the enzymes involved in PGE2 synthesis and PGE2 receptors are not influenced by hormonal status during the estrous cycle whereas PGES mRNA expression is likely associated with cervical relaxation in the bitches with pyometra.

Keywords: cervical patency, dog, prostaglandins, transcription 1 Department of Obstetrics, Gynaecology and Reproduction, 2 Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Veterinary Clinical Sciences, the Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, AL9 7TA, UK. *Corresponding author: E-mail: kaywalee.c@chula.ac.th

Original Article

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บทคดยอ

การแสดงออกของเมสเซนเจอรอารเอนเอของตวรบพรอสตาแกลนดนอ2(ซบไทป 2 และ 4) ไซโคลออกซจเนส-2 และเอนไซมพรอสตาแกลนดนอซนเทส ในคอมดลกสนขทอยในวงรอบการเปนสด และในสนขเปนมดลกอกเสบแบบมหนอง พชนนท ลฬหรตนรกษ 1 ธรวฒน สวางจนทรอทย 1 ศยามณ ศรสวฒนาสกล 2 มฮมหมด คาลด 3 เกวล ฉตรดรงค 1*

พรอสตาแกลนดนมความสาคญในการควบคมการเปดปดของคอมดลก การผลตพรอสตาแกลนดนอ2 (PGE2) ควบคมโดยเอนไซมไซโคลออกซจเนส (COX) และพรอสตาแกลนดนอซนเทส (PGES) พรอสตาแกลนดนอ2ทางานผานการจบกบตวรบฮอรโมนพรอสตาแกลนดนอ2 (EP) ซบไทป EP2และ EP4 ทาใหเกดการคลายตวของกลามเนอ การศกษาครงนมวตถประสงคเพอตรวจหาระดบการแสดงออกของเมสเซนเจอรอารเอนเอ คอ EP2 EP4 COX-2 และ PGES ในคอมดลกของสนขระยะตางๆ ของวงรอบการเปนสด และในสนขทมปญหามดลกอกเสบแบบมหนอง ทาการศกษาในสนขปกตจานวนทงหมด 28 ตว โดยแบงออกเปน 3 กลม คอ แอนเอสตรส (10 ตว) เอสตรส (7 ตว) และไดเอสตรส (11 ตว) สวนสนขกลมทมปญหามดลกเปนหนองจานวน 26 ตว แบงออกเปน 2 กลม คอ มดลกเปนหนองแบบคอมดลกเปด (18 ตว) และแบบคอมดลกปด (8 ตว) โดยดจากการพบหรอไมพบของเหลวไหลจากชองคลอด เกบคอมดลกจากชองเปดดานในถงชองเปดดานนอกหลงการทาหมนโดยตดรงไขและมดลกออก สกดอารเอนเอจากตวอยางคอมดลกแลวนามาตรวจหาระดบการแสดงออกของ EP2 EP4 COX-2 และ PGES ดวยวธ quantification real-time PCR (qPCR) จากการศกษาพบวา การแสดงออกของเมสเซนเจอรอารเอนเอทสนใจไมมความแตกตางกนในสนขในแตละระยะของวงรอบการเปนสด แตการแสดงออกของเมสเซนเจอรอารเอนเอของPGES ในกลมสนขทมปญหามดลกเปนหนองแบบคอมดลกเปดมคามากกวาในกลมคอมดลกปด (p < 0.05) ดงนน ระดบฮอรโมนทเปลยนแปลงในแตละระยะของวงรอบการเปนสด นาจะไมมผลตอการแสดงออกของยนทเกยวของกบการสรางพรอสตาแกลนดนอ2และตวรบฮอรโมนพรอสตาแกลนดนอ2ซบไทป 2 และ 4 แต PGES นาจะมสวนเกยวของกบกลไกการเปดของคอมดลกในสนขทมปญหามดลกเปนหนองแบบคอมดลกเปด

คาสาคญ: ภาวะเปดปดของคอมดลก สนข พรอสตาแกลนดน ทรานสครปชน 1 ภาควชาสตศาสตร เธนเวชวทยา และวทยาการสบพนธ คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย กรงเทพฯ 10330 2 ภาควชากายวภาคศาสตร คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย กรงเทพฯ 10330 3 Department of Veterinary Clinical Sciences, the Royal Veterinary College, University of London, UK *ผรบผดชอบบทความ E-mail: kaywalee.c@chula.ac.th

Introduction

Relaxation of the cervix is regulated by complex mechanism. Current knowledge about mechanism of cervical relaxation arises from the studies of pregnancy and parturition in humans (Timmons et al., 2010), rats (Chien and Macgregor, 2003), rabbits (Fukuda et al., 2007) and sheep (Kershaw-Young et al., 2009). In women, understanding of the control of cervical relaxation is crucial for the management of labor including its induction in case of prolonged pregnancy and treatment of undesired preterm births. In sheep, cervical relaxation may facilitate transcervical intrauterine artificial insemination (Kershaw et al., 2007). The cervical relaxation is regulated by serum hormonal concentrations, prostaglandin synthesis, and extracellular matrix remodeling (Kershaw-Young et al., 2009). In bitches, relationship between hormonal status and cervical patency is partly known. In

general, progesterone induces closure of the cervical canal and estrogen is capable of opening the canal during estrus which is important in fertilization process (Silva et al., 1995). Moreover, prostaglandin E2 (PGE2) was shown to induce cervical ripening in human when administered intravaginally (Feltovich et al., 2005). Biosynthesis of PGE2 is regulated mainly by two enzymes, cyclooxygenase (COX) which converts arachidonic acid to prostaglandin H2 (PGH2), and prostaglandin E synthase (PGES) which converts PGH2 to PGE2. There are two forms of COX, COX-1 and COX-2. COX-1 is a constitutive enzyme, whereas COX-2 is specific to inflammatory process (Tamada et al., 2012) and can be regulated by hormonal changes (Kowalewski et al., 2006). Studies in baboons and sheep revealed that cervical glandular epithelial cells were the major source of COX-2 mRNA (Wu et al., 2004; Wu et al., 2005). Moreover, a previous study in sheep showed that COX-2 mRNA was highly expressed in the cervix at estrus when serum estradiol

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concentrations were high, indicating a relationship between PGE2 and estradiol (Kershaw et al., 2007). Prostaglandin E2 exerts its role by coupling to prostaglandin E receptors (EP); EP1, EP2, EP3 and EP4. EP1 and EP3 are involved in the contraction of smooth muscle, whereas EP2 and EP4 function to induce smooth muscle relaxation (Narumiya et al., 1999). EP4 mRNA expression in the rat cervix was shown to be involved in cervical remodeling at the term time (Feltovich et al., 2005).

In bitches, pyometra is a common uterine disease which affects 25% of adult animals before they reach 10 years of age (Verstegen et al., 2008).The disease is classified into two categories; open- and closed-cervix pyometra. Clinical signs of pyometra depend primarily on whether the cervix is patent enough to allow the drainage of purulent fluid in uterus (Pretzer, 2008). Bitches with closed-cervix pyometra suffer from enlarged uteri and risk of uterine rupture. Cervical patency is a key to decide whether medical treatment is possible for pyometra bitches. To release pus from the uterus, aglepristone, an antiprogestin along with prostaglandins are reported to successfully induce cervical relaxation (Fieni et al., 2001). However, the exact mechanism of the induction of cervical relaxation in the bitch is unknown. In our previous study, the number of neutrophils was significantly higher in the bitches with open-cervix compared to that with closed-cervix pyometra (Kunkitti et al., 2011). Moreover, induction of cervical relaxation by neutrophil infiltration was also demonstrated in rabbits at the term time and was reported to be mediated by EP4 (Fukuda et al., 2007). Taken together, we hypothesized that there were associations between PGE2 [via its receptors (EP2 and EP4)], enzymes involved in PGE2 synthesis (COX-2 and PGES), and various stages of the estrous cycle which were influenced by sex steroid hormones. Moreover, the corresponding relationships might exist also in bitches affected with open- and closed-cervix pyometra. The present study aimed to investigate mRNA expressions (EP2, EP4, COX-2 and PGES) and protein expressions (EP2, EP4, and COX-2) in normal cyclic bitches during different stages of the estrous cycle and those with pyometra.

Materials and Methods Animals: Twenty eight bitches of various breeds with an average age of 2.1±0.8 years (ranging from 1-4 years) subjected to routine spaying were divided into 3 stages of estrous cycle; anestrus (n = 10), estrus (n = 7), and diestrus (n = 11), on the basis of presence or absence of large follicles or corpora lutea on the

ovaries, serum progesterone concentrations and vaginal cytology (Table 1). Another group of 28 bitches diagnosed as having uterine content by ultrasonography were divided into open-cervix (n = 18) and closed-cervix pyometra (n = 10) according to presence or absence of vaginal discharge, respectively. The bitches affected with pyometra had an average age of 7.0±3.9 years (ranging from 2-15 years).

Cervical tissue collection: Cervical tissue from each animal was collected immediately after ovariohysterectomy and prepared as previously described (Kunkitti et al., 2011). In brief, after collection the cervices were longitudinally cut into 2 parts. The cervical tissue was then cut into 0.1 g pieces and frozen in liquid nitrogen immediately and stored at-800C until used for RNA and protein extraction.

RNA extraction, reverse transcription and real-time qPCR: Cervical tissues were pulverized with sterile mortar and pestle. Total RNA was extracted from frozen cervical tissue using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) at room temperature following the manufacturer's instructions. The RNA concentration and purity were accessed using spectrophotometer at 260 and 280 nm (Nanadrop ND-2000, Wilmington, Delaware, USA). After extraction RNA was stored at -800C. To ensure complete removal of any trace amounts of genomic DNA, DNA digestion was performed with RNase-free DNase set (1 U/µg RNA, Promega, Madison, USA) at 370C for 30 min. Subsequently, single-stranded complementary DNA (cDNA) synthesis was performed using Omniscript First-Strand cDNA Synthesis Kit according to the guidelines supplied by the manufacturer (Invitrogen, Carlsbad, CA, USA). Ten µl of extracted RNA was incubated with 2 µl of random primer (100 µM), 2 µl of 5 mM deoxynucleotide triphosphates (dNTP) mix, 2 µl of 10x Buffer RT, 0.25 µl of RNase inhibitor (40 units/µl), 1 µl of Omniscript RT and 2.75 µl of RNase free water at 370C for 60 min. Selected negative control samples were prepared by including all reagents as above, minus the reverse transcriptase. A master mix of RT reagent was prepared once to minimize potential variation.

For quantification of mRNA expression of EP2, EP4, COX-2, and PGES, the quantitative real-time PCR (qPCR) standard curve method was employed as previously described (Swangchan-Uthai et al., 2011). To standardize the quantification method, an endogenous RN18S1 was chosen as reference gene. The primer pairs of interested genes were inferred from published canine sequences as shown in Table 2. T h e p r i m e r s w e r e t h e n t e s t e d b y

Table 1 Criteria used to determine the stage of estrous cycle, ovarian structure, appearance of vaginal cytology, serum progesterone level, and number of healthy cyclic bitches.

Anestrus Estrus Diestrus Dominant ovarian structure Absence of ovarian activity Follicles Corpora lutea Majority of cells in the vaginal cytology Basal cells Cornified cells Non-cornified cells Serum progesterone (ng/ml) < 1 2-15 > 15 (n) 10 7 11 n indicates the number of animals within each group

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conventional PCR amplification using Platinum PCR supermix (22 U/ml complexed recombinant Taq DNA polymerase with Platinum® Taq Antibody, 22 mM Tris-HCl (pH 8.4), 55 mM KCl, 1.65 mM MgCl2, 220 μM dGTP, 220 μM dATP, 220 μM dTTP, 220 μM dCTP, and stabilizers) containing Tag polymerase (Invitrogen; Invitrogen Ltd. UK), 50 ng cDNA and 20 µM primers. The hot-start PCR reaction was run for 2 min at 950C (activation of Taq polymerase), followed by 38 cycles, each one comprising a denaturing step at 950C for 30 sec, annealing at 62.50C (for EP2 and EP4) or 64.10C (for COX-2) or 62.50C (for PGES) for 30 sec, extension at 720C for 20 sec and final elongation at 720C for 5 min. The amplification products were separated by electrophoresis through a 2% (w/v) agarose gel (Bio-Rad, CA, USA). The desired PCR products of all interested genes (EP2, EP4, COX-2, PGES and RN18S1) were visualized as a single band. The identity of the PCR products was confirmed by DNA sequencing (GATC Biothech, London, UK).

In order to optimize real-time qPCR assay, serial dilutions of external standards and sample to be used in melting curve and annealing temperature analysis were prepared. The purified PCR product of each gene was produced from a conventional PCR amplification and then purified by QIAquick PCR Purification kits in accordance with guidelines supplied by Qiagen (QIAGEN, Valencia, CA, USA).The concentration of purified PCR product was determined by NanoDrop ND-1000 spectrophotometer.In the following stage, the optimal annealing temperature and melting curve analysis were assessed using a temperature gradient feature ranging from 50-650C.

The transcripts of each gene were determined by an optimized qPCR procedure with a single-plex SYBR Green I assay (CFX 96 Real-Time PCR Detection System, Bio-Rad Lab, Inc, CA, USA). The amplification mixes contained 10 μl of KAPA SYBR FAST qPCR Kits (GRI, Braintree, Essex, UK), 0.5 μl of 20 μM forward and reverse primers mix, 4.5 μl of nuclease free water and 5 μl of unknown sample (50 ng of cDNA).The qPCR reactions for both standards and samples were performed in duplicate in PCR plate. Thermal cycling conditions applied to each assay consisted of an initial Taq activation step at 950C for 5 min followed by 38 cycles of denaturation at 950C for 15 sec, annealing temperature as shown in

Table 2, extension at 720C for 20 sec followed by amplicon-specific fluorescence acquisition reading (range 74-840C).Absolute concentrations of the PCR product were calculated by comparing the Cq values of the unknown samples to a standard curve using CFX ManagerTM Software Version 1.0.1035.131 (Bio-Rad Lab, Inc) and expressed as fg/µg reverse-transcribed RNA.

Western Blot analysis: Frozen cervical tissues were ground to powder with sterile mortar and pestle and treated with lysis buffer containing Tris-Glycine-SDS buffer, sodium, orthovanadate (200mM), and protease inhibitor cocktail set 1 (Calbiochem, Germany). The sample was vortexed and centrifuged at 13,000 rpm at 40C for 10 min, supernatant were collected and the sample was boiled at 1000C for 5 min. Two µl of cervical lysate solution were used for determination of the protein concentration by spectrophotometry using Protein A280 technique (NanoDrop® ND1000 spectrophotometer, NanoDrop Technologies Inc, Wilmington, Delaware, USA). The rest of lysate was mixed with 5% β-mercaptoethanol (Sigma-Aldrich GmbH, Steinheim, Germany) and 0.02% bromophenol blue (Sigma-Aldrich GmbH, Steinheim, Germany) and stored at -200C. Protein extracts (30 μg) were resolved on a 12% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel) with a 4% stack. Proteins were transferred to a polyvinylidine difluoride membrane (BioTraceTM PVDF transfer membrane, MI, USA) and blocked with 5% BSA in TBS-T at room temperature for 1 hour. Immunoblots were performed by using goat polyclonal antihuman EP2 (sc-22196), goat polyclonal antihuman EP4 (sc-16022) (Santa Cruz, California, USA), rabbit polyclonal antimurine COX-2 (catalogue 16016 Cayman Chemecal, Ann Arbor, Mich), and mouse monoclonal beta actin (AC-15, ab6276, abcam®, Abcam plc, San Francisco, USA) at dilution of 1 : 250, 1 : 250, 1 : 250 and 1 : 5,000, respectively, in clocker solution and incubated at 40C overnight. Horseradish peroxidase (HRP-)-conjugated horse anti-goat IgG (PI-9500, Vector Laboratories, California, USA) and polyclonal rabbit anti-mouse immunoglobulin (DAKO, Glostrup, Denmark) were used as the secondary antibodies at dilution of 1 : 500 and 1 : 1.000 in TBS-T, respectively, and incubated at room temperature for 5 hours with intensive washing in TBS-T between each step.

Table 2 Primer sequences for EP2, EP4, Cox-2 and PGES, expected product’s length, and specific annealing temperature used for

real-time PCR

Primer Accession number Primer sequence Annealing (˚C)

Product-length (bp)

EP2 NM_001003170.1 F: 5’-TTC TCC TGG CTA TTA TGA CC-3’ 62.5 273 R: 5’-ATC TAC TGG CGT TTG ACT G-3’ EP4 NM_001003054.1 5’F: -GGT ACG GGT GTT CAT CAA C-3’ 62.5 323 5’AGA R: AGA GGA GGG TCT GAG ATG TG-3’ COX -2 NM_001003354.1 5’F: -ACA GGA GAG AAG GAA ATG GC-3’ 64.1 250 5’R: -GGA TTG AGG CAG TGT TGA TG-3’ PGES NM_001122854.1 5’F: -ACC ATC TAC CCC TTC CTG T-3’ 62.5 214 5’R: -CTG CTT CCC AGA CGA TCT-3’

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Bound antibodies were developed with diaminobenzidine (DAB) (ImmPACTTM DAB Peroxidase Substrate, Vector Laboratories, California, USA) for 5 min until the brown color of protein band developed. Membrane was scanned with HP scanner (HP Scanjet 2400c, HP, USA) to get the image. The optical density of EP2, EP4, and COX-2 bands were measured by using Quantity One Software Program version 4.4.0 (Bio-Rad Laboratories). The specific density of each sample was calculated by subtraction of the background density and divided by the specific density of quality control band.

Statistical analyses: Statistical test of analysis of

variance (ANOVA) using SPSS for Microsoft Windows software (version 19; SPSS Inc., Chicago, IL, USA)was performed to analyze the differences of mRNA and protein expression in bitches during different stages of the estrous cycle (anestrus, estrus and diestrus). Data were tested for homogeneity of variance using Levene’s test. Post hoc comparisons were performed using Bonferroni correction to test for significances. All data are expressed as mean±SEM. For pyometra groups, t-test was used to analyze differences of mRNA expression between open-cervix and closed-cervix pyometra groups. Values were considered to be statistically significant at p ≤ 0.05.

Figure 1 Mean (±SEM) mRNA expressions of EP2, EP4, COX-2 and PGES in cervices of healthy bitches during different stages of the

estrous cycle

Figure 2 Mean (±SEM) mRNA expression of EP2, EP4, COX-2 and PGES in cervices of bitches with open- or closed-cervix pyometra. Bars with different superscripts show significant (p < 0.05) differences.

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Results Cervical expression of EP2, EP4, COX-2, and PGES mRNA in cyclic and pyometra bitches: The mRNA for EP2, EP4, COX-2 and PGES was expressed in cervical tissue of the normal cyclic bitches during all stages of the estrous cycle. However, no differences were observed in the expression of any gene studied among the stages of the estrous cycle when analyzed either as relative number (after normalized with RN18S1) or absolute concentrations (fg cDNA/μg RT RNA). The data are presented as absolute numbers in Fig 1. In the bitches with pyometra, PGES mRNA expression in the cervical tissue of open-cervix pyometra was higher than closed-cervix pyometra (p < 0.05) whereas the expression of EP2, EP4, and COX-2 did not differ between the two groups (Fig 2).

Cervical expression of EP2, EP4, and PGES proteins in cyclic and pyometra bitches: The results from western blot confirmed the protein expressions of EP2, EP4, and COX-2 in canine cervical tissue during estrous cycle and in bitches with pyometra. EP2, EP4, and COX-2 proteins were expressed in anestrus, estrus and diestrus although the differences among them were not observed (p > 0.05) (Fig 3). Similarly, there were no differences of EP2, EP4, and COX-2 protein expressions between open-cervix and closed-cervix pyometra (p > 0.05) (Fig 4).

Figure 3 Relative density (Mean±SEM) of EP2 receptor

protein expression of canine cervix at estrous cycle

Figure 4 Relative density (Mean±SEM) of EP4 receptor

protein expression of canine cervix in bitches with open-cervix pyometra and closed-cervix pyometra

Discussion

In the present study, the cervical mRNA and protein expressions of EP2, EP4, COX-2, and PGES were studied in bitches during the various stages of the estrous cycle and those affected with pyometra with the aim to help the understanding of the prostaglandin associated cervical relaxation.

Estradiol and progesterone are two major steroids functioning during the estrous cycle to regulate cervical patency possibly through prostaglandins production (Kershaw et al., 2005; Kershaw et al., 2007; Kershaw-Young et al., 2009). In the present study mRNA expression of COX-2 in the cervix was not influenced by dominated hormones during the estrous cycle. Our results are concordant with the study of COX-2 mRNA expression in the bitch uterus, demonstrating no differences between anestrus and diestrus (Silva et al., 2009), whereas they differ from the studies in sheep, in which estradiol was shown to significantly elevate the COX-2 mRNA expression in the cervix (Zhang et al., 2007; Kershaw-Young et al., 2010). The protein expression of COX-2 in this study did not differ among the stages of estrous cycle. The study in sheep cervix demonstrated that COX-2 protein expression did not increase in cervix treated with estradiol compared to control group (Zhang et al., 2007), which is concordant with our result. On the contrary, the high level of COX-2 protein expression in pre-LH surge was found compared to luteal phase (Falchi and Scaramuzzi, 2013). One possible explanation is that the modulation of COX-2 expression may involve multifactorial regulation. Although the differences in EP2 mRNA expression were not statistically significant between the estrous and diestrous periods, the considerably lower EP2 mRNA expression during estrus might resulted from a down regulation by the estradiol during the estrous stage. Similar findings were demonstrated in the baboon cervix in which EP2 mRNA was lower during labor when the cervix was relaxed than the animals that were not in labor (Smith et al., 2001). Such differences were also observed in the rat uterus (Brodt-Eppley and Myatt, 1998). Interestingly, the EP2 mRNA expression was regulated by changes in estrogen and progesterone in the mouse uterus in the fashion that the estrogen down-regulated the level of EP2 mRNA whereas progesterone up-regulated the expression of this receptor (Lim and Dey, 1997). This was also supported by the report in the rat, showing the increase of EP2 mRNA expression in the myometrium after treatment with progesterone (Dong and Yallampalli, 2000). However, the protein expression of EP2 did not differ between estrus and diestrus, suggesting that the regulation of EP2 expression might be modulated at the translation stage. Our results of a lack of differences in EP4 mRNA expression throughout the estrous cycle are similar to the previous study in the sheep (Kershaw-Young et al., 2009). Although the circulating estradiol concentrations did not seem to influence mRNA and

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protein expression of EP4 in the bitch cervix in the present study, the immunohistochemical study revealed a higher EP4 expression in the luminal epithelium of the bitch cervix during estrus than anestrus in our previous study (Linharattanaruksa et al., 2013). Moreover, the protein expression of EP4 in rat cervical tissue increased with advancing gestation and was found highest on the day of parturition when cervix relaxed (Chien and Macgregor, 2003). These lead us to propose that the differential expression of EP4 during the different stages of the estrous cycle are localized in certain regions and with the whole cervix together tissue, such differences in EP4 expression become masked.

It has been known that PGE2 plays an important role in various inflammatory responses (Rocca and FitzGerald, 2002) including cervical relaxation during labor by modification of extracellular matrix, i.e. a decrease in collagen content and an increase in collagen remodeling in the cervix (Rodriguez et al., 2003). In mammalian cervix, proinflammatory cytokines responsible for the changes in extracellular matrix are secreted by various cells including cervical fibroblasts, endothelial cells, and leukocytes such as neutrophils. Although the protein expressions of EP2 and EP4 protein did not differ between open- and closed-cervix pyometra in this study, our previous study revealed that immunolocalization of EP4 expression was higher in the open-cervix than closed-cervix pyometra (Linharattanaruksa et al., 2013). These finding support the notion that EP4 protein expression in specific tissue layer (in this case luminal epithelium) may be involved in cervical relaxation in the bitches.

In the present study, expression of COX-2 mRNA and protein did not differ between open- and closed-cervix pyometra while the PGES mRNA did, suggesting that a post-COX enzyme mechanism is likely to be involved in the determination of final prostaglandin product (Hafez and Smith, 1982).

In our previous study, the number of neutrophils in the cervix was higher in bitches with open- than closed-cervix pyometra (Kunkitti et al., 2011). In this study, higher COX-2 and PGES mRNA levels were observed in the cervix of bitches with pyometra compared to the normal cyclic bitches, indicating an up-regulated gene transcription. This was also reported in the endometrium of the bitches with pyometra (Silva et al., 2009). COX-2 and PGES are said to be stimulated by proinflammatory cytokines and endotoxins such as lipopolysaccharides (LPS) (Murakami et al., 2000; Helliwell et al., 2004). Moreover, the main proinflammatory cytokine in cervical relaxation is interleukin-8 (IL-8) (Uchiyama et al., 1992; el Maradny et al., 1994; El Maradny et al., 1996), which has chemotactic effect on neutrophils which are the major source of matrix metalloproteinases (MMPs) (Stygar et al., 2002). Furthermore, IL-8 stimulates the activation and degranulation of neutrophils, thus provoking the release of MMPs (van Engelen et al., 2009). MMPs are a family of enzymes that are classified according to substrates such as collagenase, gelatinase, and

stromelysin (Sorsa et al., 2004) that can degrade components of the extracellular matrix. The expression of IL-8 in the cervix is stimulated by PGE2 (Ito et al., 1994) and the increase in IL-8 concentrations stimulates degranulation of MMPs from neutrophils (Kelly, 2002; Tamada et al., 2012). This, in turn, results in the changes in collagen turn over in extracellular matrix and leads to cervical relaxation. From our results, we propose that the increase in PGES mRNA expression is stimulated by proinflammatory cytokines, resulting in the enhanced production of PGE2 that subsequently stimulates neutrophil infiltration through the IL-8 and releases MMPs to degrade extracellular matrix causing cervical relaxation.

In conclusion, the cervix of normal bitches during various stages of the estrous cycle expressed the same levels of EP2 and EP4 mRNA. Moreover, the biosynthesis of PGE2 by COX-2 and PGES was not influenced by the stages of the estrous cycle. In addition, PGES seems to control the production of PGE2 to regulate cervical relaxation in the bitches with open-cervix pyometra.

Acknowledgements

The study was financially supported by the

Royal Thai Golden Jubilee (RGJ) Ph.D. program, Thailand Research Fund and the Research Fund Project, Faculty of Veterinary Science, Chulalongkorn University. We thanks the Research Unit of Obstetrics and Reproduction in animals for providing parts of laboratory equipment. We would like to thank Dr. Thanida Sanammuang for her laboratory technical assistance.

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Development and Application of Reverse Transcription

Loop-mediated Isothermal Amplification (RT-LAMP) for

Feline Coronavirus Detection

Somporn Techangamsuwan* Araya Radtanakatikanon Roongroje Thanawongnuwech

Abstract

Mutated feline coronavirus (FCoV) causes a highly fatal disease in cats, named feline infectious peritonitis

(FIP). Common FIP clinical signs represent an accumulation of fluid in the thorax and/or abdomen. Screening tests currently rely on fluid analysis with cytologic examination to differentiate cellular components. Accordingly, accurate ante-mortem diagnosis is prerequisite for effective treatment strategies. This study focused on developing a rapid diagnostic tool for FCoV by using reverse transcription loop-mediated isothermal amplification (RT-LAMP). Clinical samples composed of bodily fluids, plasma and feces were obtained from FIP-suspected cats (n = 63), apparent healthy cats living with (n = 12) and without (n = 10) clinically ill cats. Following RNA extraction and quantification, the modified RT-LAMP targeting the highly conserved 3’-untranslated region (3’UTR) was thoroughly performed and results were compared to reverse transcription polymerase chain reaction (RT-PCR). The analysis focused on sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). The results showed that all submitted feline fluid samples were 38% (24/63) positive by RT-PCR and 44% (28/63) positive by RT-LAMP. Cats living in the same household with FIP-suspected cats strikingly displayed 98% (11/12) positivity when detected from plasma and/or feces. For cats with no history of previous FIP exposure, the results demonstrated that 30% (3/10) tested positive by RT-PCR compared to 50% (5/10) by RT-LAMP. Additionally, RT-LAMP represented a diagnostic sensitivity and NPV at 100%, while specificity and PPV were over 80%. In conclusion, RT-LAMP assay is applicable to confirm FIP infection in clinical case and to monitor FCoV carrier in healthy cats.

Keywords: 3’UTR, feline coronavirus, feline infectious peritonitis, RT-LAMP, RT-PCR Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand *Corresponding author: E-mail: somporn62@hotmail.com

Original Article

230 Techangamsuwan S. et al. / Thai J Vet Med. 2013. 43(2): 229-233.

บทคดยอ

การพฒนาและประยกตใชเทคนค Reverse transcription loop-mediated isothermal amplification (RT-LAMP) ในการตรวจหาเชอโคโรนาไวรสในแมว สมพร เตชะงามสวรรณ * อารยา รตนกถกานนท รงโรจน ธนาวงษนเวช

โรคเยอบชองทองอกเสบในแมว (FIP) เกดจากเชอโคโรนาไวรสในแมว (FCoV) ทมการกลายพนธ สตวทปวยจะมการสะสมของของเหลวในชองอก และ/หรอ ชองทอง การตรวจคดกรองเบองตนมกอาศยการตรวจตวอยางของเหลวเพอจาแนกชนดของเซลล นอกจากน การตรวจวนจฉยทแนนอนในขณะทสตวยงมชวตอยนบวามความสาคญตอการวางแผนการรกษา การศกษาในครงนมวตถประสงคเพอพฒนาวธการวนจฉยเชอ FCoV ในประเทศไทย ดวยวธการ Reverse transcription loop-mediated isothermal amplification (RT-LAMP) ตวอยางทางคลนกประกอบดวยของเหลวจากชองวางภายในรางกาย พลาสมา และอจจาระของแมวทสงสยวาปวยเปน FIP จานวน 63 ตว แมวปกตทอยรวมกบแมวปวยจานวน 12 ตว และแมวทอยตวเดยวจานวน 10 ตว หลงจากการสกดและวดปรมาณสารพนธกรรมจากตวอยางดงกลาว นามาทดสอบดวยเทคนค RT-LAMP โดยเปรยบเทยบกบเทคนค RT-PCR ตอจนในตาแหนงอนรกษ 3’-untranslated region (3’UTR) ผลการวเคราะหพบแมวทสงสยวาปวยเปน FIP ใหผลบวกรอยละ 38 และ 44 เมอตรวจดวยเทคนค RT-PCR และ RT-LAMP ตามลาดบ แมวปกตทอยรวมกบแมวปวยใหผลบวกรอยละ 98 ในทงสองวธ และแมวทอยตวเดยวใหผลบวกรอยละ 30 และ 50 ตามลาดบ เมอนามาวเคราะหคาความไวของการทดสอบ (sensitivity) และคาพยากรณลบ (NPV) ของเทคนค RT-LAMP พบวาใหผลรอยละ 100 ในขณะทคาความจาเพาะของการทดสอบ (specificity) และคาพยากรณบวก (PPV) ใหผลมากกวารอยละ 80 ดงนนเทคนค RT-LAMP จงสามารถนามาใชตรวจคดกรองแมวปวยและแมวทเปนพาหะได

คาสาคญ: 3’UTR ไวรสโคโรนาในแมว เยอบชองทองอกเสบตดตอในแมว RT-LAMP RT-PCR ภาควชาพยาธวทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย กรงเทพฯ 10330 *ผรบผดชอบบทความ E-mail: somporn62@hotmail.com

Introduction

Loop-mediated isothermal amplification (LAMP) is a powerful innovative gene amplification tool developed for early detection and identification of microbial disease. It is a simple, rapid, specific and cost-effective nucleic acid amplification method. Amplification and detection of each specific gene can be completed in a single tube by incubating mixture of samples, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature (about 60-650C). The whole procedure can be completed in less than an hour. Because of its high specificity, the presence of amplified product indicates the presence of the target gene. The products can be visualized by naked eyes, agarose gel electrophoresis or with hydroxynaphthol blue dye addition (Parida et al., 2008; Cardoso et al., 2010).

Coronaviruses are enveloped, positive-sense RNA viruses belonging to the subfamily Coronavirinae in the family Coronaviridae. Coronaviruses cause varied ranges of diseases in broad host spectrum not only found in humans but also in wild and domesticated animals including severe acute respiratory syndrome (SARS) in humans,

transmissible gastroenteritis (TGE) in pigs, infectious bronchitis virus (IBV) in chickens, canine coronavirus (CCoV) in dogs and feline coronavirus (FCoV) in cats. Although reverse transcription-LAMP assay is developed for the detection of several coronaviruses during the last decade (Pyrc et al., 2011; Ren and Li, 2011; Qiao et al., 2012), there has not been an attempt to use this technique on companion animal‘s coronaviruses.

Feline coronavirus (FcoV) consists of two biological types resulting in different forms of clinical manifestations. Infected cats with subclinical or mild diarrhea are usually caused by feline enteric coronavirus (FECV), while those with effusions (wet form) or caseous abscesses (dry form) in their bodily cavities are particularly mediated by feline infectious peritonitis virus (FIPV). Recently, incidence of FCoV isolates among cat populations in Thailand during 2010-2011 has been reported (Techangamsuwan et al., 2012). Cats with FIP clinically suspicions showed 46% positivity by RT-PCR targeting the highly conserved 3’-untranslated region (3’UTR). However, even the superior sensitivity and specificity of the PCR; it remains quite expensive for routine diagnosis. The objective of this study was to establish a simple, rapid diagnostic tool for feline infectious peritonitis disease

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detection in Thailand using the reverse transcription loop-mediated isothermal amplification (RT-LAMP) compared to the RT-PCR.

Materials and Methods Clinical specimens: All samples were collected at The Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University. Three categories of specimens comprised 70 thoracic and/or abdominal fluids collected from suspected FIP-infected cats (n = 63), 17 plasma and 21 feces collected from healthy cats living with (n = 12) and without (n = 10) FIP-suspected cats, respectively. Fluid, EDTA-anticoagulated whole blood as well as sterile phosphate buffer saline (PBS)-diluting feces were centrifuged at 3,000 rpm for 10 min. Supernatants and plasma were harvested and kept at -800C until used.

Nucleic acid extraction and RT-PCR assay: Viral RNA extraction using NucleoSpin® RNA virus (Macherey-Nagel, DÜren, Germany) following the manufacturer’s instruction was done on 150 µl of each specimen. The concentration of RNA was quantified using NanoDrop spectrophotometer (Thermo Scientific, USA). Initially, the reverse transcription (RT) was performed using 500 ng RNA, random primers (Promega, Mannheim, Germany) and Omniscript RT-PCR kit (Qiagen, Hilden, Germany). The reaction was incubated at 600C for 1 hour using the thermoregulator ATC 401 (NYX Technik Inc., USA). Specific oligonucleotide primers for feline cDNA qualification (feline GAPDH) and FCoV detection (FCoV-3’UTR) were chosen from previous studies (Table 1) (Herrewegh et al., 1995; Penning et al., 2007).

Subsequently, the PCR reaction was carried out based on 2 µl cDNA, 0.4 µM (each) primers, 1.25 U Taq polymerase (Invitrogen), 1 mM MgSO4, and 0.2 mM (each) dNTPs with GoTaq Flexi buffer (Promega, Mannheim, Germany). The annealing temperatures were 500C (feline GAPDH) and 560C (FCoV-3‘UTR). The PCR products were run on 1.5% agarose gels compared with the positive control (fresh tissue from a FIP necrosied cat diagnosed by histopathology) and the negative control (reaction omit the genomic RNA).

Positive amplicons were purified with Nucleospin® extract II kit (Macherey-Nagel, DÜren, Germany) and submitted for sequence analysis (1st

BASE Pte Ltd, Singapore). Data were analyzed by Bioedit Sequence Alignment Editor version 7.0.5.3 (Hall, 1999) and compared with previously reported sequences available in GenBank.

LAMP primer designing and establishment of LAMP amplification: Based on results of sequencing analysis above, the conserved region of 3’UTR gene of FCoV (approximately 200 bp) was selected for the LAMP primer set designing by PrimerExplorer V4 software freely available at http://primerexplorer. jp/elamp4.0.0/index.html (Fig 1, Table 1).

The LAMP reaction was performed in 25 µl mixture containing 2 µl cDNA, 0.2 µM (each) outer primers (FCoV-F3 and FCoV-B3), 1.6 µM (each) inner primers (FCoV-FIP and FCoV-BIP), 8 U Bst DNA polymerase (New England Biolabs), 8 mM MgSO4, and 0.8 M Betaine (Sigma Aldrich, 1.4 mM (each) dNTPs with ThermolPol buffer (New England Biolabs). Amplification was done at 600C for 2 hours and terminated at 800C for 2 min. The products were inspected on 2% agarose gels with a pattern of multiple bands of different molecular weights.

Data analysis: All data from the RT-LAMP assays were comparatively analyzed with the data from the RT-PCR assays considered as a gold standard. The evaluation included sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).

Results

Identification the apparently healthy FCoV carriers by detection of 3’UTR gene: The detection of FCoV RNA in various kinds of specimens including effusion from FIP-suspected cats, plasma and/or feces from apparently healthy cats living with those sick and living alone were thoroughly performed by RT-PCR targeting the 3’UTR gene. The specific amplicon was detected at 223 bp on agarose gel electrophoresis (Fig 2, Table 2). In cats with bodily accumulating fluid and highly suspected of FIP disease, 38% (24/63) were tested positive, while the healthy cats that lived in the same household with FIP-suspected cats strikingly displayed 98% (11/12) positivity when detected from plasma and/or feces. For the cats that lived alone, implying no previous FIP exposure, 30% (3/10) were tested positive.

Table 1 Oligonucleotide primers used.

Primer name Type Sequence (5’ 3’) Length (bp) Feline GAPDH Forward primer AGTATGATTCCACCCACGGCA 21 Reverse primer GATCTCGCTCCTGGAAGATGGT 22 FCoV-3’UTR Forward primer GGCAACCCGATGTTTAAAACTGG 23 Reverse primer CACTAGATCCAGACGTTAGCTC 22 FCoV-F3 Forward outer AACCCGATGTTTAAAACTGG 20 FCoV-B3 Reverse outer CCATTGTTGGCTCGTCAT 18 FCoV-FIP Forward inner ACTACACGTGCTTACCATTCTGTACATTCCGGGGAATTACTGG 43 FCoV-BIP Reverse inner CAAGCAACCCTACTGCATATTAGGAAGCGGATCTTTAAACTTCTCT 46

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Figure 1 Different types of primers used in RT-LAMP reaction were designed based on the 210-bp conserved region of FCoV-3‘UTR gene. Forward outer primer (FCoV-F3) located at nucleotide (nt.) 4th-23rd. Forward inner primer (FCoV-FIP) spanned 2 regions consisted of the F2 region (F2; nt. 25th-43rd) and the complement to the F1 region (F1C; nt. 88th-65th). Reverse/Backward inner primer (FCoV-BIP) spanned 2 regions consisted of the complement to the B1 region (B1C; nt. 97th-121st) and the B2 region (B2; nt. 181st-161st). Reverse outer primer (B3) located at nt. 199th-182nd

LAMP primer design: After verifying the sequencing results, the 210-bp selective conserved fragment of 3’UTR gene of FCoV was used for LAMP primer design via web-based interactive PrimerExplorer V4 software (Fig 1). The primers including outer primers (F3 and B3) and inner primers (FIP and BIP) are demonstrated in Table 1. Detection of FCoV by a modified RT-LAMP: The modified RT-LAMP was performed in the same samples and directly compared with the conventional RT-PCR. After visualization by 2% agarose gel

electrophoresis stained with ethidium bromide, the presence of a ladder-like pattern indicating different molecular weights products was considered a positive result (Fig 3, Table 2). By parallel comparison with the RT-PCR, the RT-LAMP increased the positivity up to 44% (28/63) and 50% (5/10) in suspected FIP cats and in apparently healthy cats, respectively. In addition, the RT-LAMP demonstrated its diagnostic sensitivity and negative predictive value (NPV) at 100%, while specificity and positive predictive value (PPV) were over 80% (*, Table 2).

Figure 2 Analysis of FCoV by RT-PCR with 1.5% agarose gel

electrophosis. M: 100 bp DNA ladder marker, N: negative, E: effusion, F: feces, P: plasma

Figure 3 Analysis of FCoV by RT-LAMP with 2% agarose gel

electrophosis. M: 100 bp DNA ladder marker, N: negative, E: effusion, F: feces, P: plasma

Table 2 Comparison between RT-PCR and RT-LAMP assays

RT-PCR

Bodily fluids (63 FIP-suspected cats)

Plasma/Feces (12 cats living with FIP)

Plasma/Feces (10 cats living singly)

Positive Negative Positive Negative Positive Negative Total Positive 24 4 11 0 3 2 44 RT-

LAMP Negative 0 35 0 1 0 5 41 Total 24 39 11 1 3 7 85 Sensitivity1 100% 100% 100% 100%* Specificity2 89.7% 100% 71.4% 87.2%* PPV3 85.7% 100% 60% 81.9%* NPV4 100% 100% 100% 100%* 1 Sensitivity related to the RT-LAMP's ability to identify positive results when compared to the gold standard RT-PCR 2 Specificity related to the RT-LAMP's ability to identify negative results when compared to the gold standard RT-PCR 3 Positive predictive value (PPV) or precision rate is the proportion of subjects with positive test results correctly diagnosed. 4 Negative predictive value (NPV) is the proportion of subjects with negative test results correctly diagnosed. * Mean value of all groups

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Discussion

One of the most life-threatening infectious diseases in cats is feline infectious peritonitis (FIP) disease caused by the mutated feline coronavirus (FCoV). The major obstacle to gain successful treatment is a lack of rapid and accurate ante-mortem diagnosis. Even though the distinguished symptom of FIP-suspected effusive form is an accumulation of pleural and/or abdominal fluids, several etiologies are needed to be differentially diagnosed including suppurative pleuritis/peritonitis, traumatic hemothorax/chylothorax, nephropathy and lymphoma-induced ascites (Sharif et al., 2010). Several laboratory aids have been implemented to overcome this burden, particularly molecular technique such as RT-PCR.

The RT-PCR has been developed for the detection of RNA genomic FCoV and for the differentiation of FCoV genotypes in clinical samples by emphasizing on the 3’UTR and S genes, respectively (Herrewegh et al., 1995; Lin et al., 2009; Techangamsuwan et al., 2012). However, the application of RT-PCR method is limited only to the best-equipped laboratory. To establish a simple rapid diagnostic tool for FIP disease, the reverse transcription loop-mediated isothermal amplification (RT-LAMP) is developed. We showed that RT-LAMP assay for FCoV provided better positivity rate (44-50%) with high sensitivity and specificity. This is in accordance with previous studies particularly in swine transmissible gastroenteritis coronavirus and turkey coronavirus showing the comparable detection limit, sensitivity and specificity of RT-LAMP assay with the gold standard PCR (Cardoso et al., 2010; Chen et al., 2010).

Although a better positivity rate was achieved, the method was not as simple as expected. From our experience, RT-LAMP assays still yielded a high rate of false positive incidence in the negative control group. In general, the LAMP assay is found to be 10-100 folds more sensitive than the routine PCR with a detection limit of 0.01-0.001 plaque forming unit (pfu) of virus (Parida et al., 2008). Accompanied by the set of 4 primers spanning 6 distinct sequences of target gene, only a minute amount of target sequence could be amplified. The detection of amplified products still required agarose gel electrophoresis due to the fact that the detection of turbidity by naked eyes was scarcely observed. Taken together, we suggested that the modified RT-LAMP assay for FCoV diagnosis could be used for the screening cases but could not be used for RT-PCR replacement.

Acknowledgements

This study was financially supported by the

Ratchadapiseksompotch Fund R_014_2553 from Chulalongkorn University and Grants for the Development of New Faculty Staff (GDNS 55-011-31-003). The authors have no conflict of interest to report.

References Cardoso TC, Ferrari HF, Bregano LC, Silva-Frade C, Rosa

AC and Andrade AL 2010. Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye. Mol Cell Probes. 24: 415-417.

Chen Q, Li J, Fang XE and Xiong W 2010. Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification. Virol J. 7: 206.

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Herrewegh AA, de Groot RJ, Cepica A, Egberink HF, Horzinek MC and Rottier PJ 1995. Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR. J Clin Microbiol. 33: 684-689.

Lin CN, Su BL, Wang CH, Hsieh MW, Chueh TJ and Chueh LL 2009. Genetic diversity and correlation with feline infectious peritonitis of feline coronavirus type I and II: a 5-year study in Taiwan. Vet Microbiol. 136: 233-239.

Parida M, Sannarangaiah S, Dash PK, Rao PV and Morita K 2008. Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol. 18: 407-421.

Penning LC, Vrieling HE, Brinkhof B, Riemers FM, Rothuizen J, Rutteman GR and Hazewinkel HA 2007. A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues. Vet Immunol Immunopathol. 120: 212-222.

Pyrc K, Milewska A and Potempa J 2011. Development of loop-mediated isothermal amplification assay for detection of humancoronavirus-NL63. J Virol Methods. 175: 133-136.

Qiao J, Meng Q, Cai X, Chen C, Zhang Z and Tian Z 2012. Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated isothermal amplification assay. J Vet Diagn Invest. 24: 174-177.

Ren X and Li P 2011. Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus. Virus Genes. 42: 229-235.

Sharif S, Arshad SS, Hair-Bejo M, Omar AR, Zeenathul NA and Alazawy A 2010. Diagnostic methods for feline coronavirus: a review. Vet Med Int. 7 pages. doi:10.4061/2010/809480.

Techangamsuwan S, Radtanakatikanon A and Purnaveja S 2012. Molecular detection and genotype differentiation of feline coronavirus isolates from clinical specimens in Thailand. Thai J Vet Med. 42: 413-422.

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Welfare Assessment of Flight-restrained Captive Birds:

Effects of Inhibition of Locomotion

Shawn Jen-Lung Peng1 Fang-Chia Chang1 Judy Sheng-Ting I2 Andrew Chang-Young Fei1*

Abstract

Morphological and physiological adaptations have allowed birds to utilize flying as part of the primary

locomotion. However, birds in captivity are often deprived of this natural skill due to limited living space or wing amputation. This article aims to investigate the essentialness of flight and welfare assessment of flight restraint in captive birds, using animal-based measurement. Ten wild-caught great mynahs (Acridotheres grandis) were chosen as test subject and evaluated in three separate studies. In pathology study, bird wings were partially amputated to assess possible neuroma formation at the site of amputation. The birds involved in this study were evaluated on day 20 and day 40 post surgery. We found that wing amputation did not result in neuroma formation. In ethology study, spatial preference of the captive birds was evaluated by providing cages with different structures. We noticed that the birds indicated higher preference for spaces that were bigger or with higher vertical space with or without food provided. In physiology study, strength of pectoralis muscle was evaluated with electromyography in both wild birds and birds that were flight restrained for 40 days. The results indicated that strength of pectoralis muscle diminished significantly in birds with flight restraint. This research suggested that captive birds were still highly inclined to use flight as means of locomotion, and provided useful information as to how enclosures of captive birds should be carefully designed for better animal welfare.

Keywords: animal welfare, captive birds, electromyography, flight restraint, great mynah, wing amputation 1 School of Veterinary Medicine, National Taiwan University, No.1, Sec.4, Roosevelt Road, Taipei, Taiwan 2 Institute of Veterinary, Animal, Biomedical Science, Massey University, Private Bag 11222, Palmerston North, New Zealand. *Corresponding author: E-mail: fei@ntu.edu.tw

Original Article

236 Peng S J-L. et al. / Thai J Vet Med. 2013. 43(2): 235-241.

บทคดยอ

การศกษาสวสดภาพสตวในนกทถกกกขง: ผลทเกดจากการจากดการบน Shawn Jen-Lung Peng 1 Fang-Chia Chang 1 Judy Sheng-Ting I 2 Andrew Chang-Young Fei 1*

ลกษณะทางกายภาพและการปรบตวทางสรรวทยา ทาใหนกสามารถใชการบนเปนสวนหนงของการเคลอนไหวหลก แตอยางไรกตามนกในกรงมกจะขาดโอกาสนเนองจากพนทจากด หรอถกตดปก การศกษานมวตถประสงค เพอศกษาความสาคญของการบนและเพอประเมนสวสดภาพสตวในนกทถกขง โดยใชการวดจากสตว ทาการคดเลอกนกเอยงหงอนปา (Acridotheres grandis) จานวน 10 ตว และทาการประเมนแยกกนในสามการทดลอง ในการศกษาดานพยาธวทยา ทาการตดปกนกบางสวนเพอประเมนโอกาสการเกดเนองอกของเสนประสาททจดททาการตดปก และทาการประเมนผลในวนท 20 และ 40 วนหลงการผาตด พบวาการตดปกไมไดสงผลใหเกดการเกดเนองอกของเสนประสาท ในการศกษาพฤตกรรมตามธรรมชาต ศกษาความชอบของพนทการกระจายของนกทถกขงโดยสรางกรงทมโครงสรางทตางกน สงเกตเหนวานกชอบบนในบรเวณทใหญกวา หรอมพนทในแนวดง โดยอาจจะมหรอไมมอาหารกได ในการศกษาทางสรรวทยาถงความแขงแรงของกลามเนออกดวยการวดคลนไฟฟากลามเนอในนกปาและนกทจากดการบนเปนเวลา 40 วน ผลการทดลองพบวาความแขงแรงของกลามเนออกลดลงอยางมนยสาคญในนกทถกจากดการบน การวจยครงนชใหเหนวานกทถกกกขงยงคงมแนวโนมสงทจะใชการบนเปนการเคลอนไหวหลก และใหขอมลทเปนประโยชนตอการออกแบบกรงสาหรบนกทถกกกขงทควรออกแบบอยางระมดระวงเพอสวสดภาพสตวทดขน

คาสาคญ: สวสดภาพสตว นกทถกกกขง การวดคลนไฟฟากลามเนอ การบงคบไมใหบน นกเอยงหงอน การตดปก 1 School of Veterinary Medicine, National Taiwan University, No.1, Sec.4, Roosevelt Road, Taipei, Taiwan 2 Institute of Veterinary, Animal, Biomedical Science, Massey University, Private Bag 11222, Palmerston North, New Zealand. *ผรบผดชอบบทความ E-mail: fei@ntu.edu.tw

Introduction

Locomotion is a fundamental part of life for nonsessile animals. It is responsible for a range of actions such as finding food, encountering a mate, and escaping from predators. Vertebrates can move in various ways, including swimming, crawling, walking and flying. Morphological and physiological adaptations in birds have allowed them to fly, which makes them unique in comparison with most other vertebrates. The ability and the need for flight will require the bird to reach almost its maximum pulmonary efficiency in order to take off and sustain flight. However, majority of captive birds such as flamingos, geese, ducks or swans are unable to fly. Birds’ ability to fly may be restricted because they may be wing clipped or pinioned in order to be kept on display at a zoo exhibition (WAZA, 2007) or they are kept in small cages as pet birds (Engebretson, 2006). In China, Taiwan and other Asian countries, it is very common to keep song birds or ornamental birds in a small 30 cm cube around cage, allowing pet shop to fit in more cages of birds, and the pet bird household can easily display, carry and relocate the cage. However, this small, with only one perch cage

would never allow birds to fly, the only thing a bird can do within the cage would be jumping between bottom layer and the perch.

Research on effects of inhibition of animals’ natural locomotion has been well-documented on: Farm animals’ stereotypies; higher level of plasma cortisol, clinical signs, other abnormal behaviours and immune indicators (Mason et al., 2001; Broom, 2007); abnormal behaviors in captive wildlife (e.g. regurgitation of most great apes (Struck et al., 2007), stereotypies of wild-ranging carnivores (Clubb et al., 2004), infant mortality, low reproduction rate and glucocorticoid responses (Barry et al., 2005).

Proponents (Bautista et al., 2001) of wing amputation claimed that birds in captivity did not need to fly since they had no enemies to escape from and did not have the need to migrate or obtain food. Also, walking is at low-cost, low-yield, whereas flying is the opposite (Bautista et al., 2003). However, the denial of flight can cause physical (Graham, 1998), reproductive (Farrell, 2000) and behavioural abnormalities in birds, (van Hoek et al., 1998; Garner et al., 2003; Meehan et al., 2003) such as feather-plucking in parrots (Forbes et al., 1999; Hesterman et al., 2001).

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Since there is no single indicator to determine the welfare of an animal, many studies have found that a combination of measures can be used as a tool to assess captive animals’ welfare (Shepherdson et al., 2004, Hill et al., 2009). There are non-invasive physiological measures that can measure stress responses, individual behavior and temperament have great potential to assess and monitor animals. In birds, assessment typically involves measuring heart rate, glucocorticoid concentrations, adrenal gland weight and responses to ACTH challenge (Verkerk et al., 1998). The short term physical stress includes handling and transportation stress, as well as psychological stress such as social disturbance or fear. These stimuli can be assessed readily by measuring the increase in corticosterone levels in blood or by other body fluids (Shepherdson et al., 2004). For long term stress or prolonged chronic pain in birds, behavioral change can provide useful information and studies performed after debeaking of farm hens indicated that hens might suffer from chronic pain because of neuroma formation (Cheng et al., 2004).

The non-invasive methods can provide a bird access to more than one environment, with a variety of resources such as the availability of food, water or space and the opportunity to express its natural behavior. There is an assumption that they will choose in accordance with their best interest (Mason et al., 2001). Such methods are closely related to operant conditioning techniques, where birds have to work to obtain or avoid according to their environment (Dawkins, 2004).

In this study, we aimed to assess captive birds’ welfare and to understand and investigate whether or not flight is essential for birds. We tried to approach these questions through animal-based measurements rather than non-animal centered-measurements.

Animal-based measurements are:

1) Pathology study: We attempted to examine neuroma formation in animals with previously partial amputated wings. The most common type is the formation of traumatic neuroma following nerve injury (often as a result of surgery). Neuromas occur at the end of injured nerve fibers as a form of uneffective, unregulated nerve regeneration; they occur most commonly near a scar, either superficially (skin, subcutaneous fat) or deep (e.g. after a cholecystectomy).

2) Ethology study: We used preference tests to find out pet birds’ strategy for choosing different sizes of space and vertical and horizontal usage.

3) Physiology study: The pectoralis muscle is proportionately large in birds up to 35% of body weight and powers the down stroke of flight (Andrew, 2011). However, pectoralis muscle could degenerate if flight is inhibited or in a restraint

environment. In previous study, the muscle strength focused on muscle mass and required the animal to be euthanised. In human, electromyography (EMG) is applied to study muscle functions to examine the muscle’s electric signals. In this research, we analyzed EMG performance instead of measuring muscle mass through stimulation response of the pectoralis muscle and potentially reduced animal euthanasia.

Materials and Methods Selecting a bird species: We found great mynahs (Acridotheres grandis) to be the best subject for this study as they are the most common wild bird species found in both urban and rural areas in Taiwan. Great mynah has the intelligence to be trained for behavioral study. To reduce the number of animals used in the study, ten wild-caught birds from pet bird shop were used in this study while only five of them were successfully managed in the experiment; the birds were then released after the trial. Two pinioned birds were sent back to the pet shop.

Data collection: Data were collected between January and February, 2009.

Pathology study: Two bird wings were pinioned by certificated veterinarian under general anesthesia using isoflurane. Each individual surgical site was plucked and aseptically prepared, and hemostatic forceps were placed at the level of the carpus before the amputation of outer wing. Hemostasis was achieved with cauterization and appropriate post-operative antibiotics and analgesics were given. Surgical wounds were regularly monitored for signs of post-operative infection and bleeding. The scar tissue of cut stumps from wing surface was examined after pinioning at days 20 and 40. Neuroma formation was expected both outside the tissue and on the biopsy sample.

Ethology study: Two wild-caught birds were caged in a restricted area with one way door made of two Polyvinyl chloride (PVC) pipes. These pipes were designed with an exclusion device, and the animals were not able to re-enter through the same pipe because of the special design of the one way door. The animals were required to enter through the pipe in order to gain access to food in the restricted area. Each time the birds passed through the PVC pipe, they were recorded by video camera as one event. By calculating these events, we were able to measure their time budgets at different locations. Data were recorded for 24 hours a day, 30 days a month for standard comparison.

In this investigation we asked: If birds have preference among same-size spaces with different heights. 2) Does food motivate birds to enter the restricted area? 3) Does space influence and impact birds’ appetite?

We designed this model based on fur-farmed mink preference study by Mason’s 2001 research.

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In preference test 1 (Diagram 1), the cage is divided into two sections, the left being larger in size and the right being smaller in size with food and water available. In preference test 2 (Diagram 2), the cage is divided into three sections, with two areas (left lower and right) being the same size and the right being higher in height but shorter in width. Both left and right sections have food, water and a perch available.

Physiology study: In this study, EMG performance was applied to analyze pectoralis muscle strength from: 1st day caught wild bird and the same bird in a space less than wing span for 40 days. The amplitude and frequency spectrum of the EMG signal is affected by the location of the electrode with respect to the innervation zone (top electrode), the myotendonous junction (bottom electrode) and the lateral edge of the muscle (middle right electrode). The location is in the pectoralis muscle midline between the nearest innervation zone and the myotendonous junction, where the probe was attached. In this location the EMG signal with the greatest amplitude is detected. The bird was measured without sedation but physically restrained. The EMG amplifier for recording was model V75-01 (Colbourn Instruments, Lehigh Valley, Penn) and the EMG signals were analyzed by AxoScope (Axon Instruments, Union City, CA).

Results Pathology study: The traumatic neuroma, which follows nerve injury (often as a result of surgery), usually occurs at the end of injured nerve fibers as a form of uneffective, unregulated nerve regeneration. It occurs most commonly near a scar, either superficially (skin, subcutaneous fat) or deep (e.g. after a cholecystectomy). In the pinion wing dissection study, the tissue from the same bird did not show any formation of neuromas at days 20 and 40 after partial wing amputation. The biopsy samples observed under a microscope 400x (Fig 1, 2) did not show any pathological changes on days 20 and 40 post surgery.

Ethology study: In the preference test 1 (Fig 3), the results revealed that the birds showed preferences for space and size. The birds preferred to stay longer in the bigger space (without food, y+) compared with the smaller space (with food, y-), given that food was

provided in the smaller area. These birds tended to stay mostly in the large area and only entered the small area for quick feeding. Each dot represents a

Figure 1 20 days post pinion. (400X)

Figure 2 40 days post pinion. (400X)

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period of time when a bird chooses to stay in bigger area(y+) or smaller area(y-).

Preference test 2 (Fig 4) showed that with enclosure being the same size and with food and perch available, the birds preferred to stay in the higher vertical space (y-) in comparison with the low vertical space ( y+).

Analyses of birds’ preference between space, food and altitude (Fig 5) indicated that the big space of sequence 1 (x = 327.09 sec.) without food had higher mean value than sequence 2 (x = 129.7 sec.) with smaller space and food. Sequence 4 (x = 507.33 sec.) with altitude of 85 cm had higher mean value than sequence 3 (x = 59.6 sec.) of 54 cm altitude.

Figure 3 Preference test 1- Relationship between space and food against time

Figure 4 Preference test 2 - Vertical altitude effects on bird’s time budget

Figure 5 Analyses of birds’ preference between space, food and altitude

Physiology study: A photo of the cross-sectional view of the breast muscle of one wild-caught great mynah at days 1 and 40 after flight restraint was taken. It showed that the pectoralis muscle significantly

diminished after 40 days of flight restraint (Fig 6). EMG performance was recorded using a 2mV continuing electric signal to stimulate the bird’s breast muscle. To prevent birds from further and unnecessary electric stimulation, we only recorded the first and second minutes after the EMG signal. The same EMG test was applied to four birds to measure pectoralis muscle, but only one bird was physically restrainted to get the data. The muscle strength decreased significantly after 40 days of flight restraint (Fig 7, 8). The first (1.506 : 0.177) and second minute (0.163 : 0.044) were measured with 0.01 volt.

Figure 6 Cross-sectional view of wild great mynah (left) after 40 days of flight restraint

Figure 7A EMG showing wild caught birds at day 1 from 0 to 20 seconds B EMG showing wild caught birds at day 1 from60 to 80 seconds

Figure 8A EMG showing birds at day 40 after flight restraint at 0 to 20 seconds B EMG showing birds at day 40 after flight restraint at 60 to 80 seconds

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Table 1 Activity of wild-caught birds at day 1 from 0 to 60 seconds

ID Time (sec.) Activity (0.01 volt) Ws1 0 1.506 Ws1 60 0.163

Table 2 Activity of flight restraint birds at day 40 from 0 to 60 seconds

ID Time (sec.) Activity (0.01 volt) As1 0 0.177 As1 60 0.044

Discussion

Animals in captivity, under proper care and management, generally live longer and healthier than their conspecifics that have the freedom to live in their natural environment. They receive stable and sufficient food and water supply, veterinary attention when needed, protection from predation, and avoidance of inter or intra-specific conflicts (Mason, 2010). However, not all species adapt well in confined setting due to variation in susceptibility to stress and subsequent physical and psychological behavioral changes. Captive birds are often kept in confined cages or flight restrained through various procedures to restrict their ability to fly in open display areas such as in parks or zoos (Hesterman et al., 2001). Scientific measurements and tools can be used to reveal how animals adapt and cope with their environment (Broom, 2006). As documented, the ability to cope is associated with behavioral, physiological, immunological and emotional components of the animal (Broom, 2011). However, there is limited information on captive bird’s welfare, and whether or not flight is essential in the captivity. The present study was based on Broom’s welfare methodology and theology, where animal welfare measurements, assessments, decisions and concerns were successfully documented and discussed. The present experiment examined the pathology, ethology and physiology methods to assess the welfare of captive birds. This information may assist in the understanding of animal welfare and point to new methods in the future.

The current study revealed that the formation of neuromas did not appear after 40 days of wing amputation. A study conducted by Cheng (2004) found that neuromas were formed between days 9 to 40 in beak trimmed hens. This result was seen in hens that constantly pecked food after beak trimming, causing friction around the debeak section. The existence of neuromas can be assessed as pain in physiological measures (Broom, 2007). However, in the pathology part of this study, the pinioned wings only had direct contact with the air, which potentially results in less friction and less pain and leading to better welfare and outcome.

The preference test revealed that the captive birds had motivation towards basic needs such as food and water. From the result, it is revealed that the amount of space does alter the birds’ preference. The experiment showed that the birds preferred larger cages and higher vertical altitude cages compare to the smaller one. Similarly, Broom (2007) indicated that positive preferences by an animal were referred to as a good indicator of good welfare. Therefore, these preference test results should be incorporated into enclosure design, in order to provide captive birds with adequate space and to reduce stereotypic

behavior. Asher et al. (2008) also suggested that cage shape played an important role in determining the quantity of stereotypic behavior, especially in medium-sized cage. In addition, based on these findings, regulations on minimum enclosure size requirement in zoological and other similar settings may be established to improve animal welfare. Providing adequate space for flight and other natural behaviors may also prevent captive birds from showing fear and distress from over-handling and stress associated with visitors.

Although EMG is applied to test human muscle or nerve abnormality, as far as we know, it is the first time EMG was used as the indicator of muscle strength in captive birds. The results verify that the response of the pectoralis muscle to electric stimulation and the muscle quantity decrease along with the flight restraint of the experimental bird. During the study period, the birds’ weight remained 5% different from its 1st day weight despite losing great amount of the pectoralis muscle at the end, indicating that flight restraint did not affect their basic survival needs as long as the birds were well fed. However, the preference test clearly responded to Mason’s mink study (2001) that both birds and fur-farmed minks were still motivated to perform the same activities as wild animals. Access to the swimming pool (for minks) and being able to fly (for birds) rated as much the same by getting food. Most of all, we believe EMG could be a good tool to examine other captive animals muscle strength, especially wild-caught animals. It may serve as a good indicator to evaluate successful bird release or reintroduction rates of injured birds after long term rehabilitation.

It is highly debatable whether flight is essential in captivity. Hesterman et al. (2001) indicated that although defighting practice deprived birds of the natural behavior of flight, but then allow them to express other behaviors that would be otherwise suppressed in a confined setting. However, it does not equate to better welfare, as flight itself is beneficial to physical wellbeing of the birds as it provides cardiovascular exercise and prevents injuries when falling from high perches. Defighting practice may also cause irritation and elicit undesirable behaviors such as feather plucking and self-mutilation in birds. Engebretson (2006) also suggested that deflighting a bird did not necessarily eliminate its natural instinct to fly, and it would usually attempt to fly once its physical ability regained. Therefore, defighting only alters their physical ability to fly, but not their interest in doing so. Our study also indicates that pectoral muscle strength rapidly deteriorates once the freedom to express natural behaviors is compromised.

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The conclusion from this study is that despite the fact that companion birds or zoological birds have been captivated for generations, birds are highly inclined to fly as part of essential locomotion even though basic needs are provided. How birds are being displayed and how enclosures are designed should be considered carefully, especially the size and the vertical altitude of the cage, to allow birds to fulfill their needs to fly. This result coincides with Broom (2007), who stated that for an animal to cope, it implies that the individual has control of mental and bodily stability. Nonetheless, more bird samples are required for more reliable and significant results and further research is needed to conclude our study.

Acknowledgements

We would like to thank Prof. Pu HF from

Institute of Physiology, N.Y.U. for physiology study assistance.

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242

Thai J Vet Med. 2013. 43(2): 243-249.

Mutations in Topoisomerase Genes and Expression of AcrAB

Multidrug Efflux System in Fluoroquinolone-Resistant

Salmonella enterica from Pork and Patients

Wechsiri Wannaprasat1 Rungtip Chuanchuen1*

Abstract

The aim of this study was to further examine the mechanisms of ciprofloxacin resistance in Salmonella

enterica isolates from pork and patients. Twenty-four isolates including 18 ciprofloxacin- resistant isolates and six susceptible strains were used. Mutations in the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE were examined. Expression of AcrAB-TolC efflux pump was quantitatively determined using qRT-PCR. While nucleotide modifications were found in gyrA, gyrB and parC, all mutations identified were novel. All 18 ciprofloxacin resistant isolates carried mutations in at least one target gene and all six isolates susceptible to ciprofloxacin did not carry mutations in their QRDRs. There were no associations between the number and type of mutations and ciprofloxacin resistance level among the ciprofloxacin Salmonella isolates. All the Salmonella isolates produced AcrB, ranging from 1 to 430 folds. A Salmonella strain susceptible to all antimicrobials tested overexpressed AcrB. The isolates with comparable AcrB demonstrated different susceptibility to ciprofloxacin. Therefore, the role of mutations in topoisomerase genes and the AcrAB-TolC efflux pump in decreased susceptibility to ciprofloxacin varied among the S. enterica clinical isolates.

Keywords: AcrAB-TolC, ciprofloxacin, fluoroquinolones, Salmonella enterica, topoisomerase genes 1 Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330 Thailand *Corresponding author: E-mail: rchuanchuen@yahoo.com

Original Article

244 Wannaprasat W. and Chuanchuen R. / Thai J Vet Med. 2013. 43(2): 243-249.

บทคดยอ

การกลายพนธในยน Topoisomerase และการแสดงออกของระบบมลตดรกซ AcrAB ในเชอแซลโมเนลลา เอนเทอรกาดอยาฟลออโรควโนโลนทแยกไดจากเนอสกรและผปวย เวชสร วรรณประสาท รงทพย ชวนชน *

การศกษาครงนมวตถประสงคเพอศกษากลไกการดอยาซโปรฟลอกซาซนในเชอแซลโมเนลลา เอนเทอรกาทแยกไดจากเนอสกรและผปวย เชอจานวน 24 isolates ประกอบดวยเชอดอยาซโปรฟลอกซาซนจานวน 18 isolates และเชอไวตอยาจานวน 6 isolates โดยตรวจหาการกลายพนธใน the quinolone resistance-determining regions (QRDRs) ของยน gyrA, gyrB, parC และ parE และตรวจการแสดงออกของระบบ AcrAB-TolC ดวย qRT-PCR พบการเปลยนแปลงของนวคลโอไทดในยน gyrA, gyrB และ parC โดยเปนการกลายพนธทยงไมเคยมรายงานมากอน เชอดอยาซโปรฟลอกซาซนทง 18 isolates มการกลายพนธในยนเปาหมายอยางนอยหนงยนและเชอทง 6 isolates ทไวตอการดอยาซโปรฟลอกซาซนไมมการกลายพนธใดๆในสวน QRDRs ไมพบความสมพนธระหวางจานวนและชนดของการกลายพนธกบระดบการดอยาซโปรฟลอกซาซน ของเชอแซลโมเนลลาทดอยาซโปรฟลอกซาซน เชอทกตวมการแสดงออกของระบบ AcrAB-TolC 1-430 เทา เชอทไวตอยาทกชนดททดสอบจานวน 1 isolate สามารถผลต AcrB ได โดยเชอทผลต AcrB ในระดบทใกลเคยงกนมความไวตอซโปรฟลอกซาซนตางกน ดงนนบทบาทของยน topoisomerase และระบบ AcrAB-TolC ตอการดอยาซโปรฟลอกซาซนในเชอแซลโมเนลลามความหลากหลาย

คาสาคญ: ระบบ AcrABC-TolC ฟลออโรควโนโลน แซลโมเนลลา เอนเทอรกา ยน Topoisomerase ภาควชาสตวแพทยสาธารณสข คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 *ผรบผดชอบบทความ E-mail: rchuanchuen@yahoo.com

Introduction

Up to date, Salmonella enterica remains one of the major causes of foodborne illness in humans worldwide (Padungtod et al., 2006). While Salmonella gastroenteritis is typically self-limited and antibiotics are not required in most cases, antibiotics are recommended for patients with increased risk of invasive salmonellosis. Initial antibiotic therapy of salmonellosis includes ampicillin, chloramphenicol and sulphamethoxazole-trimethroprim combination (Ling et al., 2003). However, failure of such conventional antibiotic therapy frequently occurs, particularly in children with severe invasive infections. Fluoroquinones (i.e. ciprofloxacin) have been the drug of choice for treating invasive salmonellosis after the failure of common antibiotic remedy. Unfortunately, fluoroquinolone-resistant Salmonella strains have rapidly developed and salmonellosis cases with fluoroquinolone treatment failures have been reported (Vasallo et al., 1998; Walker et al., 2000).

Food of animal origins, including pork, is an important protein source for humans in most parts of the worlds. In response to increasing demand for pork consumption, several antibiotics have been introduced to modern pig production farm.

Fluoroquinolones is among antibiotics that are used for disease treatment and prevention in pigs. It has been shown that fluoroquinolone-resistant Salmonella is present in pigs and pork products (Padungtod et al., 2006) and, therefore, poses the risk of causing salmonellosis that does not responsed to the antibiotic treatment in humans.

Resistance to fluoroquinolones in S. enterica is mediated by several mechanisms, including mutations of topoisomerase genes, active efflux and topoisomerase protection by plasmid-encoded Qnr protein (Giraud et al., 2006). However, such resistance initially arises from mutation (s) in one or more topoisomerase genes (Piddock, 2002). While the mutations in the quinolone resistance-determining region (QRDR) of gyrA have mainly contributed to reduced susceptibility to fluoroquinolones in the clinical Salmonella human and animal isolates (Eaves et al., 2004), those in gyrB, parC and parE have rarely been reported. It was suggested that combination of mutations in the same or different topoisomerase gene resulted in increased fluoroquinolone-resistance level. For this instance, the Salmonella isolates with reduced susceptibility contained a single mutation in gyrA and the resistant isolates carried at least two mutations in gyrA and/or gyrB and/or parC and/or parE (Eaves et al., 2004).

Wannaprasat W. and Chuanchuen R. / Thai J Vet Med. 2013. 43(2): 243-249. 245

In addition to topoisomerase mutations, overexpression of the AcrAB-TolC efflux pump has been shown to mediate reduced-susceptibility to fluoroquinolones in salmonellae (Chen et al., 2007). The efflux system comprises three main functional components (i.e. AcrA, a periplasmic membrane fusion protein; AcrB, an inner membrane transporters; and TolC, a universal outer membrane protein) as seen in most pumps in the Resistance-Nodulation-Cell-Division family. As the AcrAB-TolC efflux pump was originally found in Escherichia coli, there are considerable structural and functional similarities between the pump in E. coli and Salmonella (Pomposiello and Demple, 2000).

As seen in other antibiotics, fluoroquinolone usage differs greatly as to drug formulations, target animal species, label indications, and geographic regions. This could result in difference in type and expression of resistance mechanisms among clinical isolates of bacteria, including Salmonella. The Salmonella strains in the present study are partly described in our previous study, where the presence of mutations in the QRDR in gyrA and parC were examined in all ciprofloxacin-resistant isolates (MICs = 4-8 µg/ml) (Wannaprasat et al., 2011). In the present study, we aimed to further study the mechanisms mediated fluoroquinolone resistance, including mutations in other topoisomerase genes and expression of the AcrAB-TolC efflux system. The ciprofloxacin-susceptible isolates (MICs = 0.125 µg/ml) were additionally included for better comparison.

Materials and Methods Bacterial isolates and antimicrobial susceptibility testing: Twenty-four S. enterica isolates previously isolated were included in this study (Wannaprasat et al., 2011). They were isolated from pork (n = 8) and humans (n = 16) in northern Thailand during 2005-2007. All of the pork isolates were originated from raw pork in retail markets, and all of the human strains were isolated from patients’ stools at Suandok hospital of the Faculty of Medicine of Chiang Mai University. All the strains were isolated as described in ISO6579:2002 (E) (ISO, 2002) and determined for serovars. Only one colony of each serotype was collected for each positive sample.

All the Salmonella isolates were tested for their susceptibilities to antimicrobials by determining minimum inhibitory concentrations (MICs) using a two-fold agar dilution in the former study (Wannaprasat et al., 2011). Eighteen isolates were resistant to ciprofloxacin (MICs = 4-8 µg/ml) while six isolates were susceptible (MICs = 0.125µg/ml).

Determination of mutations in QRDRs of gyrA, gyrB, parC and parE genes: PCR-template DNA was prepared from all the Salmonella isolates (n = 24) using the whole cell boiled lysate protocol (Levesque et al., 1995). All the PCR amplifications were performed in Thermo-StartTM 2X Reddymix™ PCR MasterMix (Thermo Fisher Scientific, CA, USA). The mutation(s) in the QRDRs of gyrA, gyrB, parC and parE were investigated using PCR with specific primers as

follows: gyrA, gyrAsalF(5′- GCTGAAGAGCTCCTA TCTGG-3′) and gryAsalR, (5′-GGTCGGCATGACG TCCGG-3′); gyrB, gyrBF (5′-GCGCGCTCGATTTAGC CG-3′) and gryBR, (5′-TGATAGCGCAGCTTGTCCG); parC, parCF (5′-GTACGTGATCATGGATCGTG-3′) and parCR (5′-TTCCTGCATGGTGCCGTCG-3′); and parE, parEF (5′-GCGATCGCGAATATCAGGCG-3′) and parER, (5′-CAGTTGTTCCAGTACGCCC-3′) (Chuanchuen and Padungtod, 2009).

All PCR amplicons were purified using Nucleospin Gel Extraction kit (Nucleospin®, Gutenburg, France) and submitted for nucleotide sequencing at Molecular Informatic Lab, NT, Hong Kong. Both strands of DNA sequence were compared with the published DNA sequence in GenBank (Genbank accession numbers AE008801, AE008878 and AE008846 for gyrA, gyrB, parC and parE, respectively) and analysed by Edit seq and Seqman (DNA-STAR) program.

Detection of acrB expression: All the Salmonella isolates (n = 24) were firstly screened for acrB transcription using reverse transcription-PCR (RT-PCR). Total RNA was extracted using QIAGEN RNeasy mini kit (Qiagen® Hilden, Germany) as described by the manufacturer and treated with DNaseI (Fermentas®, Mainz, Germany) according to the manufacturer’s protocol. Transcription of acrB was determined by using conventional RT-PCR. Firstly, cDNA was synthesized from one µg of DNaseI treated RNA sample using ImProm-IITM Reverse Transcriptase (Promega, Madison, USA) as described by the manufacturer and PCR primers acrBF (5′-TGAAAAAAATGGACCCGTTCTTC-3′) acrBR primer (5′-CGAACGGCGTGGTGTCA -3′) (Nishino et al., 2006). The cDNA was stored at -200C and used as DNA template for PCR.

Relative quantitation of acrB transcripion level: All the Salmonella isolates expressing AcrB were tested for transcription level of acrB by quantitative real-time PCR (qRT-PCR) using Biotools QuantiMix EASY SYG Kit (Biotools B&M Labs S.A., Madrid, Spain) and primer pairs acrBF and acrBR. Firstly, cDNA was synthesized from one µg of total RNA as described above and was measured for its concentration. The cDNA was 1:100 diluted to yield the threshold cycle (Ct) within the limits of standard curve (r2 > 0.990). The chromosomal DNA template from S. Typhimurium ATCC 13311 was used to make standard curve, plotting the average Ct values on the X axis and the log of given concentrations on the Y axis. The housekeeping gene, rrs, served as internal control and its transcription was determined using primers rrsF (5′-CCAGCAGCCGCGGTAAT-3′) and rrsR (5′-TTACGCCCAGTAATTCCGATT-3′). For each sample, the PCR amplifications were performed in triplicate in two separate experiments (n = 6, SD < 0.1). The Ct values were used for calculation of the average acrB cDNA copy number that was individually normalized using the average rrs copy number of the same sample. The acrB transcription level was expressed by comparison with that of S. Typhimurium ATCC 13311.

246 Wannaprasat W. and Chuanchuen R. / Thai J Vet Med. 2013. 43(2): 243-249.

Results Antimicrobial susceptibilities: All the Salmonella isolates exhibited resistance to at least one antibiotic. Most (n = 18) were resistant to ciprofloxacin (MICs = 4-8 µg/ml). Six isolates were susceptible to ciprofloxacin (MICs = 0.125µg/ml) and all other antibiotics tested. The resistance phenotype of all the isolates is shown in Table 2.

Mutations in the QRDRs of gyrA, gyrB, parC and parE: Three topoisomerase genes; i.e. gyrA, gyrB, and parC, were found to contain mutations. Of all twenty-four isolates, 18 ciprofloxacin-resistant isolates were found to contain mutations in at least one gene (Table 1). Two point mutations in gyrA (i.e. G-144-T and C-133-T leading to Met-48-Ile and Pro-45-Ser amino acid changes in GyrA, respectively) were observed. Only one point mutation in gyrB (i.e. T-188-C leading to Val-63-Ala in GyrB) was identified. All the resistant isolates carried parC mutations, of which up to 13 different nucleotide changes were found. None of the isolates carried mutations in parE. All six isolates susceptible to ciprofloxacin did not carry mutations in their QRDRs.

Eleven groups (Gr. I to Gr. XI) were defined based on type and number of genes carrying mutations (Table 2). None of the isolates carried mutations only in gyrA. Five isolates with a point mutation in gyrA additionally harbored mutations in gyrB and/or parC (Gr. I and Gr.VI to IX). Two isolates (one in Gr.1 and the others in Gr. VII) carried mutations in all three genes. Eight isolates (Gr. III, IV, VI and X) possessed mutations only in parC.

The Salmonella isolates with point mutations in all three genes (i.e. SA463 in Gr. I and SA614 in Gr. VIII, MIC = 8 µg/ml) were not more resistant to ciprofloxacin than those with mutations only in gyrB and parC (i.e.SA613 and SA622in Gr.II and SA603 in Gr. V, MIC = 8 µg/ml). Similarly, the isolates with gyrA and parC mutations (i.e. SA608 and SA609 in Gr. VI and SA615 in Gr.IX) exhibited the same ciprofloxacin resistance level (MIC = 4 µg/ml) with those carrying only parC mutations (Gr. III, IV, VI and X).

When considered mutations in gyrB and parC, the isolates with mutations in both genes (i.e. SA671 in Gr.II and SA603 in Gr. V) and those with parC mutation only (Gr. III, IV and VII) showed the same ciprofloxacin MIC (4 µg/ml).

Among those with parC mutation only, the ciprofloxacin MIC (4 µg/ml) of the Salmonella isolates with one mutation (i.e. SA578 and SA579 in Gr. III) was equivalent to that of the isolates with at least six point mutations (i.e. SA601 and SA602 in Gr. IV and SA610 and SA611 in Gr. VII). Likewise, SA624 in Gr. IV, SA612 in Gr. VII and SA616 in Gr. X showed the

comparable ciprofloxacin MIC (8 µg/ml).

Expression of AcrAB-TolC efflux system: All the Salmonella isolates produced AcrB. The transcription level varied from 1 to 430 folds of that of serovar Typhimurium ATCC 13311 (Table 2). Among the ciprofloxacin resistant isolates, AcrB expression level was diverse, i.e. those with MIC of 8 µg/ml expressed 24-131 folds while those with MIC 4 µg/ml expressed 1-430 folds. Two isolates with the highest AcrB expression SA671 (i.e. 430 folds) and SA608 (i.e. 273 folds) demonstrated ciprofloxacin MIC of 4 µg/ml comparable to those with the lowest AcrB expression, e.g. SA601 (i.e. 2 folds) and SA602 (i.e. 3 folds). Likewise, four isolates producing AcrB equivalent to ATCC 13311 (i.e. SA609, SA610, SA611 and SA578) showed ciprofloxacin MIC of 4 µg/ml. Five ciprofloxacin susceptible strains (Gr. XI) overproduced AcrB (i.e. 6-221 folds). Among these isolates, SA736 was susceptible to all antimicrobials tested and produced AcrB up to 221 folds.

The isolates with comparable AcrB demonstrated different susceptibility to ciprofloxacin. SA666 and SA741, producing AcrB of 18 folds, were susceptible to ciprofloxacin while SA579 (19 folds) and SA612 (24 folds) were resistant to the antibiotic. When considered resistance to other antimicrobials, not all the AcrB-overexpressing strains were multidrug resistant. For example, SA721 was resistant to only tetracycline and SA736 was susceptible to all antimicrobials tested overproduced AcrB 18 and 221 folds, respectively.

Table 1 Mutations in gyrA, gyrB and parC genes in the ciprofloxacin-resistant Salmonella isolates (n = 18)

Mutation Gene Nucleotide

change Amino acid substitution

No.(%)

gyrA G-144-T C-133-T

Met-48-Ile Pro-45-Ser

5 (27.8) 1 (5.6)

gyrB T-188-C Val-63-Ala 6 (33.3) parC T-2-C

A-13-C G-31-C C-62-T T-86-C C-92-T C-152-T G-173-A C-182-G A-212-G T-230-C T-230-G C-245-T

Met-1-Thr Ser-5-Arg Ala-11-Pro Ser-21-Leu Ile-29-Thr Thr-31-Met Thr-51-Ile Gly-58-Glu Ala-61-Gly Gln-71-Arg Leu-77-Pro Leu-77-Arg Thr-82-Met

6 (33.3) 1 (5.6) 12 (66.7) 12 (66.7) 12 (66.7) 7 (38.9) 6 (33.3) 1 (5.6) 6 (33.3) 12 (66.7) 7 (38.9) 4 (22.2) 14 (77.8)

Wannaprasat W. and Chuanchuen R. / Thai J Vet Med. 2013. 43(2): 243-249. 247

Table 2 Antimicrobial resistance characteristics of the Salmonella isolates (n = 24)

Mutation Gr Strain gyrA gyrB parC

CIP MIC

(μg/ml) Resistance patterna Expression

level of acrB

I II III IV

SA463 SA613 SA622 SA671 SA578 SA579 SA601 SA602

Pro-45-Ser - - - - - - -

Val-63-Ala Val-63-Ala Val-63-Ala Val-63-Ala - - - -

Ser-5- Arg, Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro, Thr-82-Met Thr-82-Met Thr-82-Met Thr-82-Met Thr-82-Met Thr-82-Met Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Gln-71-Arg, Leu-77-Pro, Thr-82-Met, Met-1-Thr, Thr-51-Ile, Ala-61-Gly Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Gln-71-Arg, Leu-77-Pro, Thr-82-Met, Met-1-Thr, Thr-51-Ile, Ala-61-Gly

8 8 8 4 4 4 4 4

CHPC-CIP-SPC-SUL-TET-TRI AMP-CHPC-CIP-STR-SPC-SUL-TET-TRI AMP-CHPC-CIP-STR-SPC-TET-TRI CIP-SPC-SUL-TET-TRI AMP-CHPC-CIP-SPC-TRI AMP-GEN-CHPC-CIP-STR-SPC-SUL-TET-TRI AMP-GEN-CHPC-CIP-STP-SPC-TET-TRI

131 49 63 430 1 19 2 3

V VI VII VIII

SA624 SA603 SA608 SA609 SA610 SA611 SA612 SA614

- - Met-48-Ile Met-48-Ile - - - Met-48-Ile

- Val-63-Ala - - - - - Val-63-Ala

Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Gln-71-Arg, Leu-77-Pro, Thr-82-Met, Met-1-Thr, Thr-51-Ile, Ala-61-Gly Thr-82-Met, Gly-58-Glu Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro

8 4 4 4 4 4 8 8

AMP-GEN-CHPC-CIP-STR-SPC-TET-TRI AMP-CHPC-CIP-STR-SPC-TET-TRI AMP-GEN-CHPC-CIP-SPC-TET-TRI AMP-CHPC-CIP-SPC-SUL-TET-TRI AMP- CHPC-CIP-SPC-STR-SUL-TET-TRI AMP-CHPC-CIP-SPC-TET-TRI AMP-CHPC-CIP-STR-SPC-SUL-TET-TRI AMP-GEN-CHPC-CIP-STR-SPC-SUL-TET-TRI

26 55 273 1 1 1 24 58

IX X XI

SA615 SA616 SA545 SA666 SA717 SA721 SA734 SA736

Met-48-Ile - - - - - - -

- - - - - - - -

Ala-11-Pro, Ser-21-Leu, Ile-29-Thr, Thr-31-Met, Gln-71-Arg, Leu-77-Pro, Thr-31-Met, Gln-71-Arg, Leu-77-Pro, Thr-82-Met Ala-11-Pro, Ser-21-Leu, Ile-29-Thr - - - - - -

8 8 0.125 0.125 0.125 0.125 0.125 0.125

AMP-CHPC-CIP-STR-SPC-TET-TRI AMP-CHPC-CIP-STR-SPC-SUL-TET-TRI AMP-TET-SUL AMP-CHPC-SPC-SUL-TET-TRI AMP-SPC-SUL-TET-TRI TET AMP-GEN-CHPC-CIP-STR-SPC-TET Susceptible to all antimicrobial tested

58 106 101 18 1 18 6 221

a Amp: ampicillin; Chp: chloramphenicol; Gen: gentamycin; Spc: spectinomycin; Str: streptomycin; Sul: sulfamethoxazole; Tet: tetracycline; Tri: trimethoprim

Discussion

In comparison to other pathogenic Enterobacteriaceae, Salmonella infrequently exhibit high fluoroquinolone resistance level and it was suggested that such limitation was associated with a prohibitive fitness cost (Giraud et al., 2003; Giraud et al., 2006). This agreed with this study, where the ciprofloxacin-resistant isolates showed the MIC value of 4-8 µg/ml. Fluroquinolone resistance in Salmonella is commonly associated with a single point mutation between nucleotides 67 to 122 in the QRDR of gyrA (Giraud et al., 1999), leading to the more common GyrA amino acid changes at position Gly81, Ser83 or Asp87 (Marimon et al., 2004). In contrast, the strains in this study lacked these prominent mutations. However, our strains carried mutations in gyrA, gyrB and/or parC. Based on our knowledge, all the mutations identified are novel in ciprofloxacin-resistant S. enterica. Since these mutations have never been found, their actual contribution may not be stated. Further experiment such as site specific mutagenesis or study in a larger population may worth it.

In Gram-negative bacteria, a single gyrA

mutation confers low ciprofloxacin resistance level and the additional mutations in gyrA or within the other topoisomerase genes results in the increased-resistance level (Liebana et al., 2002; Dimitrov et al., 2009). However, none of the isolates in the present study harbored only gyrA mutations. Therefore, it may not precisely assess the contribution of mutations in gyrA and other two genes.

No nucleotide changes in parE were identified, consistent with previous studies (Marimon et al., 2004). This is not beyond expectation and supports that parE mutation is much less common than mutations in the others. Mutations in parC are seldom seen in the ciprofloxacin-resistant S. enterica (Eaves et al., 2004) and role of these mutations is still not apparent. In contrast, parC mutations were identified in all the resistant strains in this study. While up to 13 different point mutations were observed in parC, some single strains simultaneously carried 9 parC mutations. Still, it cannot ascertain that all these mutations contributed to ciprofloxacin resistance of the bacterial hosts. However, the susceptible isolates did not carried the same mutations in any of the three topoisomerase genes, supporting that nucleotide changes observed were not

248 Wannaprasat W. and Chuanchuen R. / Thai J Vet Med. 2013. 43(2): 243-249.

likely associated with strain variation and indicating the significance of mutations in topoisomerase genes in ciprofloxacin resistance. Taken together, there are no associations between the number and type of mutations and ciprofloxacin resistance level among the ciprofloxacin-resistant Salmonella isolates in this collection.

The isolates with the same ciprofloxacin MIC expressed AcrB at different level. Vice versa, the isolates with the same AcrB expression exhibited different ciprofloxacin MICs. These data indicated that there was no correlation between expression of the AcrAB-TolC efflux pump and ciprofloxacin resistance level and also the existence of other uncharacterized mechanisms. In this case, the presence of the plasmid-borne qnr genes was demonstrated but not examined in this study. Transcription of acrB was detected in all 24 Salmonella isolates, supporting the constitutive expression of the AcrAB-TolC efflux pump in Salmonella. The AcrAB-TolC pump was identified as a major mechanism in fluoroquinolone resistance in E. coli (Piddock et al., 2000). It was shown that inactivation of the pump resulted in loss of fluoroquinolone resistance in the strains with gyrA mutations (Oethinger et al., 2000). This may not always be the case since some ciprofloxacin resistant isolates in this study (i.e. SA578, SA609, SA610 and SA611) did not overproduce AcrB when compared to the reference strain. Concurrently, the ciprofloxacin susceptible isolates in Gr.5 (MIC = 0.125 µg/ml) produced AcrB from 6 to 221 folds. The contribution of the AcrAB-TolC pump in these strains should be minimal (if any) and may not be accurately determined due to high susceptibility to ciprofloxacin of the strains.

Based on the observations in this study, the role of AcrAB-TolC of the Salmonella isolates is greatly different from previous studies (Baucheron et al., 2002; Giraud et al., 2000). This discrepancy could be explained by differences in bacterial sources and settings of fluoroquinolone-resistance phenotype. In most previous studies, the contribution of the AcrAB-TolC pump was compared between the parent strains and their isogenic-resistant mutants originated from in vitro exposure to fluoroquinolones. In contrast, all the Salmonella strains in this collection were of clinical origins and their resistance phenotype was associated with several factors within the cells and in their environment. Therefore, AcrAB-TolC expression dynamics and its contribution to fluoroquinolone resistance level may somewhat vary.

In addition to fluoroquinolone resistance, the AcrAB-TolC efflux pump plays a major role in multiple antibiotic resistance in Salmonella (Baucheron et al., 2002; Guerra et al., 2003). Deletion of the acrAB operon resulted in increased susceptibility to multiple drugs, e.g. chloramphenicol, ceftiofur, tetracycline, cephalothin, trimethoprim-sulfamethoxazole (Giraud et al., 2000; Eaves et al., 2004). Despite these previous reports, the same way may not be true in our strains. The best evidences were overproduction of AcrB in SA736 that was susceptible to all antimicrobials (221 folds) and SA721 that was resistant to tetracycline

only (18 folds).

Particular attention was paid to SA736 that was susceptible to ciprofloxacin and all other antimicrobials tested lacked mutation in topoisomerase genes and overproduced AcrB up to 221 folds. Its genetic and phenotypic property illustrated the inconsistent role of the AcrAB-TolC efflux pump, while substantiated the role of mutations in the target genes in fluoroquinolone resistance in the Salmonella clinical isolates. Therefore, further studies are warranted to elucidate the actual involvement of these mechanisms in the clinical isolates.

In conclusion, decreased susceptibility to ciprofloxacin in S. enterica clinical isolates seems to be associated with the accumulative effect of many resistance mechanisms and the role of the well-characterized mechanisms may not always be in harmony. As fluoroquinolones are one of the best choices for salmonellosis treatment, increasing resistance to this antibiotic class may devastate their future use in medical therapy. The latter could be true for not only Salmonella infection but also other pathogenic bacterial infections. Judicious use of antibiotics is mandatory and needs to be initiated with no delay.

Acknowledgements

This work was supported by the 90th

anniversary of Chulalongkorn University fund and partly by TRF-CHE Research Grant MRG5180154, co-funded by Thailand Research Fund (TRF) and the Commission on Higher Education (CHE). WW is a recipient of the Royal Golden Jubilee Ph.D. program PHD/0039/2551.

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Cloeckaert A 2002. The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium phage type DT204. Microb Drug Resist. 8(4): 281-289.

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250

Thai J Vet Med. 2013. 43(2): 251-258.

Effect of Polysaccharide Gel Extracted from Durio zibethinus

Rind on Immune Responses, Bacteria Counts and

Cholesterol Quantities in Chickens

Nustha Kitprathaung1 Nattaya Ngamrojanavanich1

Piyarat Chansiripornchai2 Sununta Pongsamart3 Niwat Chansiripornchai4*

Abstract

Polysaccharide gel (PG) from the rind of durian (Durio zibethinus Murr.) is a soluble powder that gives good

results as an excipient in pharmacological and food preparation. The aim of the present study was to evaluate the effects of PG as a feed-supplement diet on body weight gain, immune stimulation, total bacteria and Salmonella in feces, and cholesterol levels in broilers. Eighty, one-day-old broiler chicks were divided into 4 groups. Three experimental groups were fed a commercial diet coated with PG 1, 2 and 3 g/100 g, respectively, and the control group was fed a commercial diet without PG. The study was performed for 42 days. Chicken weight gain in the treatment and control groups was not significantly different. At six weeks old, the hemagglutination inhibition and ELISA antibody titers against Newcastle disease (ND) virus and infectious bursal disease (IBD) virus, respectively, were significantly different (p < 0.05). The chickens fed commercial feed with 3 g/100 g PG revealed the highest antibody titers against the ND and IBD. There was no significant difference in heterophil : lymphocyte ratio between the treatment groups and the control group. The total bacteria count in chicken feces was significantly reduced, 81-97%, in the experimental groups compared to the control group (p < 0.05). Moreover, Salmonella suspected colonies were significantly reduced in the experimental groups compared to the control group (p < 0.05) and no Salmonella suspected colonies were detected in the experimental groups. The cholesterol levels in the plasma of the chickens in the treatment groups were lower than those of the control group. Furthermore, the cholesterol content of the muscles of the broilers fed on the diet with 3 g/100 g PG was significantly lower than those of the chickens fed on the diet without PG (p < 0.05). Therefore, polysaccharide gel in the diet benefited health promotion in broiler chickens as an antibacterial activity, immunostimulant and in cholesterol reduction.

Keywords: cholesterol, dietary fiber, immunostimulant, polysaccharide gel 1Program of Biotechnology, Faculty of Sciences, Chulalongkorn University; 2Department of Veterinary Pharmacology, Faculty of Veterinary Science, Chulalongkorn University; 3Faculty of Pharmaceutical Sciences, Chulalongkorn University; 4Avian Health Research Unit, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand. *Corresponding author: E-mail: cniwat@chula.ac.th

Original Article

252 Kitprathaung N. et al. / Thai J Vet Med. 2013. 43(2): 251-258.

บทคดยอ

ผลของสารสกดเจลพอลแซกคาไรดจากเปลอกทเรยน Durio zibethinus ตอการตอบสนองทางภมคมกน จานวนแบคทเรยและปรมาณโคเลสเตอรอลในไก ณษฐา กจประเทอง 1 นาตยา งามโรจนวณชย 1 ปยะรตน จนทรศรพรชย 2 สนนท พงษสามารถ 3 นวตร จนทรศรพรชย4*

พอลแซกคาไรดเจล (PG) จากเปลอกของผลทเรยน (Durio zibethinus Murr.) มลกษณะเปนผงละลายนาไดซงเปนกระสายยาทดในทางเภสชวทยาและการเตรยมอาหาร การศกษานมวตถประสงคเพอประเมนผลของ PG ในฐานะอาหารเสรม ในดานการเพมนาหนกตว การกระตนภมคมกน จานวนแบคทเรยโดยรวมและซลโมเนลลาในมลของไกเนอ และการลดโคเลสเตอรอลในไกเนอ ไกเนออาย 1 วน จานวน 80 ตว แบงออกเปน 4 กลม คอ 3 กลมทดลองทไดรบอาหารทจาหนายเชงพาณชยทเคลอบดวย PG ขนาด 1, 2 และ 3 กรม/100 กรมตามลาดบ และกลมควบคมทไดรบอาหารทจาหนายเชงพาณชยทไมเคลอบ PG จากการศกษาเปนเวลา 42 วน ไมพบความแตกตางอยางมนยสาคญของนาหนกไกทเพมขนระหวางกลมทดลองและกลมควบคม เมอไกอาย 6 สปดาห พบความแตกตางอยางมนยสาคญทางสถต (p < 0.05) ของแอนตบอดไตเตอรตอวธการยบยงการตกตะกอนเมดเลอดแดงและวธอไลซาตอไวรสนวคาสเซล (ND) และไวรสเบอรซาอกเสบตดตอ (IBD) ตามลาดบ โดยไกทไดรบอาหารเชงพาณชยทเคลอบ PG ขนาด 3 กรม/100 กรม มระดบแอนตบอดไตเตอรสงสดตอ ND และ IBD ไมพบความแตกตางอยางมนยสาคญของ heterophil : lymphocyte ratio ระหวางกลมทดลองทไดรบอาหารทม PG และกลมควบคมทไดรบอาหารทไมม PG พบวาจานวนแบคทเรยในมลไกลดลงอยางมนยสาคญทางสถต คอ ระหวางรอยละ 81-97 เมอเปรยบเทยบระหวางไกกลมทดลองและกลมควบคม (p < 0.05) นอกจากน พบวาจานวนโคโลนทสงสยวาเปนซลโมเนลลาลดลงอยางมนยสาคญทางสถตเมอเปรยบเทยบระหวางไกกลมทดลองและกลมควบคม (p < 0.05) และไมพบโคโลนทสงสยวาเปนซลโมเนลลาในกลมทดลอง ระดบโคเลสเตอรอลในพลาสมาในไกกลมทดลองมระดบตากวาไกกลมควบคม นอกจากนปรมาณโคเลสเตอรอลในกลามเนอของไกทไดรบ PG 3 กรม/100 กรม มระดบตากวาไกกลมควบคมทไมไดรบ PG อยางมนยสาคญทางสถต (p < 0.05) ดงนน PG ในอาหารมประโยชนในการสงเสรมสขภาพไกเนอในดานการตานแบคทเรย การกระตนภมคมกนและการลดลงของโคเลสเตอรอล

คาสาคญ: โคเลสเตอรอล เสนใยอาหาร การกระตนระบบภมคมกน พอลแซกคาไรดเจล 1หลกสตรเทคโนโลยชวภาพ คณะวทยาศาสตร จฬาลงกรณมหาวทยาลย 2ภาควชาเภสชวทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย 3คณะเภสชศาสตร จฬาลงกรณมหาวทยาลย 4หนวยปฏบตการวจยสขภาพสตวปก คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย กรงเทพฯ 10330 ประเทศไทย *ผรบผดชอบบทความ E-mail: cniwat@chula.ac.th

Introduction

Polysaccharides extracted from the rind of durian (Durio zibethinus Murr.), the most popular fruit of Thailand, were studied. Polysaccharides are composed of soluble fiber (polysaccharide gel) and insoluble fiber (polysaccharide fiber) (Girddit et al., 2002; Hokputsa et al., 2004). Polysaccharide gel (PG) is composed of long chain α (1 4) linked polygalacturonan with side chains of neutral sugars with terminal non-reducing end fructose and glucan (Hokputsa et al., 2004) whereas insoluble polysaccharide fiber is composed of long chain glucan as α-cellulose (Umpray et al., 1990). Polysaccharide gel can be absorbed and slowly dissolves in water. The gelling properties of PG are of benefit for utilizing as pharmaceutical aids. The soluble and insoluble

durian polysaccharides are useful for tableting (Sithipirojsakul et al., 2002). Polysaccharide gel is resistant to acid and enzyme α-amylase hydrolysis. Polysaccharides cannot be completely digested and absorbed in the gastrointestinal tract. No toxic effects have been found in acute and subchronic toxicity studies, which confirm the consumptive safety of durian polysaccharides (Pongsamart et al., 2002). The immunomodulating properties of PG can be estimated by complement fixation assay (Hokputsa et al., 2004). Polysaccharide gel has a beneficial effect in improving the immune systems of black tiger shrimp (Penaeus monodon) (Pholdaeng and Pongsamart, 2010). Moreover, a preliminary study by Chansiripornchai et al. (2008) reported that adding PG to broiler chicken diet, as a feed additive, stimulated the humoral immune responses and could reduce cholesterol in the pectoral muscles of chickens. According to

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Chansiripornchai et al. (2008), PG in feed can reduce the level of cholesterol in muscle measured by the colorimetric method. Polysaccharide gel can well confine liquid such as cholesterol and fatty acid by absorbing lipids and cholesterol within PG molecule when passing through the alimentary canal, resulting in the low absorption of lipids and cholesterol in chickens (Tippayakul et al., 2002). Moreover, nutrient absorption rate depends on the rate at which nutrients are in contact with the absorptive epithelium layer (Beseres et al., 2005; Pholdaeng and Pongsamart, 2010). Thus, such a high PG concentration may cause interference in the rate of nutrient absorption. Increased viscosity of a diet can likely retard nutrient absorption in the same way as the effect of dietary fiber that is not absorbed in the stomach (Fair et al., 1980). Currently, broilers are not only produced at an industrial level but also at small holding level. Small holders serve as an important source of food supply in remote and poor areas. An outbreak of infectious disease (such as Newcastle disease, Infectious bursal disease, etc.) certainly causes severe economic damage to all levels of broiler production. Many pathogenic bacteria are found in chicken feces, such as coliform, Salmonella and pathogenic E. coli. Generally, bacteria in feces are 100-1000 times of anaerobic bacteria compared to aerobic bacteria and most pathogenic bacteria are aerobic bacteria (Simon and Gorbach, 1984). The procedure of total bacteria count only focuses on the aerobic bacteria and this method generally characterized the contaminated or pathogenic bacteria in feces. The present study aimed to investigate the effect of PG as a feed additive on body weight gain, immunostimulation, reduction in meat and plasma cholesterol and also reduction in fecal bacteria in chickens fed commercial feed with PG in different ratios and chickens fed commercial feed without PG.

Materials and Methods Polysaccharide gel extraction from rind of durian: Extraction of PG was previously reported (Pongsamart and Panmaung, 1998). Briefly, polysaccharide gel was extracted from the dried rind of durian with boiling water. The polysaccharide gel extracted water was concentrated under reduced pressure and precipitated by its addition into acidified aqueous ethanol, filtered, dried and ground. The pale beige colored powder of durian polysaccharide was used in the experiment.

Preparation of polysaccharide gel feed-supplement diet: To make a ten percent PG stock feed additive diet, 10 g of the purified PG was dispersed in water and then spray-coated onto 100 g of feed pellets of a commercial broiler diet (Betagro, Thailand), in a pan coating machine (Yinrich, China). To make one, two and three g% PG, 10, 20 and 30 g of stock feed additive diet were mixed with 90, 80 and 70 g of commercial feed, respectively.

Experimental designs: Eighty, unvaccinated one-day-old, female Cobb 500 broiler chicks obtained from a commercial hatchery (Krungthai, Thailand) were randomly divided into 4 groups (20 chickens each); a

negative control group (0 g% PG), and 1, 2 and 3 g % PG groups (Pholdaeng and Pongsamart, 2010). Each group was randomly divided into 2 replicates. Each group of chicks was maintained in a separate unit and each replicate of 10 chicks was raised in a metallic cage at environmental temperature. All the chicks were fed ad libitum. All the chicks at 1, 7 and 14 days old were vaccinated with live Newcastle disease (ND) vaccine (Merial, France) by eye drops, inactivated ND vaccine (Fort Dodge®, Brazil) by subcutaneous injection and infectious bursal disease (IBD) vaccine (Merial, France) by oral drops, respectively. Blood was collected from the wing vein at 1, 7, 14, 28, 35 and 42 days of age and tested to measure blood cholesterol, heterophil:lymphocyte (H:L) ratio, hemagglutination inhibition (HI) titers for ND and ELISA titers for IBD. The antibody titers of IBD were determined using ELISA test kits (Synbiotic Corp, USA). Chickens at one day old and six weeks old in each group were weighed and the amount of feed intake was recorded for the calculation of weight gain. Delivery box-liners were swabbed for total bacteria and Salmonella suspected colony test. Feces were also collected to determine total bacteria count and Salmonella suspected colony count.

Heterophil : lymphocyte (H:L) ratio: Bloods were collected and mixed with EDTA as anticoagulant and carried out to measure the ratio of heterophil : lymphocyte (H:L). The bloods were smeared and stained with Wright-Giemsa (Hauptmanova et al., 2002), approximately 2 to 4 hours after preparation by methyl alcohol fixation. One hundred leukocytes, including granular (heterophils, eosinophils and basophils) and nongranular (lymphocytes and monocytes), were counted under light microscope and the heterophil to lymphocyte ratio was calculated.

Isolation and determination of numbers of bacteria count Preparation of samples: Fecal sampling was performed from each replicate. Two pooled, fecal samples were collected in each group. A sample collection technique was modified from Andreatti Filho et al. (2007). Samples of chicken feces were collected from 5 points of a fecal tray; 4 points at the corner and 1 point at the center of the tray and then pooled into 1 sample. One gram fecal samples were taken and mixed with 9 ml buffered peptone water (BPW) pH 7.5. The fecal suspension samples were used for determination of the total number of bacteria and Salmonella suspected colony counts.

Total plate count and Salmonella suspected colony count: The fecal suspended samples were diluted by ten fold serial dilution with BPW from 10-2 to 10-13 dilution. Plate count agar medium (Merck KGaA, Darmstadt, Germany) was melted and poured into a petri dish, cooled to room temperature until solidified and then a 0.1 ml sample pipetted from the 10-2 to10-13 dilution was added into each petri dish, spreaded on the plate and incubated at 350C for 24 hours. A plate that contained between 30 and 300 colonies was selected for counting (ISO, 2002). For the Salmonella suspected colony count, 1 ml of the fecal suspension

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of each sample, obtained by mixing the fecal sample with BPW (1:9 w/v), was transferred into 9 ml of BPW; this suspension mixture was serially diluted to 10-1. A sample of 0.1 ml of the diluted solution was transferred to a plate of xylose lysine tergitol 4 agar (Merck KGaA, Darmstadt, Germany) and incubated for 24 hours at 370C. Then, the black Salmonella suspected colonies were counted (Xiong et al., 1998).

Determination of cholesterol Extraction of cholesterol from plasma: Cholesterol was extracted from 0.5 ml of chicken plasma at one day old and six weeks old with 0.5 ml cold methanol (Fisher Scientific, Loughborough, UK) and 2.5 ml hexane (Fisher Scientific, Loughborough, UK). The samples were vortexed and then centrifuged 80 x g at 40C for 5 min. The hexane layer was removed and dried under N2 and re-dissolved in a mobile phase composed of 1 ml acetonitrile : isopropanol (75 : 25, v/v) 1 ml (Fisher Scientific, Loughborough, UK) (Tippayakul et al., 2002) and transferred to a HPLC vial and then analyzed by HPLC instrument (Shimadzu Corp, Japan).

Extraction of cholesterol in breast muscles: Four chickens from each experimental group were randomly selected for euthanasia by cervical dislocation. The breast muscles of each chicken were collected and cholesterol extracted by the method of Folch et al. (1957). Briefly, one hundred grams of breast muscle were saponificated at 930C with ethanol and potassium hydroxide. Later, the samples were extracted with distilled water and hexane until the layers separated. Then, the cholesterol was diluted in a hexane layer with N2 gas and diluted with 1 ml of acetonitrile:isopropanol (75 : 25, v/v) and analyzed by the HPLC method (Shimadzu Corp, Japan).

Assay of cholesterol detection: The cholesterol content was assayed using the HPLC technique (Araki et al., 1990; Seta et al., 1990), a column symmetry C18 (3.9 x 150 mm, 5 µm) was used and each of the 15 µl samples was injected into the column. The mobile phase for HPLC was acetonitrile : 2-propanol (7 : 3), which had previously been filtered through a 0.45 µm membrane filter before use. The column was eluted at a flow rate of 1.5 ml/min at ambient temperature and the UV detector was monitored at 210 nm.

Hemagglutination inhibition and ELISA test: The hemagglutination inhibition titers of ND were tested in a U-shaped, 96-well, microtiter plate (Corning, USA) as modified by Alexander (2000). In brief, a dilution series of sera was incubated with 4 hemagglutination units of ND virus, La Sota strain, at room temperature for 30 min. The hemagglutination unit was titrated before each assay. Thereafter, chicken erythrocytes were added and agglutination was monitored after incubation at room temperature for 45 min. The hemagglutination inhibition titer was defined as the reciprocal of the highest serum dilution completely inhibiting agglutination. The antibody titers of IBD were determined using ELISA test kits (Synbiotic Corp, USA). Briefly, ELISA plates were

coated with IBDV specific antibodies. Sample serum diluted 1/10 to 1/25 (w/v) in a dilution buffer was incubated in the coated wells. Unbound antigens were discarded at the end of the incubation period by washing with a washing buffer. The captured antigens were then revealed, as in an indirect ELISA, with a detection antibody, followed by an enzyme conjugate that bound to the detection antibody only followed by the enzyme substrate. Finally, optical densities, which paralleled the amount of captured IBDV antigens, were read with an ELISA reader (Eterradossin et al., 1997).

Statistical analysis: The antibody titers, cholesterol levels and weight gain were analyzed using ANOVA and Duncan’s multiple range test with SPSS software (SPSS Inc, Chicago). Differences between groups were considered significant at p < 0.05.

Results

At forty two days old, the body weight gain (gram) (mean±SE) of chickens in groups 0, 1, 2 and 3 g% PG was 1,856.44±2.21, 1,873.67±10.29, 1,929.63± 8.71 and 1,918.58±5.63, respectively. The feed intake (gram) (mean±SE) of chickens in groups 0, 1, 2 and 3 g% PG was 4,024.40±2.12, 3,890.29±2.28, 3,714.83±1.36 and 3,922.86±2.36, respectively. The results indicated that PG at a different ratio in a feed-supplement diet did not affect the chickens’ weight gain in addition to total feed intake. The results revealed no significant difference between the group fed on the commercial feed without PG (control) and the groups fed on the commercial feed coated with PG 1, 2 and 3 g% PG.

At six weeks old, hemagglutination inhibition antibody titers against ND virus (Fig 1) and ELISA antibody titers against IBD virus (Fig 2) of the chickens in the 3 g% PG group were significantly different (p < 0.05). The chickens in the 3 g% PG group revealed higher titers against ND and IBD antibody than those of the other groups. At week 1 until week 5 the results revealed no significant difference between the group fed on the commercial feed without PG (control) and the groups fed on the commercial feed coated with PG 1, 2 and 3 g% PG.

Figure 1 Hemagglutination inhibition antibody titers against Newcastle disease virus were determined weekly in chickens receiving different concentrations of PG in feed. Each data set represents the geometric mean titer (log2±SD) of twenty serum samples

Kitprathaung N. et al. / Thai J Vet Med. 2013. 43(2): 251-258. 255

Figure 2 ELISA antibody titers against infectious bursal

disease virus were determined weekly in chickens receiving different concentrations of PG in feed. Each data set represents the arithmetic mean titer (mean±SD) of twenty serum samples.

Figure 3 Heterophil : Lymphocyte ratio of white blood cell

fraction in chickens were determined weekly in chickens receiving different concentrations of PG in feed. Each data set represents the arithmetic mean (mean±SD) of twenty serum samples.

The results of H:L ratio at the beginning until the end of the experiment revealed no significant difference between the group fed on the commercial feed without PG (control) and the groups fed on the commercial feed coated with PG 1, 2 and 3 g% PG (Fig 3). At five weeks old, the H:L ratio of chickens in each group was higher than the other weeks. Chickens in 0 g% PG group showed a higher H : L ratio than those of the other groups.

The swabs of delivery box-liners showed contamination of total bacteria and Salmonella suspected colony. The chickens in groups of 1, 2 and 3 g% PG exhibited lower colonies of total bacteria counts than the chickens in the control group (0 g% PG). At six weeks old, the total bacteria count of the chickens in groups 1, 2 and 3 g% PG was significantly reduced 81, 88 and 97%, respectively, compared to the group 0 g% PG (p < 0.05) (Table 1). The polysaccharide gel reduced the number of bacteria in chicken fecal samples and the increased PG in feed resulted in a decrease in the total bacteria count in chicken feces. The polysaccharide gel in the feed-supplement diet exhibited a killing effect on the bacteria in chicken feces. For the Salmonella suspected colony count, the amount of Salmonella suspected colonies in chicken feces was significantly lower in the groups fed on PG coated diets (p < 0.05) compared to that of the control group, at 5 and 6 weeks old (Table 2). The feed with PG showed zero Salmonella suspected colony count in 1 and 2 g% PG group at 5 weeks old. No Salmonella suspected colony was found in the chickens fed on a PG coated diet at 6 weeks old.

The average cholesterol levels in the plasma of chickens in groups 0, 1, 2 and 3 g% PG group at 1 day old and 6 weeks old are shown in Fig 4. The results revealed no significant difference between the group fed on commercial feed without PG (control) and the groups fed on commercial feed coated with PG 1, 2 and 3 g% PG (p < 0.05). The plasma cholesterol levels of the PG treated groups showed lower cholesterol levels compared to those of the control group. For the muscle cholesterol, the cholesterol level in the muscle of chickens in the 3 g% PG group was significantly reduced compared to that of the chickens in the control group (0 g% PG) (p < 0.05). The more the PG in feed the greater the cholesterol reduction in the chicken’s muscle. Recalculated to a 100% cholesterol level in the muscle of the chickens in the control group (0 g% PG), the cholesterol level in the muscle of chickens in group 4 was reduced to 75.78% (Table 3).

Table 1 Total bacteria count (11 log 10 colony forming unit/ml) in fecal samples of chickens in each group/week determined weekly

Average total bacteria count (mean±SD) Groups

1 week 2 week 3 week 4 week 5 week 6 week 0g% PG 171.25±14.43ab 290.00±7.83a 36.50±7.59 301.50±5.45a 160.00±12.25a 137.00±19.71a 1g% PG 156.25±7.41bc 155.00±26.04c 45.00±4.97 120.50±56.04b 32.00±15.43b 26.00±18.24c 2g% PG 126.50±2.12c 128.00±16.43c 87.50±62.04 111.00±15.14b 11.00±6.22c 16.25±4.65c 3g% PG 200.75±31.91a 236.50±42.61b 36.75±31.08 55.75±15.84c 4.75±2.22c 4.50±1.73c

a, b, c Different superscripts indicate a significant difference (p < 0.05) between groups

Table 2 Salmonella colony count (2 log10 colony forming unit/ml) in fecal samples of chickens in each group/week determined weekly

Average Salmonella suspected colony count (mean±SD) Groups

1 wk 2 wk 3 wk 4 wk 5 wk 6 wk 0g% PG 272.75±25.75a 299.00±24.25a 350.00±14.00a 30.00±15.00 27.50±6. 50a 10.00±1.00a 1g% PG 112.50±2.75b 196.25±7.50b 105.00±4.50b 0 0b 0b 2g% PG 47.25±23.63b 113.50±1.50b 89.75±22.25b 5.00±2.00 0b 0b 3g% PG 24.75±6.25b 100.50±39.75b 16.50±6.50b 0 2.00±1.00b 0b

a, b, c Different superscripts indicate a significant difference (p < 0.05) between groups

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Table 3 Cholesterol levels in 6 chicken muscle samples in each group analyzed at 6 weeks old.

Group Cholesterol level in

chicken muscle (mg/100 g)

Percentage of cholesterol level in chicken muscle

compared to control group

0g% PG 61.73±1.77a 100.00 1g% PG 50.85±1.70ab 82.37 2g% PG 54.31±4.59ab 87.98

3g% PG 46.78±6.94b 75.78

a, b Different superscripts indicate a significant difference (p < 0.05) between groups

Figure 4 Cholesterol level in chicken plasma before and after

being fed on different concentration of PG in feed for 6 weeks was determined. Each data set represents the arithmetic mean (mean±SD) of twenty serum samples.

Discussion

The body weight gain of the chickens in the control group was not significantly lower than those of the treatment groups. Therefore, the polysaccharide gel in the feed did not produce any adverse effects to the chickens. At six weeks old, the chickens in the group receiving 3 g% PG revealed the highest antibody titers against ND and IBD immunity and a more significant difference (p < 0.05) than the other groups (Fig 1 and 2). This is in accordance with Chansiripornchai et al. (2008) who reported that chickens fed on PG showed a better immunity against ND than non PG feeding group. Moreover, a preliminary study by Chansiripornchai et al. (2008) reported that adding PG to broiler chicken diet, as a feed additive, stimulated the humoral immune responses. Hokputsa et al. (2004) reported that the PG inhibited immunomodulating activity when estimated by complement fixation assay.

No significant difference was found in the H:L ratio between the experimental groups fed on PG and the control group (Fig 3). The Heterophil: Lymphocyte ratio is the ratio of heterophils on lymphocytes. An alteration of this value depends on the types and ages of animals and environment changes. When chickens are stressed, glucocorticoid hormone increases resulting in a decrease in lymphocytes (Puvadolpirod and Thaxton, 2000). In contrast to lymphocytes, when the heterophils increase, the H:L ratio also increases. The normal range of the H:L ratio of poultry is 0.33-0.5 (Gross,

1988). In this experiment, the H:L ratios were rather high, which might have been caused by the disturbance of the chickens by frequent serum collection and weighing in every week. Weighing chickens every week could cause stress, so glucocorticoid hormone increased and lymphocytes will decreased. At five weeks old, the H:L ratios of the chickens in all groups increased. This might have been caused by the noise from building work. Under stress condition, animals will release corticosteroids in the body, resulting in an increase in the mature neutrophil numbers (neutrophilia) with reductions in both the lymphocytes (lymphopenia) and eosinophils (eosinopenia) (Bush, 1991) or termed a stress leucogram. This is a transient change that occurs because there are shifts in the neutrophil from the marginal pool to the circulating pool. The stress leucogram may also be seen with corticosteroid administration (Barry, 1998). However, the chickens fed on 0 g% PG showed a higher H:L ratio than those chickens in the experimental groups. Thus, the chickens fed on a PG diet tended to have a better ability to resist stress than those chickens fed on a non PG diet.

At five and six weeks old, the total bacteria count and Salmonella suspected colony count in the chicken feces of the control group were significantly higher than those of the experimental groups (p < 0.05) (Table 1 and 2). Polysaccharide gel reduced Salmonella suspected colonies in chicken feces even at lower amounts of PG (1 g% PG). The polysaccharide gel has an antibacterial activity that restrains both Gram positive and Gram negative bacteria (Lipipun et al., 2002; Pholdaeng and Pongsamart, 2010). Therefore, the polysaccharide gel could reduce bacteria in chicken feces and the higher PG in the diet showed a better reduction in the total bacteria count in the feces. Polysaccharide gel is not absorbed through the stomach (Tippayakul et al., 2002), so it passes through the small intestine where it is firstly infected with Salmonella (Bangtrakulnonth, 2002). In addition, the physical properties of PG include an intrinsic acid condition with pH at 2.2-2.6 due to its acidic sugar component, especially galacturonic acid. The mechanism of inhibition of bacteria by PG may be related to the property of its acidic polygalacturonic acid chain in addition to adhesion reaction with neutral sugar side chains in the pectic polysaccharide (Hokputsa et al., 2004). Polysaccharide gel has an intrinsic viscosity and adhesive properties due to the electronegativity and branch chain neutral sugars of the pectic polysaccharide, which probably adhesively bind on the cell’s outer surface (Lipipun et al., 2002; Pongsamart et al., 2005). Moreover, polysaccharide gel may cause adhesion interference to the cell’s normal function and alter the membrane permeability (Tsai and Su, 1999).

No significant difference in cholesterol in the chickens’ plasma between the control and the experimental groups was found (Fig 4). However, the chickens fed commercial feed with PG tended to have a lower cholesterol level in the plasma than the chickens fed on normal feed. This result agrees with

Kitprathaung N. et al. / Thai J Vet Med. 2013. 43(2): 251-258. 257

Chansiripornchai et al. (2008) who discovered that mixing PG in the chicken diet could reduce the cholesterol level in serum. Moreover, the cholesterol levels in the muscle of the chickens fed on 3 g% PG was significantly lower than those of the chickens in the control group (p < 0.05) (Table 3). A preliminary study indicated that the level of cholesterol in muscle reduced when PG was added in feed as determined by a colorimetric method (Chansiripornchai et al., 2008). This method is based on the determination of cholestenone after enzymatic cleavage of the cholesterol ester, conversion of cholesterol, and the consequent formation of a red dyestuff after the reaction of 4-aminophenazone with phenol. The color intensity is directly proportional to the concentration of cholesterol and is determined photometrically (Allain et al., 1974).

Although the analysis method was different in this case, the result was the same. It was confirmed that PG in the chicken diet could potentially reduce the cholesterol in the chickens’ muscle. Polysaccharide gel can well confine liquid such as cholesterol and fatty acid by absorbing lipids and cholesterol in the alimentary canal resulting in less confinement of lipids and cholesterol in chickens (Tippayakul et al., 2002). The nutrient absorption rate depends on the rate at which nutrients are in contact with the absorptive epithelium layer (Pholdaeng and Pongsamart, 2010). Thus, such a high PG concentration may cause interference in the rate of nutrient absorption. The increased viscosity of the diet can likely retard nutrient absorption in the same way as the effect of dietary fiber (Fair et al., 1980). Too high a level of PG in the diets may not be appropriate because of the retardation of nutrient absorption in the digestive tract. For this reason, the cholesterol in the chickens fed a diet with PG was lower than that of the chickens fed a diet without PG. Therefore, polysaccharide gel can reduce cholesterol in chickens. Polysaccharide gel may be used as a feed-supplement for broiler chickens in order to reduce contaminated bacteria in the gastrointestinal tract and also decrease cholesterol in chickens’ muscle. In the future, polysaccharide gel in chickens may promote human health by reducing cholesterol level and cardiovascular diseases.

Acknowledgements

This work was financially supported by the

90thAnniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), Chulalongkorn University. We would like to thank the staff of the Program of Biotechnology, Faculty of Science, the Faculty of Pharmaceutical Sciences and Avian Health Research Unit, Faculty of Veterinary Sciences, Chulalongkorn University for their support.

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Thai J Vet Med. 2013. 43(2): 259-263.

Production of Black Goat Using Laparoscopic Artificial

Insemination and Embryo Transfer

Nitira Anakkul1 Junpen Suwimonteerabutr1 Theerawat Tharasanit1 Saritvich Panyaboriban1

Sarawanee Khunmanee2 Natthakarn Thanomsuksinchai2 Mongkol Techakumphu1*

Abstract

Laparoscopic artificial insemination (LAI) and embryo transfer (ET) were used to produce cross-bred black-

colored goat. In Experiment 1, LAI with frozen-thawed semen of black buck (Australian Melaan) was performed in 75% Saanen cross-bred does (white color, n = 70). The total numbers of 68 kids were born from 50 does. The skin colors of kids born were black (10.29%), white (39.71%) and other colors (50%). In Experiment 2, two cross-breeding programs were tested including program I: frozen semen of Australian Melaan inseminated to Black Bengal female (n = 7) and program II: frozen semen of Black Bengal inseminated to 50% Australian Melaan (n = 7). For embryo transfer program, the donors were superovulated and inseminated through laparoscopy with frozen semen at 21 hours after estrus. Thirty embryos at 4-8 cell stages (day 3) were surgically collected and transferred into 30 recipients (75% Saanen cross-breed) at approximately 60 hours following LAI. Pregnancy rates were 30%. Nine kids born from both programs were black in color with 2.56±0.95 kg birth weight. It is concluded that laparoscopic insemination and embryo transfer can be successfully combined to produce and sustain the genetic potential encoding the black colored skin.

Keywords: black goat, embryo transfer, laparoscopic artificial insemination 1Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand, 2The Office of the Commission on Agricultural Resource Education, Chulalongkorn University, Nan 55000, Thailand *Corresponding author: E-mail: tmongkol@chula.ac.th

Original Article

260 Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 259-263.

บทคดยอ

การผลตแพะดาดวยการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปและการยายฝากตวออน นธรา อนคกล 1 จนทรเพญ สวมลธระบตร 1 ธรวฒน ธาราศานต 1 สฤษฎวชญ ปญญาบรบาลบ 1 ศราวณ ขนมณ 2 ณฐกานต ถนอมสขสนชย 2 มงคล เตชะกาพ 1*

การศกษาในครงนไดนาเอาการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปและการยายฝากตวออนมาใชในการผสมขามพนธเพอผลตแพะสดา ในการทดลองท 1 ทาการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปโดยใชนาเชอแชแขงจากพอแพะสดาพนธออสเตรเลยนเมลานกบแพะสขาวพนธซาเนน (75%) จานวน 70 ตว แพะตงทอง 50 ตว ไดลกแพะจานวนรวม 68 ตว โดยเปนลกแพะสดารอยละ 10.29 สขาวรอยละ 39.71 และสอน ๆ รอยละ 50 ในการทดลองท 2 ทาการศกษาการผสมขามพนธใน 2 รปแบบ คอ รปแบบแรกทาการผสมเทยมแพะเพศเมยสดาพนธแบลคเบงกอลจานวน 7 ตวดวยนาเชอแชแขงจากพอแพะสดาพนธออสเตรเลยนเมลาน และรปแบบทสองทาการผสมเทยมแพะเพศเมยสดาซงเปนลกผสมออสเตรเลยนเมลาน (50%) จานวน 7 ตวดวยนาเชอแชแขงจากพอแพะสดาพนธแบลคเบงกอล โดยในการยายฝากตวออน แพะเพศเมยตวใหดงกลาวจะถกกระตนการตกไขและไดรบการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปท 21 ชวโมงหลงแสดงอาการเปนสด ทาการผาตดเกบตวออนภายหลงการผสมไปแลวประมาณ 60 ชวโมง ไดตวออนจานวน 30 ใบทระยะ 4 ถง 8 เซลล (อาย 3 วน) อตราการตงทองจากการยายฝากไปยงแพะตวรบจานวน 30 ตวเทากบรอยละ 30 ลกแพะทงหมด 9 ตวทผลตไดจากทงสองรปแบบการผสมมสดา มนาหนกแรกเกดเฉลย 2.56±0.95 กก. สามารถสรปไดวาการนาเอาการผสมเทยมดวยเทคนคสองกลองลาพาโรสโคปมาใชรวมกบการยายฝากตวออนประสบความสาเรจในการผลตและรกษาศกยภาพทางพนธกรรมในการรกษาลกษณะการแสดงออกของสดา

คาสาคญ: แพะสดา การยายฝากตวออน การผสมเทยมดวยเทคนคสองกลองลาพาโรสโคป 1 ภาควชา สตศาสตร เธนเวชวทยา และ วทยาการสบพนธ คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลยกรงเทพฯ 10330 2 สานกงานคณะกรรมการการศกษาวจยทรพยากรการเกษตร จฬาลงกรณมหาวทยาลย จงหวดนาน 55000 *ผรบผดชอบบทความ E-mail: tmongkol@chula.ac.th

Introduction

In Thailand, black goats are in demand and more expensive compared to other colors. However, when black goats are mated with goats in other colors, coat colors of offspring are remarkably variable (Asdell and Buchanan Smith, 1928). This is due to the fact that white, brown and tan colors are dominant over black color (Asdell and Buchanan Smith 1928; Adalsteinsson et al., 1994; Sponenberg and LaMarsh, 1996). Skin color is genetically controlled by the presence of two subtypes of melanin pigments (eumelanin and phaeomelanin). The eumelanin is predominantly produced from tyrosine and represents black color while the other pigment typically expresses reddish brown or tan color. Although melanocytes can produce both types of pigments, eumelanin is genetically recessive (Asdell and Buchanan Smith, 1928). In order to produce black offspring, mating between two breeds of black goats should be performed (Sponenberg et al., 1998). Australian Melaan (AM) is a black goat breed developed in Australia. This breed is considered hardy, disease resistant and high production. Black Bengal (BB) is a common name for a small breed black goat found in Bangladesh and Northeast India. Its

skin softness and excellent meat quality are main characteristics (Amin et al., 2000). Genetic improvement of the BB goat could be achieved by selection and cross-breeding (Amin et al., 2000).

Cross-breeding by the application of assisted reproduction technologies (ART) results in the utilization of hybrid vigor for commercial production. Artificial insemination (AI) is a basic tool for genetic improvement worldwide especially with frozen-thawed semen (Leboeuf et al., 2000). In order to get higher pregnancy rates with post-thaw semen, the spermatozoa should be deposited directly into the uterus using laparoscopic artificial insemination (LAI) (Sohnrey and Holtz, 2005). While this cross-bred LAI improves the genetics via male germplasm, superovulation followed by embryo recovery and embryo transfer (ET) is a successful method for distributing superior female genes (Isthwar and Memon, 1996). There are two techniques for collecting and transferring embryos, regarding the stage of embryo development. The embryos beyond eight-cell stage should be collected and transferred to the uterus, while early stage (embryos containing less than 8 cells) should be transferred into the oviducts (Ramon-Ugalde et al., 2008). A higher recovery rate and viability of embryos were reported when

Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 259-263. 261

embryos were collected in the early stage via oviductal flushing (Ramon-Ugalde et al., 2008). Therefore, this study aimed at examining whether or not the production of black goat offspring using LAI, superovulation as well as ET techniques were efficiently performed.

Materials and Methods Experimental animals: This study was approved by the Institutional Animal Care and Use Committee (IACUC), Chulalongkorn University (Approval No. 11310030). All female goats, aged between 2 to 4 years old, were used in this experiment. Animal facility was located at the CU-Network for Academic Opportunities and Services, Chulalongkorn University (Nan Province, Thailand, 18°N latitude and 100°E longitude). The animals were fed on concentrates containing 14% (w/w) protein, ad libitum of grass with free access to mineral salt blocks and water.

Experimental design:

Experiment 1: LAI with frozen-thawed semen of AM was performed in 75% Saanen cross-bred does (n = 70) in order to study the offspring’s coat color.

Experiment 2: Two cross-breeding programs were tested using LAI and ET techniques. These included program I: frozen semen of AM inseminated to BB female (n = 7) and program II: frozen semen of BB inseminated to 50% AM (black color does born from Experiment 1) (n = 7). Donors from both programs were inseminated using LAI technique. Embryos were collected at 60 hours after LAI and then transferred to recipients does (75% Saanen crossbred does).

Estrus synchronization: The does in experiment 1 (n = 70) were estrus synchronized using intravaginal sponge (Sincro-gest sponges; 65 mg of medroxyprogesterone, Ovejero®, Spain) for 13 days together with an injection of 300 IU pregnant mare serum gonadotropin (PMSG, Folligon®, Intervet Schering-Plough Animal Health, The Netherlands) before sponge removal.

The recipient does in experiment 2 (n = 40) were estrus synchronized at the same period as the donors. The program was as described above with an injection of 200 IU human chorionic gonadotropin (hCG; Chorulon®, Intervet Schering-Plough Animal Health, The Netherlands) when the estrus sign was detected in order to induce an ovulation.

Superovulation: The donors in Experiment 2 (n = 14) were synchronized and superovulated as previously described by Shin et al. (2008) and Lehloenya and Greyling (2010) with minor modifications. The estrous cycle of the donors was controlled by an intravaginal progesterone sponge (Sincro-gest sponges; 65 mg of Medroxyprogesterone, Ovejero®, Spain) for 13 days. A total of 200 mg of follicle stimulating hormone (FSH; Folltropin V®, Bioniche Animal Health, Vetrepharm, Canada) was divided into 7 doses (50, 25, 25, 25, 25, 25 and 25 mg). The FSH was then twice-daily administrated. The first dose was started on day 10 after the progesterone sponge insertion. Two doses of

cloprostenol (125 μg/ml, Estrumate®, Schering-Plough Animal Health, NJ, USA) were administered intramuscularly at the time of the sixth and seventh FSH injections. Estrus signs were detected every 6 hours using apronized bucks. When the does were in estrus, 200 IU of hCG (Chorulon®, Intervet Schering-Plough Animal Health, The Netherlands) was given to induce ovulation and the LAI was then performed 21 hours later.

Laparoscopic artificial insemination (LAI): All does submitted to the operation were restricted from feed for 24 hours and water for 12 hours (Mobini et al., 2012). They were intravenously anesthetized with xylazine HCl (0.1 mg/kg) and ketamine HCl (4.4 mg/kg). The analgesic drug (0.04 mg/kg phenylbutazone) and antibiotic (20,000 IU/kg penicillin-streptomycin) were also given intramuscularly. The does were positioned on a trolley in dorsal recumbency with the head tilted down at about 45° angle. After ovarian observation to confirm the presence of graafian follicles, post-thaw sperm (60 x 106 spermatozoa in 0.25 ml) were inseminated into the lumen of each uterine horn at halfway between the utero-tubular junction (UTJ) and the bifurcation (Ritar et al., 1990).

Embryo transfer (ET): Approximately 60 hours following AI, the embryos were surgically collected from the donors. The goats were anesthetized as described in LAI. The numbers of recovered embryos were recorded in relation to the number of corpus luteum (CL). The reproductive tract of a donor was accessed through a mid-ventral incision. Embryos at 4-8 cel l stages (day 3) were col lected from superovulated donors by oviductal flushing with sterile phosphate-buffered saline infiltrated through butterfly needle (25G, 19 mm) (Fig 1A and 1B). The flushing medium was collected via a polyethylene tube with outer diameter of 1.57 mm and inner diameter of 1.14 mm (Intramedic®, Becton, Dickinson a n d C o m p a n y , N J , U S A ) . T h e f l u s h i n g

Figure 1 Oviductal flushing in superovulated donors. A:

insertion of the polyethylene tube via fimbria and infundibulum and holding with fingers. B: infiltration the flushing medium at utero-tubular junction using a butterfly needle. C: insertion of an IVF catheter into the oviduct via the infundibulum of recipient to transfer the embryo. D: black kid was born by transfer the embryo to crossbred Saanen.

262 Anakkul N. et al. / Thai J Vet Med. 2013. 43(2): 259-263.

fluid was recovered in a sterile plastic Petri dish and the embryos were immediately searched and counted under a stereomicroscope (80x) (Nikon SMZ645, Japan). The recovered embryos were classified according to the gross morphology and only excellent and good embryos were used for ET. The fresh embryos were transferred into the oviducts of recipients ipsilateral to the presence of CL via the insertion of an IVF catheter (Veterinary Concepts®, Australia) into the oviduct (Fig 1C).

Pregnancy diagnosis: Pregnancy was confirmed at 45 days after LAI or ET by real time B-mode ultrasonography (HS-2000, Honda Electronics Co, Ltd, Japan). Skin color and birth weight of kids were recorded.

Statistical analysis: Data were descriptively analyzed. Percentage of goat showing estrus sign, pregnancy and embryo recovery are reported. The birth weights of kids are presented as mean±SD. The frequency of kids in each color was expressed as a percentage of the total number of kids born.

Results

Experiment 1: A total numbers of 68 kids were born from 50 does (pregnancy rates = 71.43%). The number of kids with black colored skin was lower than other colors (Table 1). Thirty-five does (70%) delivered one kid while the other 15 does gave birth to twins and triplets (12 and 3 does, respectively). Differences in colors were observed among twin and triplet. The average of birth weight was 2.87±0.61 kg.

Experiment 2: All donors and recipients does (35 from 40, 87.50%) came into estrus after synchronization. The mean number of CL (ovulation) in the donors was 13±6.6 (ranging from 8 to 32) while the recovery rate (unfertilized oocytes and embryos) varied from 0 to 100% (55.85±35.69%). The transferable embryos, at 2-8 cell stages when collected, were performed on day 3 after LAI. The number of embryos collected from two breeding programs (Table 2). Thirty good embryos from 11 donors (BB = 7, 50%AM = 4; 0% recovery rate in three donors) were transferred into 30 recipient does. The pregnancy rates were 30% (9/30). All nine kids were healthy and black in color (Fig 1D) with 2.56±0.95 kg birth weight.

Discussion

We found that the LAI and ET were successfully performed to maintain the phenotypic expression of black offspring when black goats were mated together. Therefore, when frozen semen of AM was inseminated to white Saanen does, the lower percentages of black kids were born as shown in experiment 1. This is due to the recessive eumelanin which controls black color, mating between black and black goats produces black offspring whereas mating between black goats and goats of other colors results in variable coat colors (Asdell and Smith, 1926; Adalsteinsson et al., 1994; Sponenberg et al., 1998).

In the embryo collection, the wide range of the success in recovery rate was in agreement with a

previous study, indicating that the results can vary from complete failure to extremely high success (Baldassarre and Karatzas, 2004). It was noted that high recovery rates were mostly obtained in does that had never received hormonal treatment. Moreover, we found that repeated treatments especially for PMSG and FSH (porcine origin) negatively affected the ovarian responses and also embryo recovery rate as remarked by Remy et al. (1991) and Cognie (1999). In addition, internal organ adhesion from previous LAI and surgical ET also impair the procedure during embryo collection (Suyadi et al., 2000).

All black offspring born from both breeding programs were healthy with higher birth weight (2.56±0.95) than the average birth weight of BB kids born at this breeding center (1.81±0.26 kg) or in Bangladesh (1.08±0.06 kg, Paul et al., 2011) but slightly lower than kids born in Experiment 1 (2.87±0.61). This black colored skin is controlled by the production of melanin pigments from melanocytes in response to the alpha melanocyte stimulating hormone (Jackson, 1994; Hoekstra, 2006). Although melanocytes can produce both eumelanins and phaeomelanins, the phenotypic expression of the black colored hair and skin is mediated by only eumelanin. While the mechanism that controls the hair and skin color is well described in mouse, similar mechanisms remain to be studied in goat. Practically, our study demonstrated that crossbred black goats can be produced via LAI and ET. The production of these cross-bred goats would be phenotypically and genetically beneficial means of climate adaptation, disease tolerance and also meat production. LAI and ET combined technique provides valuable practical opportunity to improve reproductive efficiency and to enhance genetic improvements.

Table 1 Skin colors of goat offspring from Australian Melaan (frozen semen) and 75% Saanen cross-bred does.

Skin Offspring

Black White Other Total

n 7 27 34 68 % 10.29 39.71 50

Table 2 Number of embryos collected from two breeding programs.

Breeding program

Frozen semen

Donor No. of donors

No. of embryos

No. of

kids 1 AM BB 7/7 18 6 2 BB 50%AM 4/7* 12 3

AM: Australian Melaan, BB: Black Bengal *Recovery rate: 0 in three donors

Acknowledgements

This work was supported by the grant of Chulalongkorn University Centenary Academic Development Project, RGJ-PhD-industrial link program, Thailand Research Fund (PHD/0156/2550) and The 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund).

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References Adalsteinsson S, Sponenberg DP, Alexieva S and Russel

AJF 1994. Inheritance of goat coat colors. J Hered. 85: 267-272.

Amin MR, Husain SS and Islam ABMM 2000. Evaluation of Black Bengal goats and their cross with the Jamunapari breed for carcass characteristics. Small Rumin Res. 38: 211-215.

Asdell SA and Buchanan Smith AD 1928. Inheritance of color, beard, tassels and horn in the goat. J Hered. 19: 425-430.

Baldassarre H and Karatzas CN 2004. Advanced assisted reproduction technologies (ART) in goats. Anim Reprod Sci. 82–83: 255–266.

Cognie Y 1999. State of the art in sheep-goat embryo transfer. Theriogenology 51: 105-116.

Hoekstra HE 2006. Genetics, development and evolution of adaptive pigmentation in vertebrates. Heredity 97: 222–234.

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Jackson IJ 1994. Molecular and developmental genetics of mouse coat color. Annu Rev Genet. 28: 189-217.

Leboeuf B, Restall B and Salomon S 2000. Production and storage of goat semen for artificial insemination. Anim Reprod Sci. 62: 113–141.

Lehloenya KC and Greyling JPC 2010. The ovarian response and embryo recovery rate in Boer goat does following different superovulation protocols, during the breeding season. Small Rumin Res. 88: 38-43.

Mobini S, Heath AM and Pugh DG 2012. Theriogenology of sheep and goats. In: Sheep and Goat Medicine. 2nd ed. DG Pugh and N Baird (eds). USA: WB Saunders Company. 129-186.

Paul S, Khandoker MAMY, Moinuddin MA and Paul RC 2011. Characterization of Black Bengal goat. J Bangladesh Agril Univ. 9: 61-66.

Ramon-Ugalde JP, Folch J, Cocero MJ, Piña-Aguilar RE and Alabart, J.L. 2008. Embryo recovery from the oviduct in superovulated ewes: A method to improve MOET systems. Czech J Anim Sci. 53: 145–151.

Remy B, Baril G, Vallet JC, Dufour R, Chouvet C, Saumande J, Chupin D and Beckers JF 1991. Are antibodies responsible for a decreased superovulatory response in goats which have been treated repeatedly with porcine follicle-stimulating hormone? Theriogenology 36: 389-399.

Ritar AJ, Ball PD and O’May PJ 1990. Examination of methods for the deep freezing of goat semen. Reprod Fertil Dev. 2: 27-34.

Shin ST, Jang SK, Yang HS, Lee OK, Shim YH, Choi WI, Lee DS, Lee GS, Cho JK, Lee YW 2008. Laparoscopy vs. laparotomy for embryo transfer to produce transgenic goats (Capra hircus). J Vet Sci. 9: 103-107.

Sohnrey B and Holtz W 2005. Technical Note: Transcervical deep corneal insemination of goats. J Anim Sci. 83: 1543-1548.

Sponenberg DP, Alexieva S and Adalsteinsson S 1998. Inheritance of color in Angora goats. Genet Sel Evol. 30: 385-395.

Sponenberg DP and LaMarsh C 1996. Dominant and recessive brown in goats. Gen Sel Evol. 28: 117-120.

Suyadi, Sohnrey B and Holtz W 2000. Transcervical embryo collection in Boer goats. Small Rumin Res. 36: 195-200.

264

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Prevalence of Salmonella enterica, Escherichia coli and

Staphylococcus aureus in Raw Meat in Thai Self-Service Style

Restaurants in Khon Kaen Municipality

Sunpetch Angkititrakul1 Arunee Polpakdee1 Rungtip Chuanchuen 2*

Abstract

One hundred and fifty eight raw meat samples were collected from 33 Thai self-service style restaurants in

Khon Kaen municipality during July-October 2011. The fresh meat included pork, beef and chicken and was provided for self-grilling or boiling. All the samples were examined for contamination of three major foodborne pathogens including Salmonella enterica, Escherichia coli and Staphylococcus aureus using standard protocol. High prevalence of S. enterica (44%), E. coli (60%) and S. aureus (25%) was observed. Seventy-seven percent of the samples were positive for at least one of the three pathogens, while only 8% were found to carry all three pathogens. The prevalence of Salmonella, S. aureus and E. coli was observed in different rates in pork (59%, 69% and 33%, respectively), beef (52%, 56% and 28%, respectively) and chicken (18%, 56% and 12%, respectively). When consider meat type, the highest contamination frequency was found in pork (87%), followed by beef (78%) and chicken (64%). The results indicate the requirement of quality and hygiene control policy for raw meat served in self-service restaurants and that public awareness of food poisoning caused by consumption of undercooked meat should be raised.

Keywords: Escherichia coli, Khon Kaen, raw meat, Salmonella enterica, Staphylococcus aureus 1 Department of Veterinary Public Health, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand. 2 Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok, Thailand *Correspondence author E-mail: rchuanchuen@yahoo.com

Short Communication

266 Angkititrakul S. et al. / Thai J Vet Med. 2013. 43(2): 265-268.

บทคดยอ

ความชกของ Salmonella enterica, Escherichia coli และ Staphylococcus aureus ในเนอดบในรานอาหารบรการตนเองแบบไทยในเทศบาลเมองขอนแกน

สรรเพชญ องกตตระกล 1 อรณ พลภกด 1 รงทพย ชวนชน 2*

เกบตวอยางเนอดบจานวน 158 ตวอยางจากรานอาหารไทยแบบปรงดวยตนเองจานวน 33 รานในเขตเทศบาลเมองขอนแกน

ระหวางเดอนกรกฎาคมถงตลาคม 2554 โดยเกบตวอยางเนอสกร เนอวว และเนอไกทพรอมเสรฟเพอใหยางหรอตมดวยตนเอง ตรวจหาการปนเปอนของเชอกอโรคอาหารเปนพษทสาคญ 3 ชนด คอ Salmonella enterica, Escherichia coli และ Staphylococcus aureus ในตวอยางทงหมดดวยวธมาตรฐาน โดยตรวจพบการปนเปอน S. enterica รอยละ 44 E. coli รอยละ 60 และ S. aureus รอยละ 25 โดยรอยละ 77 ของตวอยางใหผลบวกตอเชออยางนอย 1 ชนด ในขณะทตวอยางจานวนรอยละ 8 มเชอทง 3 ชนด ตรวจพบ Salmonella, S. aureus และ E. coli ในเนอสกร (รอยละ 59, 69 และ 33 ตามลาดบ) เนอวว (รอยละ 52, 56 และ 28 ตามลาดบ) และเนอไก (รอยละ 18, 56 และ 12 ตามลาดบ) ในอตราทแตกตางกน เมอพจารณาชนดของเนอสตวพบวาเนอสกร (รอยละ 87) มอตราการปนเปอนสงสดตามดวยเนอวว (รอยละ 78) และเนอไก (รอยละ 64) ผลการวจยชใหเหนถงความจาเปนของการควบคมคณภาพและความปลอดภยของเนอดบทจาหนายในรานอาหารไทยแบบปรงดวยตนเอง รวมทงการกระตนใหมความระมดระวงเกยวกบโรคอาหารเปนพษทมสาเหตมาจากการบรโภคเนอสตวทปรงไมสก

คาสาคญ: เอสเชอรเชย คอลย ขอนแกน เนอดบ ซลโมเนลลา เอนเทอรกา สตาฟฟโลคอคคส ออเรยส 1 ภาควชาสตวแพทยสาธารณสข คณะสตวแพทยศาสตร มหาวทยาลยขอนแกน อ.เมอง ขอนแกน 40000 2 ภาควชาสตวแพทยสาธารณสข คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330

*ผรบผดชอบบทความ E-mail: rchuanchuen@yahoo.com

Introduction Meat flesh is food of animal origin, of which consumption varies, depending on cultural or religious preferences. As seen in most parts of the world, meat particularly pork, beef and chicken is a major part of daily diet for Thai people. Meat is an excellent source of protein, various vitamins (e.g. vitamin B12, niacin and vitamin B6) and minerals (e.g. iron, zinc and phosphorus) (Romans, et al., 1994). Due to its high nutrient content, meat is perishable food that requires proper package, storage, handle and cooking to guarantee safety for consumers. Public health concern is that meat is a major source of foodborne pathogens, for example, Salmonella enterica, Campylobacter spp., Escherichia coli and Staphylococcus aureus (Nyachuba, 2010). In general, foods at high risk of contamination with these pathogens are those made by hand and require cold storage. Therefore, most cases of foodborne illness arise from improper food handling, preparation and storage. Salmonella, E. coli and S. aureus are also indicators for microbiological quality of raw meat (DMSC, 2010). Based on the current microbiological standard for raw meat described in the microbiological quality of food and relevant-food containers issued by Department of Medical Science, Ministry of Public Health in 2010, Salmonella must be absent in 25 g raw meat. The presence of E. coli must not exceed 100 MPN/g and

that of S. aureus must not exceed 100 colonies/g (DMSC, 2010).

Self-grilling (Moo-Ka-Ta) and -boiling (Moo-Joom) Thai style restaurants have gained popularity among Thai people, especially teenagers and working people due to inexpensive price and various choices of meat. A variety of raw meat flesh is served for grilling on non-stick hot pan or boiling in soup by customers; however, such raw meat may be handled with poor hygiene in certain circumstances. Undercooked meat may be occasionally eaten by consumers due to personal preference, quick-cooking and/or poor cooking pan quality. Cross-contamination between raw meat and cooked meat may also occur. These could promote microbial spread and result in an increased risk of becoming ill from contaminated foodborne pathogens. The aim of this study was to determine the presence of Salmonella, E. coli and S. aureus in raw meat available in Thai self-service style restaurants in Khon Kaen municipality where this particular type of Thai restaurant is very common.

Materials and Methods Sample collection: one hundred and fifty eight raw meat samples including pork (n = 54), beef (n = 54)

Angkititrakul S. et al. / Thai J Vet Med. 2013. 43(2): 265-268. 267

and chicken (n = 50) were obtained from 33 Thai self-service style restaurants in Khon Kaen municipality during July-October 2011. Each restaurant was visited for sample collection on one or two separate occasions, depending on permission. The samples were randomly collected from dishes served on tables or buffet bars. Each sample was placed in individual plastic bag, stored on ice and immediately delivered to the laboratory of Department of Veterinary Public Health, Faculty of Veterinary Science, Khon Kaen University for bacterial isolation and identification.

Bacterial isolation and identification: All the raw meat samples were examined for the presence of Salmonella by using ISO6579:2002(E) (ISO, 2002). Contamination of E. coli and S. aureus was determined on relevant PetriflimTM count plate (3M, 3M Microbiology, St. Paul, MN, USA) as described in AOAC Official Method 998.08 (AOAC, 2000) and 2003.11 (AOAC, 2000), respectively.

Briefly, 25 g of each sample was pre-enriched in Buffer Peptone Water (BPW, Merck, Darmstadt, Germany) at 370C overnight for Salmonella isolation. The suspension was spotted on Modified Semisolid Rappaport medium (Merck) and incubated at 420C for 24 hours. Then, Salmonella was isolated on Xylose-Lysine-Desocholate agar (Merck) and Hektoen Enteric agar (Merck) at 370C for 24 hours. Five typical colonies of Salmonella from each positive sample were biochemically confirmed using Triple Sugar Iron agar (TSI, Merck) and Motility Indole-Lysine agar (Merck).

For E. coli and S. aureus, 50 g sample was placed in BPW and the suspension was serially diluted in BPW to 10-3. One-ml was plated onto PetriflimTM E. coli/ Coliform count plate (3M) and PetrifilmTM Staph Express Count plate (3M) for determination of E. coli and S. aureus, respectively. The inoculated petrifilms were incubated at 370C for 24 hours. The samples were considered positive when there was the presence of Salmonella in 25 g raw meat and/or E. coli and S. aureus counts exceeded 100 colonies/g.

Results and Discussion One of the major findings of this study was the high prevalence of three major foodborne pathogens, Salmonella, E. coli and S. aureus in raw meat

served in Thai self-service style restaurants (Table 1) and none of the restaurants carried raw meat negative for all three pathogens. Of all the raw meat samples tested, 77% were positive for at least one of the three pathogens. Only 8% were found to carry all three pathogens tested (Table 2). Combination of Salmonella and E. coli was most commonly found (22%), followed by the presence of E. coli only (21%). These observations confirm the potential role of raw meat served in Thai self-service style restaurants as vehicle for human foodborne diseases.

When consider meat type, the highest contamination frequency was found in pork (87%), followed by beef (78%) and chicken (64%). It is not surprising to observe such high prevalence of contamination. This could be explained by inadequate sanitation in fresh meat production. Pork and beef were originated from local slaughterhouses and sold in community fresh markets. In contrast, the restaurant owners claimed that most raw chicken was frozen-flesh meat produced by poultry-production companies supplied by local distributors. Even though the contamination rate in chicken was lower than that in the others, it is still considered high for frozen chicken produced by standard poultry producers. This indicates that cross-contamination during raw meat preparation occurred and certain restaurants still purchased raw chicken that was home slaughtered and made available at community markets.

Most samples in this study carried E. coli exceeding 100 CFU/g (60%), predominantly in all types of meat i.e. 69% of the pork, 56% of the beef and 56% of the chicken. While most pork (30%) and beef (26%) carried both Salmonella and E. coli simultaneously, most chicken (36%) were positive for E. coli only. E. coli is normal flora in gastrointestinal tract and serves as a common indicator for fecal contamination. Therefore, the presence of E. coli in food suggests poor handling, poor hygiene practices and/or poor temperature control for storage. Salmonella is very common in farm animals and food contamination with this pathogen could happen throughout food chain. Regardless, such existence of Salmonella and E. coli confirmed the risk of infection through ingestion of raw meat served in Thai self-service style restaurants.

Table 1 Prevalence of Salmonella, Escherichia coli and Staphylococcus aureus in raw meat (n = 158)

No. (%) of positive samples Raw meat No. (%)

Salmonellaa E. colib S. aureusc

Pork Beef

Chicken

54 (34) 54 (34) 50 (32)

32 (59) 28 (52) 9 (18)

37 (69) 30 (56) 28 (56)

18 (33) 15 (28) 6 (12)

Total 158 (100) 69 (44) 95 (60.13) 39 (25) aNumber of samples with the presence of Salmonella in 25 g bNumber of samples with E. coli counts exceeding 100 colonies/g cNumber of samples with S. aureus counts exceeding 100 colonies/g

268 Angkititrakul S. et al. / Thai J Vet Med. 2013. 43(2): 265-268.

Table 2 Distribution of Salmonella, Escherchia coli and Staphylococcus aureus in raw meat (n = 158)

No. (%) of positive samples Raw meat No. (%)

Sa only Sa+E Sa+St E+St E only St only All None

Pork Beef Chicken

54(34) 54(34) 50(32)

5(9) 5(9) 3(6)

16(30) 14(26) 5(10)

4(7) 5(9) 0

7(13) 4(7) 4(8)

7(13) 8(15) 18(36)

1(2) 2(4) 1(2)

7(13) 4(7) 1(2)

7(13) 12(22) 18(36)

Total 158(100) 13(8) 35(22) 9(6) 15(9) 33(21) 4(3) 12(8) 37(23) Sa: Salmonella, E: Escherichia coli, St: Staphylococcus aureus

It should be noted that E. coli detection in this study derived from colony enumeration while the MPN-based method is described in the current microbiological standard. This may limit data comparison due to discrepancy between these two measurements. Comparison of samples from different analytical procedures may be made by using probabilistic models (Gronewold and Wolpert, 2008). However, it was not performed in this study.

In this study, Salmonella was present in 18% of the chicken, much less than that in pork (59%) and beef (52%). This is inconsistent with previous studies reporting the high occurrence of Salmonella in chicken (75%), pork (65%) and beef (67%) sold in local fresh market (75%) (Angkititrakul et al., 2005). Based on the claim that raw chicken used in the restaurants were supplied by frozen-chicken manufacturers, this flesh should be Salmonella free. Once again, the presence of Salmonella in raw chicken confirmed cross-contamination during meat preparation process in the restaurant kitchens and/or unhygienic chicken meat supplies.

When compared to other bacteria tested, S. aureus was found with the least frequency in all kinds of meat. The presence of S. aureus is an indicator of poor hygiene and sanitation of food handlers. Additionally, contamination of raw meat may occur during slaughtering process as shown in a previous report of the presence of S. aureus on pig skin at pig slaughterhouses and pork at fresh markets (Theerapong and Angkititrakul, 2011). S. aureus is not always pathogenic and commonly found on skin and in respiratory tract of healthy people. Its large presence in food may indicate hygiene and sanitation deficit among food handlers. However, it is not sufficient evidence to incriminate food as the cause of food poisoning because the pathogen causes disease by producing different dreadful toxins that are heat stable and not destroyed by cooking (Murray, 2005). Therefore, enterotoxins must always be tested to trace the cause of illness associated with S. aureus.

In conclusion, the results indicate that raw meat served in Thai self-service style restaurants is an important source of foodborne pathogens and necessitates the basic food safety education for food handlers and other relevant workers. Good hygiene practices must be carried out before, during, and after food preparation. Relevant governmental authorities should routinely monitor food to ensure their quality and safety. Importantly, consumers should be aware of the risks associated with consumption of undercooked meat and educated in safe food handling, particularly when eating outdoor.

Acknowledgements We thank Khon Kaen municipal officers for

sample collection. We also thank D. Sithigo, J. Phakam, C. Chuntarakoat, S. Liangchaisiri, P. Khumphui, S. Boonpluk, P. Chompukhow, C. Tessrimuang, C. Jaturakomol and P. Laobannuae, Faculty of Veterinary Science, Khon Kaen University for sample collection and laboratory assistance. This work was partially supported by Thailand Research Fund MRG5180154.

References

Angkititrakul S, Chomvarin C, Chaita T, Kanistanon K and Waethewutajarn S 2005. Epidemiology of antimicrobial resistance in Salmonella isolated from pork, chicken meat and humans in Thailand. Southeast Asian J Trop Med Public Health. 36(6): 1510-1515.

AOAC 2000. 3MTM PetrifilmTM Staph Express Count Plate Method for the Enumeration of Staphylococcus aureus in Selected Types of Meat, Seafood, and Poultry. AOAC Official Method 2003.11: AOAC International, MD, USA. 2 pp.

AOAC 2000. Escherichia coli Counts in Poultry, Meats, and Seafood, Dry Rehydratable Film Method (Petrifilm EC Plate Method). AOAC Official Method 998.08: AOAC International, MD, USA.

DMSC 2010. Microbiological quality of food and food containers. Department of Medical Science, Ministry of Public Health, Nonthaburi. volume 2. 6 pp.

Gronewold AD and Wolpert RL 2008. Modeling the relationship between most probable number (MPN) and colony-forming unit (CFU) estimates of fecal coliform concentration. Water. Res. 42(13): 3327-3334.

ISO 2002. Microbiology of food and animal feeding stuffs-Horizontal method for the detection of Salmonella spp: ISO6579. 1-27 pp.

Murray RJ 2005. Recognition and management of Staphylococcus aureus toxin-mediated disease. Intern Med J. 35 (Suppl 2): S106-119.

Nyachuba DG 2010. Foodborne illness: Is it on the rise? Nutr Rev. 68(5): 257-269.

Romans J, Costello WJ, Carlson CW, Greaser ML and Jones KW 1994. The Meat We Eat: Interstate Pub Inc, Damville, IL, USA. 1193 pp.

Theerapong J and Angkititrakul S 2011. Contamination of Staphylococcus aureus in pork and palms of butchers at fresh-food markets in Khon Kaen municipality. KKU Vet J. 21(2): 147-153.

Thai J Vet Med. 2013. 43(2): 269-272.

Coccidiosis in a Greater Slow loris (Nycticebus coucang)

Wijit Banlunara 1* Sawang Kesdangsakonwut 1 Piyaporn Kongmekee 2

Angkana Sommanustweechai 2

Abstract

Since Eimeria pachylepyron was firstly described in slow loris (Nycticebus coucang) in 1972, there has not been

any reports of the disease throughout the world. An adult male 0.8 kg bw greater slow loris (Nycticebus coucang) had diarrhea, emaciation and dead. Severe necro-hemorrhagic jejunitis with the presence of coccidian protozoa and secondary bacterial septicemia were the causes of death. Endogenous stages of E. pachylepyron were detected exclusively in the jejunal enterocytes especially in the crypt of Lieberkühn. The typical round unsporulated oocysts with homogeneous eosinophilic thick wall were closely related to E. pachylepyron. This is the first report to confirm the existence of the protozoan as the pathologic disease in the slow loris.

Keywords: Eimeria pachylepyron, non-human primate, pathology, oocyst, Thailand 1 Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330 Thailand 2 Conservation Research and Education Division, Zoological Park Organization of Thailand, Dusit, Bangkok 10300 Thailand *Correspondence author E-mail: wijit.k@chula.ac.th

Short Communication

270 Banlunara W. et al. / Thai J Vet Med. 2013. 43(2): 269-272.

บทคดยอ

โรคบดในลงลม

วจตร บรรลนารา 1* สวาง เกษแดงสกลวฒ 1 ปยะพร คงเมค 2 องคณา สมนสทวชย 2

ตงแตมการคนพบเชอบดชนด อยเมอเรย พาคเลไพรอนในนางอาย เมอป พ.ศ. 2515 ไมมรายงานการเกดโรคนในโลกอก ลงลมโตเตมวย เพศผ นาหนก 0.8 กก. แสดงอาการทางคลนก ทองเสย ผอมแหง และเสยชวต สาเหตของการเสยชวตของนางอายเกดจากลาไสเลกอกเสบแบบเนอตายปนเลอดรวมกบบดและตดเชอแบคทเรยแทรกซอนเขากระแสเลอด ระยะตางๆของเชอบด พบในเซลลเยอบของลาไสเลกสวนกลางโดยเฉพาะสวนเซลลออนทฐาน โอโอซสตทยงไมสมบรณมลกษณะเฉพาะตวทมเปลอกหนาเหมอนกบเชอบดชนด อยเมอเรย พาคเลไพรอน ผลการศกษานเปนรายงานครงแรกทยนยนการมอยจรงของโรคบดชนดน และกอรอยโรคทางพยาธวทยาในลงลม

คาสาคญ: ไอเมอเรย พาคเลไพรอน สตวตระกลลง โอโอซสต ประเทศไทย 1 ภาควชาพยาธวทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 2 สานกอนรกษ วจยและศกษา องคการสวนสตวในพระบรมราชปถมป ดสต กรงเทพฯ 10300

*ผรบผดชอบบทความ E-mail: wijit.k@chula.ac.th

Introduction Few coccidian species are identified from the family Loridae (loris) of non-human primates. There are only three species; Eimeria coucangi, E. nycticebi and E. pachylepyron, which were found in slow lorises’ (Nycticebus coucang) feces (Patnaik and Acharjyo, 1970; Colley and Mullin, 1972; Duszynski et al., 1999). The morphology of sporulated E. pachylepyron oocyst has a typically rough, irregular striated outer thick wall. However, E. lemuris oocysts that were detected from feces of Senegal babybush (Galago senegalensis) also have thick wall (Poelma, 1966). Until now, there has not been any report of coccidiosis that was caused by these coccidian protozoa in the slow loris worldwide.

This study is the first report of pathology of the E. pachylepyron infection in a greater slow loris (Nycticebus coucang).

Materials and Methods An adult male greater slow loris (Nycticebus coucang) was given by people who reared it as pet to the opened Khao Keaw Zoo, Chon buri province, Thailand. The animal was clinically emaciated with diarrhea and severe dehydration. The body weight was 0.8 kg. It died in the quarantine area in May 2009. Routine necropsy was performed. The organs were fixed in 10% buffered formalin solution and were allowed to fix for 24 hours. Fixed tissues were routinely processed for histology in paraffin embedding and 4 μm thickness tissue sections were stained with hematoxylin and eosin (H&E). The special stainings, particularly periodic acid Schiff’s reaction (PAS) and alcian blue, pH 2.5, were performed at the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University,

Thailand. The preservative solution of the case was re-collected, centrifuged and observed for oocysts.

Results and Discussion The gross pathological findings

demonstrated severe mucohemorrhagic enteritis along the jejunum and ileum that were associated with intussusception of the jejunal part (Fig 1). The carcass was dehydrated carcass with a decrease subcutaneous fat. The liver and spleen were dark red and swollen referring to congestion. Pulmonary congestion was also found.

H i s t o p a t h o l o g i c a l l y , s e v e r e n e c r o -hemorrhagic jejunitis was exclusively detected. All gametogony stages; gametocytes, zygotes and

Figure 1 The lumen of jejunum showed muco-hemorrhagic

content and congestion of intestinal mucosa. Ruler in centimeters.

Banlunara W. et al. / Thai J Vet Med. 2013. 43(2): 269-272. 271

Figure 2 Various endogenous stages of the coccidian protozoa; gametocytes, unsporulated oocysts, present in the enterocytes. Bar = 100 μm

Figure 3 The unsporulated oocysts presented among the sloughed epithelial cells, bacteria and necrotic debris in the intestinal lumen. H&E stain (inset). A formalin-fixed unsporulated oocyst is spheroid with outer thick wall. Bar = 20 μm

oocyst as well as schizonts of E. pachylepyron were presented in the enterocytes (Fig 2). Many unsporulated oocysts were presented in the intestinal lumen with severely sloughed epithelial cells, bacteria and necrotic debri. Moreover, the round unsporulated oocysts of these coccidian protozoa were distinctly irregular to knobbed homogeneous eosinophilic thick outer layer and thin, translucently, smooth inner layer of the oocyst wall (Fig 3). The diameter of round oocysts including with the thick wall in the histologic section were 32±9 μm on average (ranging from 28 to 46 μm). The outer layer and the inner layer walls were 6-8 μm and 1 μm thick, respectively with the central round unsporulated oocyst. The outer layer of oocyst wall was strongly PAS-positive despite alcian-blue-negative staining. The size of unsporulated oocysts in formalin solution was spheroid and slightly larger than theirs on histological sections. Most enterocytes throughout the villi were parasitized by immature oocysts, schizonts and macrogamonts, especially the crypt of Lieberkühn was more severely parasitized. A cluster of 2-3 immature oocysts and macrogamonts with eosinophilic wall-forming bodies were commonly found in each enterocyte. The immature oocysts and macrogamonts were approximately 11-41 and 40-46 μm in diameter, respectively. The schizonts had thick eosinophilic wall with numerous

merozoites. The size of schizonts was approximately 70-95 μm and contained numerous small, 1-1.5 μm long, sickle shaped merozoites (Fig 4).

Other histopathological findings were multifocal coagulative necrotic suppurative hepatitis, mild suppurative splenitis with moderate lymphoid necrosis and mild membrano-proliferative glomerulonephritis. There were no pathological findings in the duodenum, lung, heart and stomach.

Previously, all eimeria species in slow loris were retrieved from the feces and intestinal scraping (Patnaik and Acharjyo, 1970; Colley and Mullin, 1972; Duszynski et al., 1999). Since E. pachylepyron oocysts were reported from the fecal survey in Malaysia in 1972, there have not been further reports about this coccidian (Colley and Mullin, 1972; Duszynski et al., 1999). This present case was accidentally found in the routine necropsy. The pathomorphology of the unsporulated oocysts was closely similar to E. pachylepyron sporulated oocysts than E. lemuris sporulated oocysts (Poelma, 1966; Colley and Mullin, 1972). The thick wall of round unsporulated oocysts differed from E. lemuris (Poelma, 1966). In addition, this pet was not exposed to any apes of African origin that are the host of E. kemuris.

The unsporulated oocysts of this coccidian, which had distinctly irregular eosinophilic thick outer layer of the oocyst, were easily seen in the histopathological sections. The thick outer wall was composed of neutral mucopolysaccharides composition, which was strongly stained by PAS. The size of the unsporulated oocysts in this case was slightly larger than the previous report, which sporulated oocysts were found in the immature stage. Most parasitic organisms in the case were in the gametogony stage and the enterocytes were severely affected. The cycles of this coccidian replication were not declared from the case. Most epithelial cells were possibly damaged and also led to secondary bacterial infection and caused septicemia. Unfortunately, bacterial identification was not successfully cultured. However, the cause of death might be coccidiosis with secondary bacterial septicemia because the liver showed severe hepatic necrosis.

Figure 4 Immature oocysts (arrows), macrogamonts (arrowheads) and schizonts (S) predominantly presented in the crypt of Lieberkühn. Bar = 50 μm

272 Banlunara W. et al. / Thai J Vet Med. 2013. 43(2): 269-272.

Based on the pathomorphology of the coccidian, this report revealed that the site of E. pachylepyron infection was the jejunum, which could possibly be the primary site for replication. This is the first report to confirm the existence of the protozoan and also the pathology in a greater slow loris (Nycticebus coucang).

Acknowledgements The authors would like to thank Mr.

Supradit Wangnitham, the technician, for histopathological laboratory assistance.

References Colley FC and Mullin SW 1972. Eimeria pachylepyron sp.

n. (Protozoa: Eimeriidae) from the slow loris in Malaysia. J Parasitol. 58: 110-111.

Duszynski DW, Wilson WD, Upton SJ and Levine ND 1999. Coccidia (Apicomplexa: Eimeriidae) in the primates and Scandentia. Int J Primatol. 20: 761-797.

Mullin SW, Colley FC and Welch QB 1975. Ecological studies on coccidia of Malaysian forest mammals. Southeast Asian J Trop Med Public Health. 6: 93-98.

Patnaik MM and Acharjyo LN 1970. Eimeria nycticebi n. sp. and E. coucangi n. sp. from Indian slow loris (Nycticebus coucang), and notes on Isospora leonina from an African lion (Panthera leo leo). Orissa Vet J. 5: 13-14.

Poelma FG 1966. Eimeria lemuris n. sp., E. galago n. sp. and E. otolicni n. sp. from a Galago Galago senegalensis. J Protozool. 13: 547-549.

Thai J Vet Med. 2013. 43(2): 273-277.

Histological Characteristics of Auditory Tube Diverticulum of

Domestic Donkey (Equus asinus)

Seyed Morteza Ghazi1 Behzad Mobini2*

Abstract

The purpose of this study was to investigate histological characteristics of auditory tube diverticulum in

domestic donkeys (Equus asinus) using conventional histological and histochemical methods. Specimens were collected from twenty regions (five different locations of each compartment of auditory diverticulum). The mucosa consisted of pseudostratified columnar epithelium with brush-like cilia and forms of some non-isometric folds. The few goblet cells, which were irregularly distributed over the whole luminal surface, reacted strongly with Alcian blue pH 2.5, whereas neutral mucosubstances were not observed. The lamina muscularis mucosa was absent. The lamina propria-submucosa contained all connective tissue fibers, tubulo-acinar glands, parasympathetic ganglia, nerve bundles, numerous diffuse or nodular lymphatic tissues, blood vessels, adipose tissue and skeletal muscles. There were some differences in type of epithelium and submucosal connective tissue, frequency of goblet cell and seromucosal gland between sexes and among the various regions.

Keywords: auditory tube diverticulum, domestic donkey (Equus asinus), histology, sex 1 Graduated from School of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, P.O.Box 166, Shahrekord- Iran. 2Department of Basic Sciences, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, P.O. Box: 166. Shahrekord, Iran *Correspondence author E-mail: dr.mobini@iaushk.ac.ir

Short Communication

274 Ghazi, S.M. & MobiniB. / Thai J Vet Med. 2013. 43(2): 273-277.

บทคดยอ

ลกษณะทางจลกายวภาคของ diverticulum ของทอหลา (Equus asinus)

Seyed Morteza Ghazi1 Behzad Mobini2*

วตถประสงคของการศกษานเพอศกษาลกษณะทางจลกายวภาคของ diverticulum ของทอหลาเลยง (Equus asinus) โดยใชวธการทางเนอเยอวทยาและฮสโตเคม เกบตวอยางรวบรวมจากยสบภมภาค (จากหาตาแหนงทตางกนของชองหแตละสวนใน diverticulum) ชนเยอเมอกประกอบดวยเยอบผวประเภท pseudostratified columnar ทมซเลยแบบขนแปรงและมการพบไปมาไมสมมาตร พบการกระจายตวของ goblet cell ไมสมาเสมอทางดานในทอ โดยตดสยอม Alcian blue ท pH 2.5 โดยไมพบสาร mucosubstance ไมพบชน lamina muscularis mucosa ในขณะทชน lamina propria-submucosa ประกอบดวยเสนใยเนอเยอเกยวพน ตอม tubulo-acinar ปมประสาท parasympathetic กลมของเสนประสาท เนอเยอนาเหลองทงทกระจายตวและอยเปนตอม หลอดเลอด เนอเยอไขมนและกลามเนอโครงราง นอกจากนนพบความแตกตางบางประการในสวนของเยอบผวและเนอเยอเกยวพนในชน submucosa จานวน goblet cell และตอม seromucosal ระหวางเพศและสวนตางๆ ของทอได

คาสาคญ: diverticulum ของทอห ลา (Equus asinus) จลกายวภาค เพศ 1Graduated from school of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, P.O. Box: 166. Shahrekord, Iran

2Department of Basic Sciences, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, P.O. Box: 166. Shahrekord, Iran

*ผรบผดชอบบทความ E-mail: dr.mobini@iaushk.ac.ir

Introduction Auditory tube diverticula or guttural pouches are air-filled evaginations of the auditory tubes present in perissodactyls such as donkey (Sasaki et al., 1999; Dyce et al., 2002; Alsafy et al., 2008; Oto and Haziroglu, 2011; Ghazi et al., 2012), horse (Sasaki et al., 1999; Lepage et al., 2004; Parillo et al., 2009a,b) and tapir (Sasaki et al., 1999). The diverticula are located in the caudal area of the head, under the base of the skull and the atlanto-occipital joint (Ghazi et al., 2012). In the medial plane, the right and left diverticula are in contact with each other by a thin septum (Sasaki et al., 1999; Lepage et al., 2004; Alsafy et al., 2008; Ghazi et al., 2012). From the lateral aspect, the stylohyoid bone incompletely divides each diverticulum into lateral and medial compartments (Baptiste et al., 2000; Ghazi et al., 2012). The auditory diverticula have important anatomical relationships with other neighbouring structures such as several cranial nerves, cranial cervical ganglion, cranial sympathetic trunk, blood vessels (Manglai et al., 2000; Lepage et al., 2004; Liebich and König, 2004; Ghazi et al., 2012) and retropharyngeal lymph nodes (Lepage et al., 2004). Ghazi et al. (2012) described that the opening of the pharyngeal part of auditory tube in domestic donkey was like a triangle shape orifice. Recent evidence demonstrates that the auditory diverticula have an important function in the bray mechanism (Ghazi et al., 2012), physiology of swallowing (Hodgson, 1998; Sasaki et al., 1999) and also in brain cooling, which regulate the temperature

of arterial blood circulating to the brain (Mitchell et al., 2006), during exercise, keeping the brain from overheating (Baptiste et al., 2000). It is well-known that the domestic donkey (Equus asinus) possessed relative large symmetric auditory tube diverticula (Manglai et al., 2000; Liebich and König, 2004; Alsafy et al., 2008; Oto and Haziroglu, 2011; Ghazi et al., 2012), although their size is lesser than those in horses (Manglai et al., 2000; Liebich and König, 2004; Ghazi et al., 2012). Although some advanced studies give adequate detail on the histological characteristics of the auditory tube diverticulum of the horse (Manglai et al., 2000; Parillo et al., 2009a,b), to the author’s best knowledge, no research had been done on the histology of the auditory tube diverticula of domestic donkey. Therefore, in this work we investigated the histological characteristics of the auditory tube diverticulum of domestic donkey (Equus asinus).

Materials and Methods Four clinically healthy adult domestic

donkeys (Equus asinus) of both sexes (2 of each sex), weighing 140-155 kg, at the Department of Anatomy, College of Veterinary Medicine, Shahrekord Islamic Azad University, Shahrekord, Iran, were employed in this study. Deep anesthesia was made by using combination of xylazine hydrochloride and ketamine hydrochloride. The animals were exsanguinated and perfused with 10% neutral buffered formalin. The guidelines of the ethical committee of Shahrekord

Ghazi, S.M. & Mobini B. / Thai J Vet Med. 2013. 43(2): 273-277. 275

Azad University were strictly followed during the procedure. All the heads were cut off transversely at the level of the sixth cervical vertebra. Transverse section of the donkey’s head passed at the level of the medial canthus of the eyes, whereas median section passed longitudinally on the head. Samples from the medial and lateral compartments of each auditory diverticulum were obtained from the following five different locations: the rostral part, middle part, caudal part, dorsal part and ventral part. All the specimens were immediately fixed in neutral buffered formalin solution for 24 hours, subjected to routine tissue processing for light microscopic examination and embedded in paraffin blocks. Serial sections (5 μm) were stained with hematoxylin-eosin for routine histological examination, Van Gieson’s (for collagen fibers), Verhoeff’s (for elastic fibers) and Gomori’s staining for reticulum. To investigate the chemical character (pH) of the secretion material in the epithelial cells, periodic acid-Schiff (PAS) reaction was employed to determine neutral mucosubstance and alcian blue (AB) (pH 2.5) was used for determining acidic mucosubstances (Kiernan, 1999). Histological studies on stained sections were carried out by light microscopy.

Results and Discussion Light microscopic examination revealed that

the auditory diverticulum of the domestic donkeys was composed of tunica mucosa and tunica submucosa in all different locations (Fig 1). The tunica mucosa of auditory diverticulum was mainly lined by pseudostratified columnar epithelium with brush-like cilia (Fig 2). However, in some regions (especially lateral compartment of right auditory diverticulum in males and right medial compartment of female's auditory diverticulum) it varied from non ciliated stratified cuboidal to columnar. This variation in females was slightly higher than in males. Tunica mucosa formed some folds which appeared to be irregularly distributed over the whole luminal surface of the auditory diverticulum. The mucosal folds of varied heights were almost simple and non-isometric.

Figure 1 Auditory tube diverticulum of domestic donkey.

tunica mucosa (M), lamina propria-submucosa (L-S), epithelium (E), mucosal fold (F), lymphatic tissues (Lt), seromucosal glands (GL), skeletal muscles (Sm), adipose tissue (A), blood vessels (B), Lumen (L). H&E, x 100

Figure 2 Collagenous fibres (arrowheads) in lamina propria-

submucosa (L-S) of auditory diverticulum of domestic donkey, epithelium (E), brush-like cilia (arrows), Lumen (L). Van gieson’s, x 400

Figure 3 Acidophilic mucosubstances are present in all

goblet cells (arrowheads). epithelium (E), lamina propria-submucosa (L-S). Alcian blue, x400

Figure 4 Neutral mucosubstances are absent in all goblet

cells (arrowheads). brush-like cilia (arrows), epithelium (E), lamina propria-submucosa (L-S), Lumen (L). PAS, x 400

Figure 5 Reticular fibres (arrowheads) in lamina propria-submucosa (L-S), of auditory diverticulum of domestic donkey, epithelium (E), brush-like cilia (arrows), Lumen (L). Gomori’s staining for reticulum, x 400

276 Ghazi, S.M. & Mobini B. / Thai J Vet Med. 2013. 43(2): 273-277.

However, in some regions the shape and arrangement of these folds varied as branched, anastomotic, or constituted by foliaceous vilosities (Fig 1). The highest and lowest mucosal folds were found respectively in the right medial compartment of males and right lateral compartment of female's auditory diverticulum.

A few goblet cells were observed in the epithelium, which appeared to be irregularly distributed over the whole luminal surface of the auditory diverticulum in both sexes. The numbers of goblet cells differed between sexes and among the various regions. The majority of the goblet cells reacted strongly with AB pH 2.5 (Fig 3), whereas neutral mucosubstances was not present (Fig 4). The lamina propria-submucosa of auditory diverticulum in both sexes was formed by frame of connective tissue containing collagenous (Fig 2), reticular (Fig 5), elastic fibers (Fig 6), numerous diffuse or nodular lymphatic tissues, blood vessels, tubulo-acinar glands, adipose tissue, skeletal muscles (Fig 1), nerve bundles and parasympathetic ganglia (Fig 6). The connective tissue of lamina propria-submucosa, which varied from loose to dense irregular type in male donkeys, as well as in medial compartment, were more observed to be of loose type as compared to females and lateral compartment of auditory diverticulum. The seromucosal glands were occasionally seen in male donkeys but were numerous in the females. These glands in lateral compartment of auditory diverticulum were numerously higher than in medial compartment. The lamina muscularis mucosa was absent in all different locations of auditory diverticulum in both sexes.

The auditory diverticulum of domestic donkeys was composed of tunica mucosa and tunica submucosa, which was similar to those of horse (Manglai et al., 2000; Parillo et al., 2009a,b). Although some variations were observed in the epithelium of tunica mucosa, it was mainly lined by pseudostratified columnar epithelia with brush-like cilia similarly to the reports of Parillo et al. (2009a,b) in horse. In contrast, Manglai et al. (2000) reported that the mucosa of the horse auditory diverticulum was consisted of stratified columnar epithelia.

Previous studies demonstrated that mucosa of the horse auditory diverticulum provided foreign substance clearance ability, but that its ability varied among different regions of the epithelium (Manglai et al., 2000).

In the present study, the histology of the auditory diverticulum revealed differences between the male and female domestic donkeys in the epithelial type, height of mucosal folds, goblet cell frequency, connective tissue type of lamina propria-submucosa and seromucosal gland frequency. Manglai et al. (2000) reported the sex effect on goblet cell frequency and the thickness of lamina propria in equine auditory tube diverticulum, whereas Parillo et al. (2009a,b) reported non-sex related differences between male and female horse. The higher mucosal folds in auditory diverticulum of male donkeys may

be due to the bray mechanism (Lindsay and Clayton, 1986).

In horse, the numerous goblet cells were reported in auditory diverticulum mucosa which were topographically located mainly on the basis of the folds (Parillo et al., 2009a,b), but in the present study, few goblet cells were observed which interspersed among the epithelial cells in the whole luminal surface of the auditory diverticulum.

On the basis of these results, the numbers of goblet cells differed between sexes and among the various regions of the epithelium which is in agreement with the results reported by Manglai et al. (2000) and Parillo et al. (2009a,b). AB-positive reactions of the majority of the goblet cells in domestic donkey auditory diverticula mirror the findings of Parillo et al. (2009b) in horse. Parillo et al. (2009b) reported that in horse the majority of the goblet cells reacted strongly with PAS. These investigators concluded that the glycosoaminoglycans provided a hydrophilic environment that prevented dehydration and desiccation of the guttural membranes during air passage. Additionally, the presence of these glycosoaminoglycans might be of importance in relation to the pathogenesis of some bacterial disease in horses such as equine strangles and involved in host defence. But in the present study, neutral mucosubstances were not observed in the goblet cells of the auditory diverticulum in domestic donkeys.

Reported submucosal glands of the auditory diverticulum in donkey (Abdel Rahman et al., 1994) and horse (Parillo et al., 2009a,b) which contained both serous and mucous secretory granules were in agreement with our results. Some researchers (Abdel Rahman et al., 1994; Parillo et al., 2009a,b) reported that the submucosal glands were tubulo-acinar type, which agrees with the results obtained from this

Figure 6 Elastic fibres (arrowheads), parasympathetic

ganglion (G) and blood vessels (B) in lamina propria-submucosa (L-S) of auditory diverticulum of domestic donkey. epithelium (E), brush-like cilia (arrows), mucosal fold (F), Lumen (L). Verhoeff’s, x 400

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study. The finding of lipids within the submucosal glands of the auditory diverticulum was reported in donkey (Abdel-Rahman et al., 1994) and horse (Parillo et al., 2009a,b). Abdel-Rahman et al. (1994) described the occurrence of lipid droplets in serous acinar cells and argued that these cells were responsible for phospholipid secretion. Baptiste and Cake (1994) demonstrated that the presence of phospholipid compounds involved in protection of the tubal membrane from corrosion, defensive mechanisms and the facilitation of fast pressure equilibration. But in the present study, lipid droplets were not observed in the submucosal glands of the auditory diverticulum in domestic donkeys.

In the present study, the submucosal glands in the lateral compartment of auditory diverticulum, as well as the females, were more numerous as compared to medial compartment and males, whereas Parillo et al. (2009a,b) reported no differences between sexes and among the various regions.

In conclusion, the present study is the first report regarding the histological structures of the auditory diverticulum in domestic donkeys. The histological properties of the auditory diverticulum in domestic donkeys were generally similar to those of horse except for the goblet cells, which were few and interspersed among the epithelial cells, the absence of neutral mucosubstances and lipid droplets in auditory diverticulum. Epithelial type, height of mucosal folds, goblet cell frequency, submucosal connective tissue type and seromucosal gland frequency differed between sexes and among the various regions studied.

References Abdel-Rahman YA, Abou-Elmagd A and Salem AO 1994.

Morphological studies on the guttural pouch of donkey: II – The subepithelial glands. Assiut Vet Med J. 32: 39-51.

Alsafy MAM, El- Kammar MH and El-Gendy SAA 2008. Topographical anatomy, computed tomography and surgical approach of the guttural pouches of the donkey. J Equine Vet Sci. 28: 215-222.

Baptiste KE and Cake MH 1994. Lipid analysis of lavage samples from the equine guttural pouch (auditory tube diverticulum). Ann Otol Rhinol Laryngol. 103: 383-388.

Baptiste KE, Naylor JM, Bailey J, Barber EM, Post K and Thornhill J 2000. Physiology: A function for

guttural pouches in the horse. Nature. 403: 382-383. Dyce KM, Sack WO and Wensing CJG 2002. Textbook of

Veterinary Anatomy. 3rd ed. WB Saunders Philadelphia: 496-501.

Ghazi SM, Mobini B and Afsharzadeh MR 2012. Topographical and morphological anatomy of the guttural pouches of the domestic donkey (Equus asinus). Global Veterinaria. 9(6): 691-695.

Hodgson DR 1998. What is the function of the guttural pouches: Selective brain cooling? Augmentation of swallowing? Still to be defined. Vet J. 155: 115-117.

Kiernan JA 1999. Histological and Histochemical Methods: Theory and Practice. 3rd ed. Butterworth Heinemann: Oxford, Boston: 103-132.

Lepage OM, Perron MF and Cadore JL 2004. The mystery of fungal infection in the guttural pouches. Vet J. 168: 60-64.

Liebich HG and König HE 2004. Vestibulocochlear organ. In: Veterinary Anatomy of the Domestic Mammals. 4th ed. HE König and HG Liebich (eds). Schattauer, Stuttgart: 577-579.

Lindsay FEF and Clayton M 1986. An anatomical and endoscopic study of the nasopharynx and larynx of the donkey (Equus asinus). J Anat. 144: 123-132.

Manglai D, Wada R, Kurohmaru M, Yoshihara T, Kuwano A, Oikawa M and Hayashi Y 2000. Histological and morphometrical studies on the mucosa of the equine guttural pouch (auditory tube diverticulum). Okajimas Folia Anat Jpn. 76: 335-346.

Mitchell G, Fuller A, Maloney SK, Rump N and Mitchell D 2006. Guttural pouches, 10 brain temperature and exercise in horses. Biol Lett. 2: 475-477.

Oto C and Haziroglu RM 2011. Magnetic resonance imaging of the guttural pouch (diverticulum tubae auditivae) and its related structures in donkey (Equus asinus). Ankara univ Vet Fak Derg. 58: 1-4.

Parillo F, Arias MP and Verini Supplizi A 2009a. Glycoprofile of the different cell types present in the mucosa of the horse guttural pouches. Tissue Cell. 41: 257-265.

Parillo F, Rossi G, Busoni V, Magi GE and Verini Supplizi A 2009b. Differentiation of glycans in equine guttural pouches. Vet J. 180: 246-252.

Sasaki M, Hayashi Y, Koie H, Yamaya Y, Kimura J, Manglai D, Kawashima S, Endo H and Yamamoto M 1999. CT examination of the guttural pouch (auditory tube diverticulum) in Przewalski’s horse (Equus przewalskii). J Vet Med Sci. 61: 1019-1022.

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Thai J Vet Med. 2013. 43(2): 279-284.

Antiparasitic Efficacy of 10% w/v Fipronil Spot-on (Fiproline

Spot-on) against Experimental Tick (Rhipicephalus sanguineus)

Infestations on Dogs

Sonthaya Tiawsirisup1* Kanokwan Thiansirikhun1 Kanoksak Thanadumkerng1

Nuttika Pastarapatee1 Nunaree Trirattananuwong1 Wilai Rattanatayarom2*

Abstract

This study was conducted to investigate the efficacy of 10% w/v fipronil spot-on for treatment and

prevention of Rhipicephalus sanguineus infestations on dogs. Twelve Beagles were randomly separated into two groups which were treatment and control group (6 dogs/group). The treatment group received 10% fipronil spot-on at the dosage of 6.7 mg/kg on day 0. A group of 60 Rhipicephalus sanguineus was released to feed on each dog on day -7, -2, 7, 14, 21, and 28. The ticks were counted and removed on days-4, 3, 10, 17, 24, and 31. Mean number of tick infestations in the control group ranged from 16.67-24.50 ticks per dog and the attachment rate ranged from 27.78-40.83%. Mean number of tick infestations in the treatment group on the days before fipronil was applied on the dogs was 21.67 ticks per dog and the attachment rate was 36.11%. Percent efficacy of fipronil used in this study was 71.77, 96.03, 100, 91.84, and 90.21% on days 3, 10, 17, 24, and 31, respectively. There were statistically significant differences between the mean numbers of tick between the control and treatment groups on days 3, 10, 17, 24, and 31. There was no significant difference of complete blood count and kidney and liver functions before and after the study. Skin rash did not show up in the treatment group after fipronil was applied on the dogs.

Keywords: brown dog tick, dog, efficacy, fipronil, spot-on 1 Veterinary Parasitology Unit, Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Pathumwan 10330 Thailand 2 Department of Pharmacology, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand *Correspondence author E-mail: sonthaya.t@chula.ac.th, wilai@swu.ac.th

Short Communication

280 Tiawsirisup S. et al. / Thai J Vet Med. 2013. 43(2): 279-284.

บทคดยอ

ประสทธภาพของฟโปรนล 10% (ฟโปรไลน สปอต-ออน) ในการกาจดและปองกนเหบ Rhipicephalus sanguineus บนสนข

สนธยา เตยวศรทรพย 1* กนกวรรณ เทยรสรคน 1 กนกศกด ธนดาเกง 1 ณฐฐกา พสตรเภท 1 ณนร ไตรรตนานวงษ 1 วไล รตนตยารมณ 2*

ศกษาประสทธภาพของฟโปรนล 10% w/v รปแบบหยดหลง ในการกาจดและปองกนเหบ Rhipicephalus sanguineus บนสนข

การศกษานไดใชสนขพนธบเกลจานวน 12 ตว โดยแบงสนขออกเปน 2 กลม คอ กลมทดลองและกลมควบคม (6 ตว/กลม) ในวนท 0 กลมทดลองจะไดรบฟโปรนล 10% ในขนาด 6.7 มก.ตอกก. ปลอยเหบสนขชนด Rhipicephalus sanguineus ลงบนตวสนขในวนท -7, -2, 7, 14, 21 และ 28 และนบเหบบนตวสนขและเกบเหบออกจากสนขในวนท -4, 3, 10, 17, 24 และ 31 จากการศกษานพบวาคาเฉลยของจานวนเหบบนสนขในกลมควบคม คอ 16.67-24.50 ตว หรอคดเปนรอยละ 27.78-40.83 และคาเฉลยของจานวนเหบบนสนขในกลมทดลองกอนทจะมการหยดยาคอ 21.67 ตว หรอคดเปนรอยละ 36.11 สาหรบประสทธภาพของฟโปรนลนนมคาเทากบรอยละ 71.77, 96.03, 100, 91.84 และ 90.21 ในวนท 3, 10, 17, 24 และ 31 ตามลาดบ และคาเฉลยของเหบในกลมทดลองและกลมควบคมในวนท 3, 10, 17, 24 และ 31 มความแตกตางกนอยางมนยสาคญทางสถต จากการศกษานไมพบความผดปกตของเมดเลอด ตบ และไตของสนขทงกอนและหลงการทดสอบ รวมทงไมพบความผดปกตของผวหนงสนขภายหลงการหยดยา

คาสาคญ: เหบสนข ประสทธภาพ ฟโปรนล รปแบบหยดหลง 1 หนวยปรสตวทยา ภาควชาพยาธวทยา คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ 10330 2 ภาควชาเภสชวทยา คณะแพทยศาสตร มหาวทยาลยศรนครนทรวโรฒ

*ผรบผดชอบบทความ E-mail: sonthaya.t@chula.ac.th, wilai@swu.ac.th

Introduction Rhipicephalus sanguineus or brown dog ticks are ectoparasites that can be found on dogs in both urban and rural areas worldwide including Thailand (Inokuma et al., 1995; Maroli et al., 1996; Tinoco-Gracia et al., 2009). This tick can also be found on cat and human (Uspensky and Ioffe-Uspensky, 2002; Uspensky, 2009). Rhipicephalus sanguineus has a three-host tick life cycle which is composed of egg, larval, nymphal, and adult stage. Larva, nymph, and adult can be found on dogs. Engorged larva and engorged nymph will leave host for molting to nymph and adult stage, respectively. Engorged female adult will also leave host for egg laying. Infestation of Rhipicephalus sanguineus is concerned by pet owners since the infestation can cause itch, irritation and serious blood loss. Infected ticks can also transmit pathogen during blood feeding process. Rhipicephalus sanguineus is an important vector for several pathogens in Thailand which are Babesia canis, Ehrlichia canis, and Hepatozoon canis (Baneth et al., 2001; Dantas-Torres, 2008; M'Ghirbi and Bouattour, 2008). Tick paralysis in dogs can also be caused by Rhipicephalus sanguineus. Therefore, the treatment and prevention of Rhipicephalus sanguineus infestation on dogs and other pet animals are very important.

Spot-on application is a convenient method for pet owners because they can do this by themselves at home. There are many spot-on products in veterinary market. Efficacy and price of these products are concerned by pet owners before purchasing. There are several chemicals that can be used as a spot-on application. Single or combination chemicals can be found in the present, for example, fipronil, imidacloprid, methroprene, permethrin, pyriprole, and selamectin (Denny, 2001; Doyle et al., 2005; Hagimori et al., 2005; Otranto et al., 2005; Dryden et al., 2006; Dryden et al., 2008; Schuele et al., 2008a,b; Bouhsira et al., 2009). Fipronil is a phenylpyrazole antiparasitic agent which can interfere brain and spinal cord of the ticks, resulting in death of the ticks. Fipronil acts by binding to gamma-aminobutyric acid (GABA) and glutamate receptors. It inhibits the opening of the chloride ion channels and leads to neuronal hyperactivity (Hosie et al., 1995; Hainzl and Casida, 1996; Narahashi et al., 2010). Fipronil has efficacy against various species of fleas, lices, and ticks, for example, Ctenocephalides canis, Ctenocephalides felis, Trichodectes canis, Dermacentor reticulatus, Rhipicephalus sanguineus, and Ixodes recinus (Cooper and Penaliggon, 1996; Ritzhaupt et al., 2000; Pollmeier et al., 2002; Oliveira et al., 2009). This study was conducted to investigate the efficacy of 10% w/v fipronil spot-on for treatment and

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prevention of brown dog tick (Rhipicephalus sanguineus) infestations on dogs in Thailand.

Materials and Methods Experimental animals: Twelve Beagles of both sexes and with various weights were used in this study. They were randomly separated into two groups which were treatment and control group (6 dogs/group). This study was approved by the Chulalongkorn University Animal Care and Use Committee (Animal Use Protocol and Approval No. 11310068).

Ticks: Thailand strain of Rhipicephalus sanguineus was used in this study. They were reared and maintained at the parasitology laboratory, Parasitology Unit, Department of Veterinary Pathology, Faculty of Veterinary Science, Chulalongkorn University. Two- to three-week-old adult ticks were used in this study.

Tested substance: 10% w/v fipronil spot-on (Fiproline Spot On, Thainaoka Pharmaceutical, Thailand, Lot. No. R&D 17/09/11 Mfg. Date 26/09/2011) was used in this study. A dose of 6.7 mg fipronil per kg bw was applied on the skin of the dogs at the base of the neck between the shoulder blades once only on day 0 in the treatment group.

Experimental design: There were 12 dogs in this study and they were randomly separated into two groups (6 dogs/group). All dogs were free from any acaricide for at least two months. The dogs were bathed with non-acaricide shampoo about two weeks before the study was started.

The control group did not received any fipronil during the study but the treatment group received Fiproline Spot On on day 0. A group of 30 males and 30 females of Rhipicephalus sanguineus was allowed to feed on each dog on days-7, -2, 7, 14, 21, and 28. The ticks were counted and removed on days-

4, 3, 10, 17, 24, and 31. Mean numbers of ticks between the control and treatment groups were compared by using t-test and fipronil efficacy was calculated as the following formula.

Fipronil efficacy (%) = ((Geometric mean control – Geometric mean treatment)/ Geometric mean control) x 100

Geometric mean control = geometric mean number of ticks on untreated control dogs at each individual assessment day

Geometric mean treatment = geometric mean number of ticks on fipronil treated dogs at each individual assessment day

Blood was collected from each dog before and after the study. It was tested for complete blood count, creatinine, BUN, SGPT, and alkaline phosphatase to indicate kidney and liver functions. Skin rash on the treatment group was also investigated. Tick infestations were taken to be successful when the attachment rates (attached/applied) were equal or more than 25%. For all analyses, a p-value threshold was set to 0.05.

Results and Discussion There were 12 Beagle in this study and they

were randomly separated into two groups (6 dogs/group). The control group did not receive any fipronil during the study but the treatment group received fipronil spot-on. Body weight of the control group ranged from 9.9-14.9 kg and body weight of the treatment group ranged from 7.3-12.4 kg.

A group of 30 males and 30 females of Rhipicephalus sanguineus was released to feed on each dog on days-7, -2, 7, 14, 21, and 28. The ticks were counted and removed on days-4, 3, 10, 17, 24, and 31. The results are shown on Table 1 and 2.

Table 1 Number of Rhipicephalus sanguineus found on the control group on days-4, 3, 10, 17, 24, and 31

Control group Tick released day Tick counted day Dog#1 Dog#2 Dog#3 Dog#4 Dog#5 Dog#6

Day-7 Day-4 7 18 15 22 19 19 Day-2 Day 3 29 19 12 16 23 25 Day 7 Day 10 19 18 16 11 28 34

Day 14 Day 17 27 10 16 8 30 28 Day 21 Day 24 27 25 21 10 36 28 Day 28 Day 31 30 18 17 20 30 28

Table 2 Number of Rhipicephalus sanguineus found on the treatment group on days-4, 3, 10, 17, 24, and 31, which

received Fiproline Spot On at the dose of 0.67 ml/10 kg body weight (equivalence to fipronil 6.7 mg/kg) on day 0

Treatment group Tick released day Tick counted day

Dog#7 Dog#8 Dog#9 Dog#10 Dog#11 Dog#12 Day-7 Day-4 12 24 21 21 25 27 Day-2 Day 3 0 7 9 6 12 1 Day 7 Day 10 0 0 0 3 2 0 Day 14 Day 17 0 0 0 0 0 0 Day 21 Day 24 2 0 4 6 0 0 Day 28 Day 31 1 0 3 4 2 4

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Mean number of tick infestations in the control group ranged from 16.67-24.50 ticks per dog and the attachment rate ranged from 27.78-40.83% (mean of 35.14%). Mean number of tick infestations in the treatment group on the days before fipronil was applied on the dogs was 21.67 ticks per dog and the attachment rate was 36.11%. Mean numbers of ticks between the control and treatment groups were compared on day-4 to indicate that there was no difference between tick feeding or attachment between the groups of the dogs. In this study, there was no statistically significant difference of mean number of ticks between the control and treatment groups on day-4 (p = 0.1307).

Percent efficacy of Fiproline Spot-on used in this study was 71.77, 96.03, 100, 91.84, and 90.21% on days 3, 10, 17, 24, and 31, respectively (Table 3). There were statistically significant differences between the mean numbers of tick between the control and treatment groups on days 3, 10, 17, 24, and 31 (p < 0.05). There was no significant difference of complete blood count and kidney and liver functions before and after the study. Skin rash did not show up in the treatment group after fipronil was applied on the dogs.

Not all released ticks would attach and feed on the dogs. In this study, we found that the mean number of tick infestations, indicating vigorous tick challenges on all assessment days (EMEA, 2007) in the control group ranged from 16.67-24.50 ticks per dog and the attachment rate ranged from 27.78-40.83%. Other studies showed that Dermacentor reticulatus attachment rate on dogs were between 29.7-59.6% and Ixodes ricinus attachment rate on dogs were between 37.0-50.7% (Bonneau et al., 2010; 2011).

The efficacy of this product against the attached ticks on dogs was 71.77% on day 3 after the product was applied on the dogs. The efficacy of this product on days 10, 17, 24, and 31 after the product was applied on the dogs were higher than 90% with the range between 90.21-100%. Accordingly, if ticks of Rhipicephalus sanguineus are present when the product is applied, all the ticks may not be killed within the first 72 hours but they may be killed within a week. The highest efficacy of this product was on day 17, after the product was applied on the dogs, and it decreased to 90.21% on day 31. The study indicated that this product had the persistent efficacy against

Rhipicephalus sanguineus infestations on the dogs for at least 4 weeks.

The study by Guerrero (2010) indicated that the arithmetic mean of Rhipicephalus sanguineus attachment for the untreated control group of dogs ranged from 18.8/50 to 34.3/50. This study also showed that the efficacy of PetArmor® (9.7% w/w fipronil) against Rhipicephalus sanguineus was 51.3, 98.4, 99.6, 99.6, and 97.5% on days 2, 9, 16, 23, and 32, respectively, and the efficacy of Frontline® Top Spot (9.7% w/w fipronil) against Rhipicephalus sanguineus was 65.0, 100.0, 98.0, 94.9, and 76.1% on days 2, 9, 16, 23, and 32, respectively.

The efficacy of fipronil reported in this study is similar to the efficacy of other formulations of the fipronil. Bonneau et al. (2010) found that the efficacy of one fipronil formulation (Frontline®) against Ixodes ricinus was 98.8, 100, 100, 100, 86.3, and 77.2% on day 2, 9, 16, 23, 30, and 37 after treatment, respectively, and the efficacy of another fipronil formulation (Effipro®) against Ixodes ricinus was 93.8, 100, 100, 98.9, 97.9, and 94.1% on day 2, 9, 16, 23, 30, and 37 after treatment, respectively.

Bonneau et al. (2011) also found that the efficacy of one fipronil formulation (Frontline®) against Dermacentor reticulatus was 99.2, 100, 99.1, 96.2, 97.3, and 89.8% on days 2, 42, 44, 51, 58, and 65 after treatment, respectively, and the efficacy of another fipronil formulation (Effipro®) against Dermacentor reticulatus was 98.3, 100, 97.4, 98.8, 96.3, and 85.6% on days 2, 42, 44, 51, 58, and 65 after treatment, respectively.

Fipronil has been used as insecticide and acaricide in the veterinary market as a spray and spot-on formulation for more than ten years. Some veterinarians and pet owners might worry about the efficacy of fipronil against tick infestations on dogs. This study not only indicated the efficacy of this fipronil product but also indicated that fipronil still had the efficacy against Rhipicephalus sanguineus infestations on dogs in Thailand. Although the addition of S-methoprene prevents flea eggs from hatching and larvae from developing into egg-laying adults, formulation containing only fipronil continues to provide excellent efficacy against fleas and ticks (Guerrero, 2010). The efficacy of fipronil against fleas and lice on dogs in Thailand still needs to be investigated.

Table 3 Percent efficacy of 10% w/v fipronil spot-on (Fiproline Spot On, Thainaoka) against Rhipicephalus sanguineus on dogs on

days 3, 10, 17, 24, and 31

Mean number of ticks Tick attachment rate Tick released day

Tick counted day Control

group Treatment

group Control group

Treatment group

Percent Efficacy p-value*

Day-7 Day-4 16.67 21.67 27.78 36.11 0.1307 Day-2 Day 3 20.67 5.83 34.45 - 71.77 0.0009 Day 7 Day 10 21.00 0.83 35.00 - 96.03 0.0002 Day 14 Day 17 19.83 0.00 33.05 - 100.00 0.0005 Day 21 Day 24 24.50 2.00 40.83 - 91.84 0.0001 Day 28 Day 31 23.83 2.33 39.72 - 90.21 <0.0001

*Comparison of mean numbers of ticks between control and treatment groups

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Acknowledgement This study was financially supported by

Thainaoka Pharmaceutical CO., Ltd., Thailand.

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Different Influence of Intracellular Glutamine Depletion on

Glutamate Uptake Mediated by Amino Acid Transport Systems;

EAATs and X-c, in Placental (BeWo) Cells

Boonrit Thongsong

Abstract

Glutamate transporters are responsible for active transport of glutamate over the cell membrane and play an

important role in the glutamate-glutamine cycle. The purpose of the present study was to investigate the influence of intracellular glutamine depletion on glutamate uptake mediated by amino acid transport systems; EAATs and X-c in placental choriocarcinoma (BeWo) cells as a model of human trophoblasts. Intracellular glutamine was depleted by culturing the cells in regular medium without glutamine and by treating with an inhibitor of glutamine synthetase (methionine sulfoximine; MSX), for 16 hours. The uptake of glutamate was measured by the use of appropriate substrates and ionic conditions. When cultured in not only the absence of extracellular glutamine but also treatment with MSX, the differential uptake of glutamate depended on the glutamate transport systems. This study concluded that the depletion of intracellular glutamine decreased the activity of EAATs; as sodium-dependent manner and at the same time significantly enhanced the activity of X-c; as sodium-independent manner for glutamate transport in the BeWo cells. Thus, intracellular glutamine was obligatory for maintenance of optimal activity of system EAATs in the BeWo cells. The depletion of intracellular glutamine caused stress to the cells and the stress leaded to the upregulation of system X-c activity.

Keywords: amino acid transport system, glutamate, glutamine, placenta Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330 *Correspondence author E-mail: Boonrit.T@chula.ac.th

Short Communication

286 Thongsong B. / Thai J Vet Med. 2013. 43(2): 285-289.

บทคดยอ

ความแตกตางระหวางอทธพลของสภาวะกลทามนภายในเซลลรกลดลงตอการนาเขากลทาเมททถกเหนยวนาโดยตวขนสงกรดอะมโนสองระบบ

บญฤทธ ทองทรง

ตวขนสงกลทาเมทชวยในการขนสงกลทาเมทผานเซลลแบบใชพลงงาน และมบทบาทในการเปนสวนประกอบทสาคญประการหนงในวฏจกรกลทาเมท-กลทามน วตถประสงคการศกษาครงน เพอศกษาอทธพลของสภาวะกลทามนภายในเซลลรกลดลงตอการนาเขากลทาเมททเหนยวนาโดยตวขนสงกรดอะมโนทงสองระบบ เซลลรกถกเพาะเลยงดวยอาหารเลยงเซลลตามปกตแตไมมกรดอะมโนกลทามนและทาการลดกลทามนภายในเซลลโดยการเตมสารททาหนาทยบยงเอนไซมกลทามนซนทเทส ไดแก เมทไธโอนนซลโฟซมน เปนระยะเวลานาน 16 ชวโมง ศกษาการนากรดอะมโนกลทาเมทผานเซลลโดยใชสารตงตนและปรบสภาวะไอออนทเหมาะสม จากการศกษาพบวาการเลยงเซลลในสภาวะกลทามนภายในเซลลลดลง ทาใหการนาเขากรดอะมโนกลทาเมทระบบทใชตวขนสงชนดตองอาศยโซเดยมไอออนลดลง แตในสภาวะเดยวกนกลบทาใหการนาเขากรดอะมโนกลทาเมทระบบทใชตวขนสงชนดไมตองอาศยโซเดยมไอออนเพมขนอยางมาก จากผลการศกษาดงกลาวแสดงใหเหนวาปรมาณกรดอะมโนกลทามนภายในเซลล มบทบาทหนาทในการคงสภาวะทเหมาะสมสาหรบระบบตวขนสงกรดอะมโนกลทาเมทชนดทตองอาศยโซเดยมไอออน และการลดลงของระดบกลทามนภายในเซลลรกน อาจทาใหเกดสภาวะเครยดซงทาใหระบบตวขนสงกรดอะมโนกลทาเมทชนดทไมตองอาศยโซเดยมไอออนทาหนาทเพมขน

คาสาคญ: ระบบขนสงกรดอะมโน กลทาเมท กลทามน รก ภาควชาสตวบาล คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย ปทมวน กรงเทพฯ *ผรบผดชอบบทความ E-mail: Boonrit.T@chula.ac.th

Introduction Most of the nutrients for fetal growth and development are supplied from maternal blood circulation while some of them are synthesized in the placenta. Both glutamate and aspartate, unlike others, are concentrated higher within the placental trophoblast than in the maternal and fetal circulations. These amino acids may provide an energy source to the placenta (Battaglia, 2000) and are thought to be important for fetal growth and development. The ability to transport glutamate across cellular membranes by glutamate transporters was evidenced in many tissues including placenta (Moe, 1995; Matthews et al., 1998; Noorlander et al., 2004). By molecular identification in mammals, at least five isoforms (Excitatory Amino Acid; EAA, transporters) of high affinity sodium-dependent glutamate transporters representing system EAATs were identified in both apical and basal membranes of syncytiotrophoblasts (Cariappa et al., 2003; Noorlander et al., 2004). These transporters can transport not only L-glutamate but also L- and D-aspartate (Kanai and Hediger, 1992). Additionally, sodium-independent glutamate transporter representing system X-c (Christensen, 1990) was identified from placenta as a transport system which exchanges glutamate and cystine (Wagner et al., 2001). The work of Noorlander et al. (2004) demonstrated that expression patterns of placental glutamate transporters were indicated in active

transport of glutamate in blood circulation between fetus and mother. Placental transport of maternal glutamine establishes the fetoplacental glutamine-glutamate cycle. In addition, placental uptake of glutamate from fetal blood circulation is important to maintain low concentration of extracellular glutamate and prevent toxic levels to reach the brain (Robinson and Dowd, 1997).

The intracellular concentration of glutamine is very high. There are two routes to obtain glutamine; taken up into mammalian cells from extracellular medium by several active and passive amino acid transport systems (Ganapathy et al., 2003) and synthesized by the amidation of glutamate. The ATP-dependent reaction is catalyzed by glutamine synthetase, an enzyme inhibited by the glutamine analog methionine sulfoximine (MSX). Knowledge concerning the intracellular glutamine depletion of glutamate uptake in trophoblast cells is necessary to understand the glutamate-glutamine regulation. In the placenta, the glutamate-glutamine cycle plays a crucial role in contributing glutamine to the fetus and excreting glutamate and ammonia from fetal circulation (Battaglia, 2000) as well as regulating the concentrations of some amino acids. It is known that glutaminase produces glutamate from glutamine in many organs. This transformation between glutamine and glutamate has an essential role in some regulations such as intracellular pH (Neu, 2001). Furthermore, glutamine synthetase is responsible

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enzyme for glutamine synthesis in the placenta to keep up with fetal demand (Battaglia, 2000). Little is known about the effect of depletion of intracellular glutamine on uptake activity of amino acid transport systems in placenta. Therefore, the purpose of the present study was to investigate the influence of intracellular glutamine depletion on glutamate uptake mediated by amino acid transport systems; EAATs and X-c in the placental choriocarcinoma (BeWo) cells as a model of the human trophoblasts. These cells express abundant and several amino acid transport systems that are subject to extensive regulation in human placenta under various physiological and pathological conditions (Sibley et al., 1997; Oehler and Roth, 2003; Thongsong et al., 2005). Therefore, in vitro study into the condition of intracellular glutamine depletion for mediating uptake of glutamate transport systems in BeWo cells may lead to a better understanding of the glutamate transporters in the maternal-placental-fetal unit.

Materials and Methods Cell culture and reagents: BeWo choriocarcinoma cell line, cell culture media, fetal bovine serum, unlabeled and radiolabeled amino acid of L-[3H] glutamic acid (40 Ci/mmol) were provided by Professor Dr. Vadivel Ganapathy and Professor Dr. Puttur D Prasad.

Cell culture and treatment: BeWo cells were cultured in 12-well culture plates for uptake assays in DMEM/F-12 (50:50) medium containing 2.5 mM glutamine and supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The culture conditions and the rationale of intracellular glutamine depletion for the study were described in my previous report (Thongsong, 2012a). Confluent cultures were treated in a glutamine-free culture medium to lead to the absence of extracellular glutamine. Under this condition, cells were treated without MSX to control availability of intracellular glutamine and with MSX for 16 hours to induce the depletion of intracellular glutamine because of the inhibition of endogenous synthesis of

glutamine. After treatment of the cells with or without MSX, the cells were used for measurement of glutamate uptake.

Glutamate uptake measurement: Uptake measurement was carried out at 370C. The medium was aspirated and the cell monolayer was washed once with the uptake buffer. Uptake was then initiated by the addition of 500 µl of uptake buffer containing 0.5 µCi of radiolabeled glutamic acid. The composition of the uptake buffer was as previously described (Thongsong, 2012b). For sodium-dependent glutamate uptake, the uptake of 5 µM glutamate was measured in the presence of sodium chloride. For sodium-independent glutamate uptake, the uptake of 5 µM glutamate was measured in the presence of N-methyl-D-glucamine chloride. The incubation was continued for a desired length of time, following which the uptake was terminated by aspirating the uptake medium. After the termination of the uptake, the cells were washed two times with 1.5 ml of ice-cold uptake buffer. The cells were then solubilized with 0.5 ml of 1% SDS/0.2 N NaOH and transferred to scintillation vials for the determination of the radioactivity associated with the cells. Experiments were made in triplicate. The results are given as means±SEM.

Results and Discussion Influence of intracellular glutamine depletion on glutamate uptake in BeWo cells

Since glutamate and glutamine are involved in many biosynthesis and metabolism processes, control of the concentration of these amino acids among the maternal-placenta-fetal unit is essential. Some studies described that maternal glutamine rather than glutamate was transferred across the placenta to the fetal circulation (Liechty et al., 1991; Vaughn et al., 1995). In the consistent study of Battaglia (2000), a large amount of glutamine was released from the placenta into fetal circulation and reversely,

Figure 1 Influence of intracellular glutamine depletion with MSX treatment on glutamate uptake mediated by amino acid transport

system EAATs and X-c. BeWo cells were treated in the absence of extracellular glutamine without (open bars) or with (filled bars) MSX (2 mM) for 16 hours. Uptake of 5 µM glutamate was then measured at 370C in the presence of sodium chloride or N-methyl-D-glucamine chloride for 5 min. Figure 1A represents the activity of the combined activity of EAATs and X-c; the uptake of glutamate measured in the sodium ion replete buffer. Figure 1B represents the activity of system X-c; the sodium-independent glutamate uptake measured in the presence of N-methyl-D-glucamine chloride. Figure 1C represents the activity of EAATs; the difference between the uptake in the presence of sodium chloride and the uptake in the presence of N-methyl-D-glucamine chloride.

288 Thongsong B. / Thai J Vet Med. 2013. 43(2): 285-289.

glutamate was taken up by the placenta from fetal blood. To examine activity of glutamate transport systems in this study, confluent cells were treated in the absence of extracellular glutamine with or without MSX for 16 hours and the uptake of glutamate was then measured in the presence of sodium chloride or N-methyl-D-glucamine chloride. The uptake of glutamate measured in the sodium ion replete buffer represents the combined activity of EAATs and X-c (Fig 1A). The sodium-independent glutamate uptake measured in the presence of N-methyl-D-glucamine chloride represents the activity of system X-c (Fig 1B). The difference between the uptake in the presence of sodium chloride and the uptake in the presence of N-methyl-D-glucamine chloride represents the activity of EAATs (Fig 1C). These data involve both sodium-dependent and sodium-independent manner of glutamate uptake in the control cells. In addition, they demonstrate and conclude that the depletion of intracellular glutamine by MSX treatment decreased the sodium-dependent glutamate uptake (Fig. 1C) as the activity of system EAATs by ~60%, whereas the sodium-independent glutamate uptake as the activity of system X-c increased by ~350% (Fig 1B) under identical conditions. To explain the decreasing system EAATs activity, Novak et al. (2001) demonstrated that EAAT2 played a vital role under some conditions, especially in conditions of amino acid depletion. This transporter was detected in the syncytiotrophoblast and could be involved in transporting into or out of this cell (Noorlander et al., 2004). Thus, intracellular glutamine is obligatory for maintenance of optimal activity of system EAATs in the BeWo cells and the depletion of glutamine in the cells induces stress leading to the increase in sodium-independent glutamate uptake via the glutamate/cystine exchanger. According to my previous reports, the influence of intracellular glutamine depletion in placental cells on the regulation of amino acid transport was not limited to specific amino acid (Thongsong, 2012a) and depended on amino acid transport systems (Thongsong, 2012b, 2013).

To speculate what the mechanism of MSX action on system X-c is, my previous experiments had shown that the kinetic pattern and uptake activity of system A inhibition were associated with MSX treatment without changes in steady-state levels of mRNA specific for system A (Thongsong, 2012b, 2013). In contrast, the steady state levels of xCT mRNA were elevated by MSX treatment (personal communication with Miyauchi), showing that the increase in X-c activity seen with MSX treatment is due to enhanced de novo synthesis of the transporter protein. Therefore, this in vitro study on mediating uptake by glutamate transport systems under condition of intracellular glutamine depletion in BeWo cells may provide more insight about the role of the glutamate transporters in transyncytial and maternal-placental-fetal nutrient transport.

Acknowledgement The author would like to thank Professor Dr.

Vadivel Ganapathy and Professor Dr. Puttur D Prasad from Georgia’s Health Sciences University, USA, for their kindness and significant advice.

References Battaglia FC 2000. Glutamine and glutamate exchange

between the fetal liver and the placenta. J Nutr. 130: 974S-977S.

Cariappa R, Heath-Monnig E and Smith CH 2003. Isoforms of amino acid transporters in placental syncytiotrophoblast: Plasma membrane localization and potential role in maternal/fetal transport. Placenta. 24: 713-726.

Christensen HN 1990. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev. 70: 43-77.

Ganapathy V, Inoue K, Prasad PD and Ganapathy ME 2003. Cellular uptake of amino acids: systems and regulation, in: LA Cynober (ed.), Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, CRC Press, New York, NY: 63-78.

Kanai Y and Hediger MA 1992. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360: 467-471.

Liechty EA, Kelley J and Lemons JA 1991. Effect of fasting on uteroplacental amino acid metabolism in the pregnant sheep. Biol Neonate. 60: 207-214.

Matthews JC, Beveridge MJ, Malandro MS, Rothstein JD, Campbell-Thompson M, Verlander JW, Kilberg MS and Novak DA 1998. Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta. Am J Physiol. 274: C603-C614.

Moe AJ 1995. Placental amino acid transport. Am J Physiol. 268: C1321-C1331.

Neu J 2001. Glutamine in the fetus and critically ill low birth weight neonate; metabolism and mechanism of action. J Nutr.131: 2585S-2589S, discussion 2590S.

Noorlander CW, de Graan PNE, Nikkels PGJ, Schrama LH and Visser GHA 2004. Distribution of glutamate transporters in the human placenta. Placenta. 25: 489-495.

Novak DA, Quiggle F, Artime C and Beveridge MJ 2001. Regulation of glutamate transport and transport proteins in a placental cell line. Am J Physiol Cell Physiol. 281: C1014-C1022.

Oehler R and Roth E 2003. Regulative capacity of glutamine. Curr Opin Clin Nutr Metab Care. 6: 277-282.

Robinson MB and Dowd LA 1997. Heterogeneity and functional properties of subtypes of sodium-dependent glutamate transporters in the mammalian central nervous system. Adv Pharmacol. 37: 69-115.

Sibley C, Glazier J and D'Souza S 1997. Placental transporter activity and expression in relation to fetal growth. Exp Physiol. 82: 389-402.

Thongsong B 2012a. The depletion of intracellular glutamine by methionine sulfoximine on amino acid uptake in placental cells (BeWo). Thai J Vet Med. 42(2): 209-212.

Thongsong B 2012b. Influence of intracellular glutamine depletion on regulation of amino acid transport

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system A in placental (BeWo) cells. Thai J Vet Med. 42(3): 267-273.

Thongsong B 2013. Mediating trophoblast uptake of methylaminoisobutyric acid and glutamine by absence of extra- or intracellular glutamine. Thai J Vet Med. 43(1): 85-90.

Thongsong B, Subramanian RK, Ganapathy V and Prasad PD 2005. Inhibition of amino acid transport system A by interleukin-1beta in trophoblasts. J Soc Gynecol Invest. 12: 495-503.

Vaughn PR, Lobo C, Battaglia FC, Fennessey PV, Wilkening RB and Meschia G 1995. Glutamine-glutamate exchange between placenta and fetal liver. Am J Physiol. 268: E705-E711.

Wagner CA, Lang F and Broer S 2001. Function and structure of heterodimeric amino acid transporters. Am J Physiol Cell Physiol. 281: C1077-C1093.

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Genetic Diversity of Spirometra erinaceieuropaei from Dogs in

Hunan province, China Based on Analyses of Two

Mitochondrial Sequences

Liang Liang* Jian Liang Hui-Shen Xiao-Lei Yao

Abstract

Sequence variability in two mitochondrial DNA (mtDNA) regions, namely NADH dehydrogenase subunit 5

(nad5) and small subunit of ribosomal RNA (rrnS) in Spirometra erinaceieuropaei from dogs in Hunan province in China was examined. A portion of the nad5 (pnad5) and rrnS (prrnS) genes were amplified separately from individual S. erinaceieuropaei by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. The sequences of pnad5 and prrnS were 531 and 328 bp in size, respectively. The intra-specific sequence variations within each of the S. erinaceieuropaei were 0-3.5% for pnad5 and 0-1.4% for prrnS, while the inter-specific sequence variations within each of the S. erinaceieuropaei were 12.2-35.7% for pnad5 and 10.4-11.8% for prrnS. Phylogenetic analysis using neighbour joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) methods, indicated that all the isolates in Hunan province represented S. erinaceieuropaei. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics studies of S. einaceieuropaei in human and animal health significance.

Keywords: genetic variation, mitochondrial DNA (mtDNA), hylogenetic analysis, Spirometra erinaceieuropaei Department of Plastic and Aesthetic, Affiliated Ruikang Hospital of Guangxi Traditional Chinese Medicine University, Nanning 530011, Guangxi Zhuang Autonomous Region, China *Correspondence author E-mail: 404877308@qq.com (LL)

Short Communication

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บทคดยอ

ความหลากหลายทางพนธกรรมของ Spirometra erinaceieuropaei จากสนขในจงหวด

หหนาน ประเทศจน โดยวเคราะหจากลาดบพนธกรรมของไมโตคอนเดรย

Liang Liang* Jian Liang Hui-Shen Xiao-Lei Yao

การศกษาครงนศกษาความแปรปรวนของลาดบพนธกรรมของไมโตคอนเดรย (mtDNA) ท NADH dehydrogenase subunit 5

(nad5) และหนวยยอยของ ribosomal RNA (rrnS) ของ Spirometra erinaceieuropaei จากสนขในจงหวดหหนาน ประเทศจน โดยสวนหนงของยน nad5 (pnad5) และ rrnS (prrnS) ถกเพมจานวนแยกตางหากจาก S. erinaceieuropaei ดวยวธ Polymerase chain reaction (PCR) โดยผลผลต amplicons ทไดนาไปวเคราะหลาดบพนธกรรมเพอประเมนหาความแปรปรวนของลาดบพนธกรรม โดยpnad5 และ prrnS มขนาดเทากบ 531 และ 328 bp ตามลาดบ พบวาความแตกตางภายในลาดบพนธกรรมของแตละตวอยางของ S. erinaceieuropaei มคาเทากบ 0-3.5% สาหรบ pnad5 และ 0-1.4% สาหรบ prrnS ในขณะทความแตกตางระหวางลาดบพนธกรรมของแตละตวอยางของ S. erinaceieuropaei มคาเทากบ 12.2-35.7% สาหรบ pnad5 และ 10.4-11.8% สาหรบ prrnS การวเคราะห phylogenetic ดวยวธ neighbour joining (NJ) maximum likelihood (ML) และ maximum parsimony (MP) ชใหเหนวาเชอทแยกไดจากจงหวดหหนานทงหมดเปนตวแทนของ S. erinaceieuropaei ผลการศกษานแสดงใหเหนอยางชดเจนถงประโยชนของลาดบพนธกรรมของไมโตคอนเดรย ในการศกษาดานพนธศาสตรประชากรของ S. erinaceieuropaei ในดานวทยาศาสตรสขภาพของคนและสตว

คาสาคญ: ความแปรปรวนของลาดบพนธกรรม ลาดบพนธกรรมของไมโตคอนเดรย การวเคราะห phylogenetic Spirometra erinaceieuropaei Department of Plastic and Aesthetic, Affiliated Ruikang Hospital of Guangxi Traditional Chinese Medicine University, Nanning 530011, Guangxi Zhuang Autonomous Region, China *ผรบผดชอบบทความ E-mail: 404877308@qq.com (LL)

Introduction Sparganosis is an important worldwide parasitic disease caused by infection with spargana, the plerocercoid larvae of various diphyllobothroid tapeworms belonging to the genus Spirometra (Ooi et al., 2000; Pampiglione et al., 2003; Wiwanitkit et al., 2005). The spargana invade mainly the brain, spinal cord, eye, subcutaneous tissues and abdominal cavity, and can cause blindness, and even death (Li et al., 2009). This disease is most frequently found in Asia countries (including China), and causes significant public health problem in humans and major economic impact in animals (Cui et al., 2011).

Molecular approaches for studying the taxonomy, systematics, and population genetics of animals are available recently. Mitochondrial DNA (mtDNA) sequences have been proven useful and reliable genetic markers due to their maternal inheritance, fast rate of evolutionary change, and relatively conserved genome structures than nuclear ribosomal genome (McManus and Bowles, 1996; Blouin, 2002). Previous studies have shown that the cytochrome c oxidase subunit 3 gene (cox3) is the preferred gene for genetic variation and phylogentic analyses (Zarowiecki et al., 2007), and NADH dehydrogenase subunits 1 and 4 genes (nad1 and

nad4) also have more characters of phylogentic information and variability (Gasser et al., 1999; Zhao et al., 2009). Therefore, these mt genes can provide markers for both phylogenetic and population studies. In the S. erinaceieuropaei, recent studies have shown that the cytochrome c oxidase subunit 1 gene (cox3) and NADH dehydrogenase subunits 1 and 4 genes (nad1 and nad4) are the preferred gene for genetic variation and phylogentic analyses (Liu et al., 2002a), but there is a paucity of information on the genetic variation of nad4 and small subunit of ribosomal RNA (rrnS) genes.

The objectives of the present study were to examine sequence variability in mitochondrial nad5 and rrnS regions, among S. erinaceieuropaei isolates from dogs in Hunan province in China. Based on the pnad5 and prrnS sequences, the phylogenetic relationships in S. erinaceieuropaei were also reconstructed.

Materials and Methods Parasites and isolation of genomic DNA: The parasite species, with their sample codes, number of samples, host species and geographical origins are listed in Table 1. Total genomic DNA was extracted from individual samples by sodium dodecyl sulphate/proteinase K treatment, column-purified

Liang L. et al. / Thai J Vet Med. 2013. 43(2): 291-295. 293

(WizardTM DNA Clean-Up, Promega) and eluted into 50 µL H2O according to the manufacturer’s recommendations.

Enzymatic amplification: The primer sets for nad5 and rrnS genes were designed by author based on sequences well-conserved in many distantly related taxa (Table 2). These primers were synthesied on a Biosearch Model 8700 DNA synthesizer (Shanghai, China). PCR reactions (25 μl) were performed in 2 mM of MgCl2, 2.5 μM of each primer, 2.5 μl 10 x rTaq buffer, 0.2 mM of each dNTPs, 1.25 U of rTaq DNA polymerase (Takara), and 1 μl of DNA sample in a thermocycler (Biometra) under the following conditions: after an initial denaturation at 940C for 5 min, then 940C for 30 sec (denaturation), 550C (for pnad5 and prrnS) for 30 sec (annealing), 720C for 30 sec (extension) for 38 cycles, followed by a final extension at 720C for 10 min. These optimized amplification conditions for the specific and efficient amplification of individual DNA fragments were obtained after varying annealing and extension temperatures. One microlitre (5~10 ng) of genomic DNA was added to each PCR reaction. Samples without genomic DNA (no-DNA controls) were included in each amplification run, and in no case were amplicons detected in the no-DNA controls (not shown). Five microlitres of each amplicon were examined by 0.8% (w/v) agarose gel electrophoresis to validate amplification efficiency. PCR products were sent to Sangon Company (Shanghai, China) for sequencing using a primer walking strategy.

Sequences analysis and phylogenetic reconstruction: Sequences of the three mitochondrial genes were separately aligned using the computer program Clustal X 1.83 (Thompson et al., 1997). Pairwise comparisons were made of the level of sequence differences (D) among and within the species using the formula D = 1-(M/L), where M is the number of alignment positions at which the two sequences have a base in common, and L is the total number of alignment positions over which the two

sequences are compared (Chiltonet et al., 1995).

The sequences of two mitochondrial genes available in this study were used for phylogenetic analyses. Three methods, namely neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) were used for phylogenetic re-constructions. Standard unweighted MP was performed using package Phylip 3.67 (Felsenstein, 1995). NJ analysis was carried out using the Dayhoff matrix model implemented by MEGA 4.0 (Tamura et al., 2007), and ML analysis was performed using PUZZLE 4.1 under the default setting (Strimmer and Haeseler, 1996). The consensus tree was obtained after bootstrap analysis, with 1000 replications for NJ and MP trees, and 100 for ML tree, with values above 50% reported. To study the genetic relationships among cestodes, other cestodes were considered into the present study (Diphyllobothrium nihonkaiense NC_009463; D. latum AB269325; Spirometra erinaceieuropaei NC_011037; Taenia asiatica NC_004826; T. crassiceps NC_002547; T. saginata NC_009938; T. solium NC_004022; Echinococcus multilocularis AB018440; E. granulosus NC_008075; Hymenolepis diminuta AF314223), with Ascaris suum (GenBank accession number HQ704901) as the outgroup. Phylograms were drawn using the Tree View program version 1.65 (Page, 1996).

Results and Discussion Genomic DNA was extracted from 30

individual cestodes representing 11 geographical locations in Hunan province, China (Table 1). Pnad5 and prrnS (~580 and 380 bp, respectively) were amplified individually and subjected to agarose gel electrophoresis. The results showed that no size variation was detected on agarose gels among any of the amplicons examined for each mtDNA region. To assess sequence variation in these two mtDNA regions within and between isolates, amplicons of

Figure 1 Phylogenetic relationship among examined cestode species inferred by maximum parsimony (MP), maximum likelihood (ML) and neighbour joining (NJ) analyses based on combined mitochondrial dataset (pnad5+prrnS) sequences, using one nematode species (Ascaris suum) as outgroup. Numbers along branches indicate bootstrap values resulting from different analyses in the order: MP/ML /NJ.

294 Liang L. et al. / Thai J Vet Med. 2013. 43(2): 291-295.

pnad5 and prrnS from samples representing different isolates were selected and then subjected to sequencing. The sequences of pnad5 and prrnS were 531 and 328 bp in size, respectively. The intra-specific sequence variations within each of the S. erinaceieuropaei were 0-3.5% for pnad5 and 0-1.4% for prrnS; while the inter-specific sequence variations within each of the S. erinaceieuropaei were 12.2-35.7% for pnad5 and 10.4-11.8% for prrnS, consistent with those recently reported (Liu et al., 2012a).

For the pnad4 and prrnS, intra-specific nucleotide variation was related mainly to changes at the third codon position, while fewer changes were detected at the first or second codon positions, consistent with results of other organisms (Li et al., 2008; Zhao et al., 2009). The combined sequences of pnad5 and prrnS representing different isolates were aligned over a consensus length of 859 bp. Topologies of all trees constructed by different methods (NJ, MP, and ML) with different building strategies and/or different distance models were identical or similar, with only small difference of bootstrap values (Fig 1). These results indicate that all the isolates in Hunan Province represent S. erinaceieuropaei. From the phylogenetic tree, parasites of genus Diphyllobothrium were sister to the genus Spirometra, and S. erinaceieuropaei and D. nihonkaiense were more closely related to the other members of the Diphyllobothrium genus (D. latum), consistent with results of recent classifications based on complete mitochondrial genome datasets (Liu et al., 2011; Liu et al., 2012b).

Table 1 Geographical origins (different locations in Hunan province) of Sparganum erinaceieuropaei samples used in the present study, as well as their GenBank accession numbers for sequences of partial mitochondrial NADH dehydrogenase subunits 5 (pnad5) and small subunit of ribosomal RNA (prrnS) genes

GenBank accession number Sample

codes Geographical origin Pnad1 Pnad4

SECS1 Changsha GU946413 GU946433 SECS2 Changsha GU946414 GU946434 SECS3 Changsha GU946415 GU946435 SECS4 Changsha GU946416 GU946436 SECS5 Changsha GU946417 GU946437 SECZ1 Chenzhou GU946418 GU946438 SECZ2 Chenzhou GU946419 GU946439 SECZ3 Chenzhou GU946420 GU946440 SECZ4 Chenzhou GU946421 GU946441 SECZ5 Chenzhou GU946422 GU946442 SECD1 Changde GU946423 GU946443 SECD2 Changde GU946424 GU946444 SECD3 Changde GU946425 GU946445 SECD4 Changde GU946426 GU946446 SECD5 Changde GU946427 GU946447 SEXX1 Xiangxi GU946428 GU946448 SEXX2 Xiangxi GU946429 GU946449 SEXX3 Xiangxi GU946430 GU946450 SEXX4 Xiangxi GU946431 GU946451 SEXX5 Xiangxi GU946432 GU946452

In conclusion, the genetic variability among S. erinaceieuropaei isolates from China could be revealed by sequences of two mitochondrial DNA genes. For the two mt DNA genes, genetic variation of pnad5 was higher than prrnS. The results of the present study also have implications for the diagnosis and control of S. erinaceieuropaei infections in animal and human health significance.

Table 2 Sequences of primers used to amplify a portion of the mitochondrial NADH dehydrogenase subunits 5 (pnad5) and small subunit of ribosomal RNA (prrnS) genes from Sparganum erinaceieuropaei in the present study.

Name of primer Sequence (5′ to 3′) For pnad5

Senad5F TCATACTGGGTCTATCAGGTGTT Senad5R ACAGCAAAGTTAGGGGGTAATAGGT

For prrnS SerrnSF TAGTTTGGCAGTGAGTTATTCCG SerrnSR GGCTACCTTGTTACGACTTACCTCA

References Blouin MS 2002. Molecular prospecting for cryptic

species of nematodes: mitochondrial DNA versus internal transcribed spacer. Int J Parasitol. 32: 527-531.

Chilton NB, Gasser RB and Beveridge I 1995. Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). Int J Parasitol. 25: 647-651.

Cui J, Lin XM, Zhang HW, Xu BL and Wang ZQ 2011. Sparganosis, Henan Province, central China. Emerg Infect Dis. 17: 146-147.

Felsenstein J 1995. PHYLIP (Phylogeny Inference Package), version 3.57c, Department of Genetics, University of Washington, Seattle, WA.

Gasser RB, Zhu X and McManus DP 1999. NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda). Int J Parasitol. 29: 1965-1970.

Li MW, Lin RQ, Song HQ, Sani RA, Wu XY and Zhu XQ. 2008. Electrophoretic analysis of sequence variability in three mitochondrial DNA regions for ascaridoid parasites of human and animal health significance. Electrophoresis. 29: 2912-2917.

Li MW, Lin HY, Xie WT, Gao MJ, Huang ZW, Wu JP, Li C, Lin RQ and Zhu XQ. 2009. Enzootic sparganosis in Guangdong, China. Emerg Infect Dis. 15: 1317-1318.

Liu GH, Lin RQ, Li MW, Liu W, Liu Y, Yuan ZG, Song HQ, Zhao GH, Zhang KX and Zhu XQ. 2011. The complete mitochondrial genomes of three cestode species of Taenia infecting animals and humans. Mol Biol Rep. 38: 2249-2256.

Liu W, Liu GH, Li F, He DS, Wang T, Sheng XF, Zeng DL, Yang FF and Liu Y. 2012a. Sequence variability in three mitochondrial DNA regions of Spirometra erinaceieuropaei spargana of human and animal health significance. J Helminthol, 86: 271-275.

Liu GH, Li C, Li JY, Zhou DH, Xiong RC, Lin RQ, Zou FC and Zhu XQ. 2012b. Characterization of the complete mitochondrial genome sequence of Spirometra erinaceieuropaei (Cestoda:

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Diphyllobothriidae) from China. Int J Biol Sci. 8: 640-649.

McManus DP and Bowles J 1996. Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics. Int J Parasitol. 26: 687-704.

Ooi HK, Chang SL, Huang CC, Kawakami Y and Uchida A 2000. Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats. J Helminthol. 74: 173-176.

Page RD 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci. 12: 357-358.

Pampiglione S, Fioravanti ML and Rivasi F 2003. Human sparganosis in Italy. Case report and review of the European cases. APMIS: Acta Pathologica, Microbiol Immunol. 111: 349-354.

Strimmer K and Haeseler AV 1996. Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol Biol Evolution. 13: 964-969.

Tamura K, Dudley J, Nei M and Kumar S 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 24: 1596-1599.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F and Higgin DG 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality Analysis tools. Nucleic Acids Res. 24: 4876-4882.

Wiwanitkit V 2005. A review of human sparganosis in Thailand. Int Infect Dis. 9: 312-316.

Zarowiecki MZ, Huyse T and Littlewood DT 2007. Making the most of mitochondrial genomes--markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea). Int J Parasitol. 37: 1401-1418.

Zhao GH, Mo XH, Zou FC, Weng YB, Lin RQ, Xia CM and Zhu XQ 2009. Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes. Vet Parasitol. 162: 67-74.

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Surgical Removal of Foreign Bodies in the Gastrointestinal

Tract of Monocellate Cobra, Naja kaouthia

Taksa Vasaruchapong* Lawan Chanhome

Abstract

A wild-captured female monocellate cobra (Naja kaouthia) had abnormal posture and move with difficulty as

consequences of swelling at the middle third of the body. The swelling area was firm on palpation with average size of 10x4 cm. The shape of the swelling area was similar to a small bottle which was obviously recognized on the dorsal recumbency. Therefore, the foreign body obstruction in gastrointestinal tract was diagnosed without radiographic examination. Digital manipulation was performed, but could not move the foreign bodies. Therefore, gastrotomy was considered. The foreign bodies were a plastic bottle and a piece of cloth which were removed. Feeding was withdrawn for one week with parenteral supportive treatment with fluid. The snake fully recovered 1 month after surgery.

Keywords: foreign body, gastrotomy, GI obstruction, Naja kaouthia Snake Farm, Queen Saovabha Memorial Institute, The Thai Red Cross Society 1871 Rama 4 Rd. Patumwan Bangkok, 10330, Thailand *Correspondence author E-mail: taksa.v@gmail.com, taksa.v@redcross.or.th

Short Communication

298 Vasaruchapong T. and Chanhome L. / Thai J Vet Med. 2013. 43(2): 297-300.

บทคดยอ

การผาตดแกไขภาวะสงแปลกปลอมอดตนในกระเพาะอาหารงเหาไทย (Naja kaouthia)

ทกษะ เวสารชชพงศ * ลาวณย จนทรโฮม

งเหาไทยเพศเมยถกจบจากแหลงอาศยในธรรมชาต ไมทราบอาย มอาการบวมบรเวณกลางลาตว ทาใหไมสามารถเคลอนไหวได

ตามปกต เมอคลาตรวจบรเวณทบวมพบวามลกษณะแนนเปนกอนขนาดประมาณ 10x4 เซนตเมตร เมอจบงนอนหงายพบวาบรเวณทบวมมลกษณะรปทรงเหมอนขวด จงวนจฉยเบองตนวามสงแปลกปลอมอดตนในทางเดนอาหารโดยไมไดทาการถายภาพรงส การบบไลดวยมอไมสามารถดนใหสงแปลกปลอมดงกลาวเคลอนออกมา จงพจารณาผาตดเปดทางเดนอาหารอาหารเพอนาเอาสงแปลกปลอมออก จากการผาพบวามขวดนมเปรยวหนงขวดและเศษผาหนงชนภายในกระเพาะอาหาร ภายหลงการผาตดไดงดใหอาหารและรกษางแบบประคองอาการดวยการฉดสารนาเขาชนใตผวหนงเปนเวลาหนงสปดาห จากนนจงเรมใหกนของเหลว อาหารขนาดเลก และอาหารขนาดปกต ตามลาดบ งหายเปนปกตภายหลงการผาตดหนงเดอน

คาสาคญ: สงแปลกปลอม การผาเปดกระเพาะอาหาร ทางเดนอาหารอดตน งเหาไทย สวนง สถานเสาวภา สภากาชาดไทย 1871 ถ.พระราม 4 แขวงปทมวน เขตปทมวน กรงเทพฯ 10330 ประเทศไทย *ผรบผดชอบบทความ E-mail: taksa.v@gmail.com, taksa.v@redcross.or.th

Introduction Snake is a carnivorous animal which swallows whole body of the prey without chewing or tearing into small pieces. Mistaken swallow of a variety of foreign bodies such as stone, golf ball, heating pad and artificial chicken egg have been reported in snakes (Smith, 1953; Jacobson et al., 1980; Zwart et al., 1986, Souza et al., 2004). These foreign bodies cause obstruction in the gastrointestinal tract. If the snake cannot pass the foreign bodies via feces or regurgitation, total obstruction and death are the consequences. In Thailand, surgery in reptile and native snake is rare and there is lack of its basic knowledge. Therefore, the aim of this case report was to describe the surgical removal of unmovable foreign bodies in the gastrointestinal tract of a snake.

Materials and Methods Case history: A wild-captured female monocellate cobra (Naja kaouthia) of unknown age was caught and donated to Snake Farm, Queen Saovabha Memorial Institute (QSMI), The Thai Red Cross Society. The snake had a 145-cm snout to vent and weighed one kg. Upon arrival, the snake was presented with abnormal posture and difficulty in moving as consequences of swelling at the middle third of the body.

Diagnosis and treatment: Physical examination revealed moderate dehydration and pale mucous membrane. The swollen area was firm on palpation with average size of 10x4 cm. The shape of the swollen area is similar to a small bottle which was obviously recognized on the dorsal recumbency (Fig 1). Therefore, foreign body obstruction in the

gastrointestinal tract was diagnosed without radiographic examination. Digital manipulation was performed, but the bottle was unmovable. Therefore, surgical removal was considered. Preoperative packed cell volume, uric acid, alkaline phosphatase and aspartate aminotransferase were in normal range (Diethelm and Stein, 2006). The snake received subcutaneously acetate ringer solution (Acetar, Otsuka, Thailand) at the dose of 20 ml/kg (Carpenter, 2005) for 2 hours before anesthesia.

The snake was anesthetized with ketamine at the dose of 40 mg/kg (Calypsol, Gedeon Richter Ltd., Budapest, Hungary) and xylazine at the dose of 2 mg/kg (Rompun, Bayer Korea Ltd., Seoul, South Korea) by intramuscular injection. The incision line was made between the first and the second dorsal scale rows on the left lateral side of the body above the midpoint of the mass. The foreign body was found in the stomach, therefore gastrotomy was performed through a longitudinal stab incision at the cranial part of the stomach. The foreign bodies were a

Figure 1 Swelling of the middle third of the snake. A foreign

body with a shape of bottle was diagnosed.

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plastic bottle and a piece of cloth which were gently removed from the stomach (Fig 2 and 3). The stomach was flushed with normal saline and sutured with 4-0 polyglyconate (Connek, Novatec Healthcare, Thailand) in Lambert and Cushing suture patterns, respectively (Fig 4). Normal saline was injected into stomach lumen to test leakage at the suture line. The skin was sutured with 2-0 nylon (Nylon, UNIK, Taipei Hsien, R.O.C., Taiwan) in an everting horizontal mattress pattern. Ceftazidime was given at the dose of 20 mg/kg (Cef-4, Siam Bheasach, Thailand) by intramuscular injection every 72 hours for 5 treatments (Carpenter, 2005).

Feeding was withdrawn for one week after surgery with parenteral supportive treatment with 20 ml/kg acetate ringer solution by subcutaneous injection, vitamin B complex (Biocatalin, Fatro, Bologna, Italy) at the dose of 10 mg/kg and vitamin C (Vitamin C, T.P. Drug Laboratories, Thailand) at the dose of 20 mg/kg given once by intramuscular injection (Carpenter, 2005). After one week, 20 ml of acetate ringer solution was given per oral for 2 days. On days 10 and 15, the snake received force feeding with 12 gram pre-killed mouse. Followed by 20 gram pre-killed mice on day 22.

Results and Discussion The suture was removed after shedding of

the skin, the incision line was completely healed (Fig 5). The snake was fully recovered within 1 month after surgery. Defecation was normal which indicated normal function of the gastrointestinal tract. The snake was active and had a good appetite and no abnormal clinical signs.

Snake uses sense of smell and taste to choose its prey (McKeown, 1996). Therefore, there could be some kind of smell that contaminates the foreign body and cause the snake to mistakenly swallow.

Radiographic examination could provide better information for diagnosis. In this case, even though the obstruction was diagnosed correctly, a piece of cloth was missed out. If there had been any foreign bodies remaining in other parts of the gastrointestinal tract, they would have been left unnoticed. Therefore, the radiographic examination of the gastrointestinal tract should be performed for better diagnosis.

Figure 2 Removal of a plastic bottle from the stomach.

Basic principle of reptile gastrointestinal surgery is similar to mammals. However some consideration should be concerned in reptile surgery. Reptile skin, especially of snake and lizard, is the primary holding layer for wound closure and likely to invert after incision. Therefore, mattress suture gives strong evertion of skin which is advantageous to wound healing. Nylon and polypropylene are the preferred non-absorbable sutures used for skin suture (Mader et al., 2006) while polyglyconate and poliglecaprone 25 are the preferred absorbable sutures with the least tissue reaction (Govett et al., 2004). Wound healing in reptiles has the same process as in mammals but slower. Optimum temperature can promote wound healing (Mader et al., 2006).

Figure 3 Removal of a piece of cloth from the stomach.

Figure 4 Suturing of the stomach.

Figure 5 Complete healing of the incision line at one month after surgery.

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Therefore, the suture is generally removed 4-6 weeks after surgery or after shedding of the skin (Mader et al., 2006). There is no report on snake gastrointestinal surgery in Thailand and Thai native snakes. Although, reptiles have become more popular in Thailand, there is still lack of veterinary practice on reptile medicine and surgery. This report could encourage reptile study in Thailand and gather the information on surgery in Thai native snake.

References Carpenters JW 2005. Exotic Animal Formulary. 3rd ed.

Elsevier Saunders. St. Louis: 55-93. Diethelm G and Stein G 2006. Hematology and blood

chemistry values. In Reptiles: Reptile Medicine and Surgery. 2nd ed. WB Saunders Co, Philadelphia: 1103-1107.

Govett PD, Harms CA, Linder KE, Marsh JC and Wyneken J 2004. Effect of four different suture materials on the surgical wound healing of Loggerhead Sea Turtles, Caretta caretta. J Herp Med Surg. 14(4): 6-11.

Jacobson E, Calderwood H and Spencer C 1980. Gastrotomy in a gulf hammock snake (Elaphe obsolete williamsi). Vet Med Small Anim Clin. 75: 879-880.

Smith HM 1953. Case history of a snake with an irregurgitable egg. Herpetologica. 9: 93-95.

Souza MJ, Hall KE, Wilson DW and Lewbart GA 2004. Surgical removal of an artificial chicken egg from the gastrointestinal tract of black rat snake, Elaphe obsolete. J Herpetol Med Surg. 14(4): 4-5.

Zwart P, Volkers V, Wijnands M and Gerritsen R 1986. Foreign body in the stomach of a snake, surgical removal. Tijdschr Diergeneeskd. 111: 925-927.

Mader DR, Bennett RA, Funk RS, Fitzgerald KT, Vera R and Hernandez-Divers SJ 2006. Surgery. In: Reptile Medicine and Surgery. 2nd ed. WB Saunders Co, Philadelphia: 581-600.

McKeown S 1996. General husbandry and management, In: Reptile Medicine and Surgery. 1st ed. WB Saunder Co, Philadelphia: 9-19.

Thai J Vet Med. 2013. 43(2): 301-306.

Implications of Cat Ownership Statistics and Social Changes:

A Longitudinal Study in Taiwan from 2001 to 2009

Li-Huan Chen1 Ting-Wei Chang1 Meng-Chih Tung2 Chang-Young Fei1 I-Tsun Chiang1,3*

Abstract

Pet ownership statistics in Taiwan had not been investigated until Animal Protection Act was announced on

November 4, 1998. This longitudinal study aims to investigate the demographic information of household cats in Taiwan and discuss its implications with social changes from 2001 to 2009 biennially. Household cats were surveyed biennially by randomly dialed telephone survey. A structured questionnaire was designed to obtain household demographics and population-associated data on pet ownership. Related social changes data were obtained from the Statistical Yearbook of the Ministry of Interior, Taiwan. Results showed that household cat ownership increased from 2001 to 2009, but the average number of household cats owned by each household decreased every year. The study concluded that the increase in cat ownership might be caused by the following reasons: (1) dramatic rise in life stress and social/environmental/economic pressures of owners who had strong needs of self-objective fulfillment and comfort, (2) less care and attention required for cats than dogs, and (3) the availability of care-giving for cat is decreasing with the declining of the number of people within a household in Taiwan.

Keywords: cat ownership, household cats, human-animal bound, pet, social change 1 Taiwan Animal Welfare Research Center, School of Veterinary Medicine, National Taiwan University, No.1, Sec. 4, Roosevelt Rd., Da’an Dist., Taipei, 10617 Taiwan, R.O.C. 2 Changhua County Government, No.416, Sec. 2, Jungshan Rd., Changhua County, 50093 Taiwan, R.O.C. 3 National Changhua University of Education, No.1, Jin-De Road, Changhua 500, Taiwan, R.O.C. *Correspondence author E-mail: john@cc.ncue.edu.tw

Short Communication

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บทคดยอ

ผลกระทบจากสถตเจาของแมวและการเปลยนแปลงทางสงคม: การศกษาระยะยาวในไตหวน 2001-2009

Li-Huan Chen1 Ting-Wei Chang1 Meng-Chih Tung2 Chang-Young Fei1 I-Tsun Chiang1,3*

สถตเจาของสตวเลยงในไตหวนไมไดรบการตรวจสอบจนพระราชบญญตคมครองสตวไดมการประกาศเมอ 4 พฤศจกายน 1998

การศกษาครงนมวตถประสงคเพอตรวจสอบขอมลประชากรของแมวในครวเรอนในไตหวนในระยะยาวและหารอเกยวกบผลกระทบตอการเปลยนแปลงทางสงคมทกสองประหวางป 2001-2009 ทาการสารวจแมวเลยงทกสองปโดยการสอบถามดวยการสมโทรทางโทรศพท แบบสอบถามถกออกแบบมาเพอใหไดขอมลประชากรในครวเรอนและขอมลทเกยวของกบประชากรจากเจาของสตวเลยง ขอมลทเกยวของกบการเปลยนแปลงทางสงคมทไดรบจากรายงานประจาปทางสถตของกระทรวงมหาดไทย ประเทศไตหวน ผลการศกษาพบวาจานวนผเลยงแมวเพมขนจากป 2001-2009 แตจานวนแมวเฉลยตอครวเรอนลดลงทกป ผลการศกษาสรปไดวาการเพมจานวนของเจาของแมวอาจจะเกดจากสาเหตดงตอไปน (1) ประชากรแมวอาจเพมขนจากความเครยดและความกดดนทางสงคม / สงแวดลอม / เศรษฐกจของเจาของทตองการบรรลเปาหมายของตนเองและความสะดวกสบาย (2) การทการดแลแมวสามารถใชเวลานอยกวาการดแลสนข และ (3) การลดลงของความพรอมในการดแลแมวลดลงพรอมกบการลดลงของจานวนคนในครวเรอนในไตหวน

คาสาคญ: เจาของแมว แมวในครวเรอน ความสมพนธระหวางคนและสตว สตวเลยง การเปลยนแปลงทางสงคม 1 Taiwan Animal Welfare Research Center, School of Veterinary Medicine, National Taiwan University, No.1, Sec. 4, Roosevelt Rd., Da’an Dist., Taipei, 10617 Taiwan, R.O.C. 2 Changhua County Government, No.416, Sec. 2, Jungshan Rd., Changhua County, 50093 Taiwan, R.O.C. 3 National Changhua University of Education, No.1, Jin-De Road, Changhua 500, Taiwan, R.O.C. *ผรบผดชอบบทความ E-mail: john@cc.ncue.edu.tw

Introduction Public data published on domestic pet demographics have been investigated and available in many countries (Downes et al., 2009) such as the US (AVMA, 2007), Australia (Baldock et al., 2003) and Brazil (Serafini, 2008). It is important to understand and analyze the trends and size of domestic pet population for government and industry to plan or make decision (Nassar and Mosier, 1991). For example, related pet-associated issues such as pollution and zoonotic disease, which become more relevant to increase in pet population (Stirling et al., 2008). Moreover, domestic pets which are abandoned and become free-roaming animals have long caused principally public-health problems and animal welfare concerns (Slater, 2000). To regulate and control pet population problems, a regularly recurrent investigation into pet population is essential. The size of pet population must be established and the information can assist in pet population control (Nassar and Fluke, 1991). Reliable estimations of the populations of dogs and cats are very crucial to efficient enforcement as well as assessment of animal welfare, veterinary epidemiology, potential risks of zoonoses, market of pet food, governmental budget, and animal policy. In Taiwan, the Animal Protection Act was announced and enforced on November 4, 1998, making Taiwan the 54th country in the world to put animal protection into legislation and mandating

that the competent authority is the Council of Agriculture, the Executive Yuan (Phipps, 2005; Animal Legal and Historic Center, 2010). However, Taiwan had not surveyed populations of pets and other related works regularly as well as lawfully until Animal Protection Act was announced. Therefore, this study complied with the law and discussed sourced data from the surveys on household cats from 2001 to 2009.

Materials and Methods In the years from 2001 to 2009, household

cats were surveyed biennially by telephone survey which was randomly dialed and followed the method developed by Murray et al. (2010). A structured questionnaire was designed to obtain household demographics and population-associated data on pet ownership. The selection methods of sample were designed by random rules based on the official annual census of national household distribution. There are 99% confidence level for all surveys and margin of error was plus and minus 3%. Calculations of the questionnaire results were made by using database Visual FoxPro version 6.0. The estimates of the total Taiwan cat populations every two years from 2001 to 2009 using the descriptions shown in Table 1. The estimates of the number of total household cats were affected by two factors, the number of total households in Taiwan and cat ownership percentage

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which is the average number of cats owned by cat-owning household. According to a study of dog population in Taiwan (Tung et al., 2010), the number of household cats per 100 people in Taiwan could be estimated as: (Total household cats/Total human population)*100%. Annual resident populations and numbers of households in Taiwan from 2001 to 2011 were cited from the Statistical Yearbook of Interior, the Executive Yuan, Taiwan, file code 2 – y02_12 in Chinese. Annual total number of national domestic violence of Taiwan from 2005 to 2011 were cited from the Statistical Yearbook of Interior, the Executive Yuan, Taiwan, file code y09_02 in Chinese.

Results and Discussion As shown in Table 1, our study investigated

household cat population biennially from 2001 to 2009, in total of five times. Our biennial investigations of cat populations from 2001 to 2009 were 238,727 cats, 276,037 cats, 266,859 cats, 345,623 cats and 346,667 cats. The corresponding percentages of cat owning households in Taiwan were 2.06%, 2.31%, 2.20%, 2.76%, and 2.80%. For every one hundred people investigated, there were 1.07 cats, 1.22 cats, 1.17 cats, 1.51 cats, and 1.50 cats. Figure 1 is summarized from Table 1, showing the trends of total cat population from 2001 to 2009, and the number of cats per household. As Figure 1 shows, total cat

population in Taiwan increased since 2007, but the average number of cats per household decrease from 1.71 cats per household in 2005 to 1.59 cats per household in 2009.

As shown in Table 2, the numbers of people per household declined from 3.29 people/household to 2.88 people/household during 2001 to 2011. Table 3 shows that the annual total number of national domestic violence of Taiwan increased every year from 2005 to 2011, as shown subsequently, 66,080 cases in 2005, 70,842 cases in 2006, 76,755 cases in 2007, 84,195 cases in 2008, 94,927 cases in 2009, 112,798 cases in 2010, 117, and 162 cases in 2011.

Figure 1 Trend of cat population and number of cats per

household in Taiwan from 2001 to 2009.

Table 1 Values of relevant origin and estimation process of the numbers of household cats in Taiwan from 2001 to 2009. Items Years

2001 2003 2005 2007 2009 No. households being surveyed = a 9,629 10,019 11,427 10,990 16,602 Confidence level 99% 99% 99% 99% 99% No. households owning cats = b 198 231 251 303 465 Percent of households owning cats = c (b/a) 2.06% 2.31% 2.20% 2.76% 2.80% Total number of cats owned by surveyed families = d 339 394 419 507 740 Average number of cats for each cat-owning family = e (d/b) 1.71 1.71 1.67 1.67 1.59 Total number of human household* = f 6,780,828 7,019,327 7,277,800 7,491,916 7,777,527 Total household cats number in Taiwan = g (c*e*f) 238,727 276,037 266,859 345,623 346,667 Taiwan human population* = h 22,368,502 22,573,965 22,744,839 22,925,311 23,086,441 Number of household cats per 100 capita = g/h/100 1.07 1.22 1.17 1.51 1.50 *Department of Household Registration, Ministry of the Interior, the Executive Yuan. Statistical Yearbook of Interior, http://sowf.moi.gov.tw/stat/year/elist.htm

Table 2 Number of resident populations, numbers of households of Taiwan, from 2001 to 2011.

Unit: person

Years Households (=a) Population (=b) Average person per household (=b/a) 2001 6,802,281 22,405,568 3.29 2002 6,925,019 22,520,776 3.25 2003 7,047,168 22,604,550 3.21 2004 7,179,943 22,689,122 3.16 2005 7,292,879 22,770,383 3.12 2006 7,394,758 22,876,527 3.09 2007 7,512,449 22,958,360 3.06 2008 7,655,772 23,037,031 3.01 2009 7,805,834 23,119,772 2.96 2010 7,937,024 23,162,123 2.92 2011 8,057,761 23,224,912 2.88

Resource: Statistical Yearbook of Interior, the Executive Yuan, Taiwan from: file code= 2 – y02_12 in Chinese.

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This study investigated the biennial total cat population and number of cats per household in Taiwan from 2001 to 2009 and noticed that the total cat population was less than total dog population (Tung et al., 2010). In Tung’s study in 2009, the total dog population was 1,565,156 in 2009, but the total cat population was 346,667 (Table 1). However, since 2001, the total cat population increased (as shown in Fig 1), while the total dog population decreased simultaneously (Tung et al., 2010).

According to recent studies of Beck and Katcher (2003), Brown (2007) and White (2009), companion animals were regarded as family members with the most desired attributes to release life and social pressure and the law shall provide that abandonment of companion animals is considered criminal act against the law. Therefore, dramatic rise in life stress and social/environmental/economic pressures of owners might be one of the major reasons of the increase in cat ownership in Taiwan. According to the U.S. Census Bureau (2012), the population density (persons per sq. km.) of Taiwan ranked 15th in the whole world in 2011. The total population and total household numbers were 23,224,912 people and 8,057,761 households. The number of people per household from 2001 to 2011 decreased every year as shown in Table 2, 3.29 people/household, 3.25 people/ household, 3.21 people/household, 3.16 people/ household, 3.12 people/household, 3.09 people/ household, 3.06 people/household, 3.01 people/ household, 2.96 people/household, 2.92 people/ household, and 2.88 people/household, respectively. Conversely, the total cases of domestic violence, as shown in Table 3, increased from 66,080 cases in 2005 to 117,162 cases in 2011. Observing the above data, life stress and social/environmental/economic pressures increased annually in Taiwan.

Recent research identified that companion animals played an important role in general well-being, including physical, psychological, and social aspects of human kinds. Several studies stated that companion animals had positive physical and emotional impacts on patients who have chronic diseases, heart diseases, dementia, or cancer (Kaminsky et al., 2002; Johnson et al., 2005; Friedmann and Tsai, 2006). Pets could also alleviate depression in the elderly and HIV patients psychologically (Jessen et al., 1996; Siegel, 2011). Furthermore, they provide assistance in children’s medicine and palliative care (Geisler, 2004). Most pet owners choose to have pets to fulfill their psychological needs rather than for physical health purposes. Based on self-psychology perspective, the cohesion of the owner’s sense of self is maintained because pets can play an important role as self-objects. Pet owners’ sense of self was affirmed by the subjective psychological reality of emotional reliance to the pet (Brown, 2004a). In other words, companion animals provide a calming effect when a pet owner encounters stress and negative emotions (Allen et al., 1991). The calming effect of companion animals to their owners may be a self-object function that helps the owner in maintaining an aspect of the

self. In addition, some studies also identified that consistent and long-term petting and stroking of a companion animal could bring relaxation and comfort to combat emotional stress (Virués-Ortega and Buela-Casal, 2006). The interaction between human and animals alleviates anxiety, depression, and loneliness; moreover, it brings a sense of social support and fulfillment (Friedmann and Tsai, 2006). A study showed that pets increased neighbor interactions and senses of community because pet owners might have more chances to interact with others by taking their pets out (Wood et al., 2007). Companion animals provide a temporary harbour to our emotions where people live under tremendous stress and economic crises (Walsh, 2009). Regardless of the age group within a family, either children, adults, or elders, they all benefit from animal interaction physically, psychologically and socially.

Highly sociable animals like dogs and cats are most suitable as self-object companion animals (Palley et al., 2010). However, because civilized cities around the world are mostly composed of apartments, cats are more chosen as pets than dogs because cats are more independent, require less space for exercising, rely less on their owners, and are less controversial, especially when it comes to barking and implicating neighbour quarrels (Schwartz, 2002; Hare, 2004; Milani, 2004; Fuh et al., 2012). Investigation in the United Kingdom by Murray and his colleagues (2010) showed that the more people in a household, the greater the possibility of possessing a pet dog; there is a strong positive correlation between the two factors (p < 0.001); however, the more people who achieve higher education in a household, the greater the possibility of possessing a pet cat than a pet dog (p = 0.001). Furthermore, in a family with pet dogs, the chance of having another pet cat doubles when there is an 11 to 15 year-old child in the family. Similarly, Westgarth (2007) and Leslie (1994) both discovered that families which had exceed five family members and no children under the age of six had greater possibility to have a dog than those families with less than five individuals and with children under age of six. Researchers believed that the major reason for these data was the greater responsibility of dog ownership in time devotion than in cat ownership. In general, the availability of time for companion animals is one of the crucial factors in having a pet (Brown, 2004b).

Table 3 Annual total number of national domestic violence of Taiwan, from 2005 to 2011

Unit: individual case Years National total cases 2005 66,080 2006 70,842 2007 76,755 2008 84,195 2009 94,927 2010 112,798 2011 117,162

Resource: Statistical Yearbook of Interior, the Executive Yuan, Taiwan from: file code y09_02 in Chinese.

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According to the high density of population and the increase in domestic violence in Taiwan, this study concluded that (1) residents with life stress and social/environmental/economic pressures in Taiwan need self-object to achieve self-fulfillment and comfort, and dogs or cats are appropriate companion animals to be self-objects, (2) Taiwanese choose cats over dogs for companion because it takes less time to give care to a cat than a dog, and (3) the availability of care-giving for pets declines because the number of people within a household has decreased over recent 11 years. These may be three conceivable reasons that caused the increasing cat population, decreasing dog population, and decreasing average numbers of dogs or cats owned by households. Further research is suggested to employ different approaches (i.e. qualitative research methodology) to examine those reasons, and possible clinical implications are outlined.

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Thai J Vet Med. 2013. 43(2): 307-311.

Comparison of Repetitive Sequence-based Polymerase Chain

Reaction (rep-PCR) and Pulsed-Field Gel Electrophoresis

(PFGE) for Genetic Characterization of Arcobacter spp.

Panvipa Phasipol 1 Nipa Chokesajjawatee 2* Taradon Luangtongkum 1*

Abstract

Arcobacter has been associated with foodborne illness in humans. Recently, this organism has been receiving

more attention as a pathogen of public health concern. The contamination of Arcobacter is frequently observed in foods of animal origin especially poultry products; however, the source of contamination as well as the molecular epidemiology of Arcobacter is not clearly understood. In the present study, we compared the use of repetitive sequence-based polymerase chain reaction (rep-PCR) and pulsed-field gel electrophoresis (PFGE) for genetic characterization of Arcobacter. Thirty Arcobacter butzleri isolates from retail chicken carcasses and 3 Arcobacter reference strains were typed with rep-PCR and PFGE. Rep-PCR yielded 27 fingerprint patterns, while PFGE yielded 29 PFGE patterns. Two pairs of Arcobacter isolates that exhibited the same rep-PCR pattern yielded different PFGE patterns. Discriminatory power determined by Simpson’s index of diversity of rep-PCR was as high as 0.989, comparable to 0.992 as obtained by PFGE. Concordance of the two methods as determined by Adjusted Rand coefficient was 0.798. Prediction of PFGE results by rep-PCR results was quantified by Wallace coefficient, which showed the value of 0.667. Together, our study shows that rep-PCR can be used as an effective screening tool for studying genetic profiles of Arcobacter.

Keywords: Arcobacter, genetic profiles, PFGE, rep-PCR 1Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Henry Dunant Road, Bangkok 10330, Thailand 2Food Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathum Thani 12120, Thailand

*Correspondence author E-mail: taradon.l@chula.ac.th, nipa.cho@biotec.or.th

Short Communication

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บทคดยอ

การเปรยบเทยบเทคนค repetitive sequence-based PCR (rep-PCR) และ pulsed-field gel electrophoresis (PFGE) ในการศกษาลกษณะทางพนธกรรมของเชออารโคแบคเตอร

พรรณวภา ภาษผล 1 นภา โชคสจจะวาท 2* ธราดล เหลองทองคา 1*

เชออารโคแบคเตอรเปนเชอแบคทเรยททาใหเกดโรคอาหารเปนพษในมนษย ในชวงระยะเวลา 2-3 ปทผานมา เชอนไดรบความ

สนใจเพมมากขนในฐานะเชอแบคทเรยกอโรคทมความสาคญทางสาธารณสข การปนเปอนของเชออารโคแบคเตอรมกพบไดในอาหารทมาจากสตว โดยเฉพาะเนอสตวปก อยางไรกตามแหลงทมาของการปนเปอนรวมถงระบาดวทยาระดบโมเลกลของเชออารโคแบคเตอรยงไมเปนททราบแนชด การศกษาในครงนเปนการเปรยบเทยบการใชเทคนค repetitive sequence-based polymerase chain reaction (rep-PCR) และเทคนค pulsed-field gel electrophoresis (PFGE) ในการศกษาลกษณะทางพนธกรรมของเชออารโคแบคเตอร โดยทาการศกษาเชออารโคแบคเตอรจานวน 30 ตวอยางทแยกไดจากเนอไก และเชออารโคแบคเตอรสายพนธมาตรฐานอกจานวน 3 สายพนธ ผลการจาแนกสายพนธดวยเทคนค rep-PCR พบวาเชออารโคแบคเตอรททดสอบใหรปแบบลายพมพสารพนธกรรมจานวน 27 แบบ สวนเทคนค PFGE นนใหรปแบบลายพมพสารพนธกรรมจานวน 29 แบบ โดยเชออารโคแบคเตอร 2 คทไมสามารถจาแนกลกษณะทางพนธกรรมออกจากกนไดดวยเทคนค rep-PCR สามารถจาแนกลกษณะทางพนธกรรมพนธออกจากกนไดดวยเทคนค PFGE คาแสดงความสามารถในการจาแนกเชอ (Simpson’s index of diversity) ของเทคนค rep-PCR และ PFGE มคา 0.989 และ 0.992 ตามลาดบ คาสมประสทธความสอดคลองของการจาแนกสายพนธ (Adjusted Rand coefficient) ของเชออารโคแบคเตอรดวยวธ rep-PCR และ PFGE มคาเทากบ 0.798 และคาสมประสทธในการทานายผลการจาแนกสายพนธ (Wallace coefficient) ของเทคนค PFGE ดวยเทคนค rep-PCR มคาเทากบ 0.667 จากผลการศกษาทกลาวมาขางตนแสดงใหเหนวาเทคนค rep-PCR สามารถใชเปนเทคนคในการศกษาลกษณะทางพนธกรรมเบองตนของเชออารโคแบคเตอรทมประสทธภาพ

คาสาคญ: เชออารโคแบคเตอร ลกษณะทางพนธกรรม PFGE, rep-PCR 1 ภาควชาสตวแพทยสาธารณสข คณะสตวแพทยศาสตร จฬาลงกรณมหาวทยาลย กรงเทพฯ 10330 2 หนวยวจยเทคโนโลยชวภาพอาหาร ศนยพนธวศวกรรมและเทคโนโลยชวภาพแหงชาต อทยานวทยาศาสตรประเทศไทย ปทมธาน 12120 *ผรบผดชอบบทความ E-mail: taradon.l@chula.ac.th, nipa.cho@biotec.or.th

Introduction Arcobacter is a genus of gram-negative bacterium belonging to the family Campylobacteraceae. This organism has been implicated in human foodborne diarrheal illness and occasionally in bacteremic infections (Ho et al., 2006). Consumption of contaminated foods of animal origin especially chicken products has been considered as the most important risk factor for transmission of this organism to humans (Collado and Figueras, 2011). Although contamination of Arcobacter in chicken meat is commonly observed (Kabeya et al., 2004; Rivas et al., 2004), the source of contamination is not clearly elucidated (Collado and Figueras, 2011). Several molecular genotyping techniques such as repetitive sequence-based polymerase chain reaction (rep-PCR), randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE), and multi-locus sequence typing (MLST) have been used for typing Arcobacter strains in epidemiological studies to trace the sources of contamination or to determine genetic relatedness of Arcobacter isolated strains

(Atabay et al., 2002; Houf et al., 2002; Rivas et al., 2004; Gonzalez et al., 2007; Miller et al., 2009). Although many molecular typing techniques have been applied, a standard typing technique for Arcobacter has not yet been established. PFGE is considered as the gold standard method for typing of several organisms including those in the family Campylobacteraceae since the technique has good discriminatory power and reproducibility (Majella et al., 2006). Nevertheless, PFGE is technically-demanding, time-consuming, and labor-intensive which may not be suitable for screening a large number of samples. Rep-PCR, on the other hand, is a simple and rapid typing technique which has high throughput ability. This technique has also been used for strain characterization of several organisms (Versalovic, 1994). Since the ability of rep-PCR to characterize Arcobacter strains has never been compared with that of PFGE, the objective of the present study was to determine the discriminatory ability and the concordance of rep-PCR and PFGE in typing of Arcobacter isolates in order to assess the use of rep-PCR as an alternative genotypic tool for studying genetic profiles of Arcobacter.

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Materials and Methods Bacterial strains and growth conditions: Thirty A. butzleri isolates used in this study were obtained from a strain collection of the Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University. These Arcobacter isolates were recovered from retail chicken carcasses in Bangkok during 2010-2011. In addition, three Arcobacter strains including A. butzleri NCTC 12481, A. cryaerophilus NCTC 11885, and A. skirrowii CV1103 were also used in this study as quality control organisms. Prior to strain characterization, each Arcobacter isolate was subcultured on blood agar (CM0271, Oxoid, Hampshire, UK) supplemented with 5% defibrinated sheep blood and incubated at 300C for 48 hours under aerobic conditions.

Rep-PCR: Whole cell lysate of the test strains was used as template for rep-PCR amplification. The cells were lysed using alkaline PEG solution as described by Chomcyznski and Rymaszewski (2006). In brief, a quarter loopful of Arcobacter colonies on blood agar were washed and resuspended in 500 µl of the alkaline PEG solution and heated at 900C for 10 min. Then, the mixture was centrifuged at 12,000 rpm for 5 min to pellet the cell debris. Two microliters of the supernatant were used as DNA template for rep-PCR.

Rep-PCR amplifications were performed in 25 µl reaction volumes. Each reaction contained 0.625 U of Ex Taq (Takara Bio Inc., Shiga, Japan), 2.5 µl of 10x Ex Taq buffer (Takara), a deoxynucleotide triphosphates mixture containing each deoxynucleoside triphosphates at a final concentration of 0.2 mM (Takara), 1 µl of 20 µM (GTG)5 primer (5’-GTG GTG GTG GTG GTG-3’), and 2 µl of DNA template. The PCR consisted of an initial denaturation at 950C for 5 min, followed by 30 cycles of 940C for 45 sec, 400C for 1 min, and 650C for 10 min, and a final extension at 650C for 20 min. To evaluate the reproducibility of the technique, A. butzleri NCTC 12481 was included in every batch of rep-PCR amplification. The PCR products were separated in 1% agarose gels (UltraPureTMAgarose, Invitrogen, California, USA) in 0.5x Tris-borate-EDTA buffer at 135 V for 2.2 hours. The gels were stained with 5 µg/ml ethidium bromide for 5 min and then destained in tap water for 20 min. The gel images were visualized and captured by gel scanner (Typhoon 9410, Amersham Pharmacia Biotech Inc., New Jersey, USA).

PFGE: PFGE was performed according to CDC’s standardized PulseNet protocol for Campylobacter jejuni (Ribot et al., 2001), except for the electrophoresis conditions that the protocol for separation of restriction fragments of Arcobacter was used as previously described (Son et al., 2006). The Arcobacter strains tested in the present study were restricted with KpnI (New England Biolabs, Canada). Salmonella Braenderup H9812 restricted with XbaI was used as a molecular marker as recommended by PulseNet.

Analysis of rep-PCR and PFGE patterns: All rep-PCR and PFGE profiles were analyzed using the program GelCompar II® version 5.10 (Applied Maths

BVBA, Kortrijk, Belgium). The gel images were normalized by aligning the bands of the size marker in each gel. The optimization and band position tolerance setting was 1%. Similarity of the band patterns was calculated using Pearson’s correlation coefficient and then clustered using dendrogram generated by unweighted pair group of arithmetic mean (UPGMA) method according to the rep-PCR results. Isolates that had a similarity value higher than 94% were considered the same rep-PCR type. Isolates that had PFGE patterns showing the same number of bands with the same size of the corresponding bands or showing less than two band differences were considered indistinguishable isolates (Tenover et al., 1995).

Determination of discriminatory power and concordance between rep-PCR and PFGE: Simpson’s index of diversity (SID), Adjusted Rand coefficient, and Wallace coefficient were calculated using the online tool for quantitative assessment of classification agreement available at http://darwin.phyloviz.net/ComparingPartitions/. The Simpson’s index of diversity demonstrates the discriminatory ability of typing techniques (Hunter and Gaston, 1988). To determine the concordance between rep-PCR and PFGE, Adjusted Rand coefficient and Wallace coefficient were calculated. The Adjusted Rand coefficient provides overall concordance of two typing techniques (Hubert and Arabie, 1985), while the Wallace coefficient shows directional information on clustering concordance between different typing methods which can be used for predicting results of one technique by results of another technique (Carrico et al., 2006).

Results and Discussion Of the 33 Arcobacter isolates analyzed, 27 rep-

PCR patterns and 29 PFGE patterns were obtained. The DNA fingerprints generated by rep-PCR consisted of 8-15 fragments with the size ranging from 300-9,000 bp, whereas PFGE profiles of KpnI-digested Arcobacter genomic DNA were composed of 10-20 fragments with the size ranging from 10-500 kbp (Figure 1). The rep-PCR profiles of A. butzleri NCTC 12481 generated from different PCR amplifications were indistinguishable and clustered together at >94% similarity value (data not shown), suggesting that the technique had good reproducibility. Figure 1 shows the dendrogram constructed based on the rep-PCR results. A. butzleri isolates examined in the present study as well as A. butzleri reference strain NCTC 12481 were grouped together. This A. butzleri cluster was only 16.2% similar to A. cryaerophilus NCTC 11885 and A. skirrowii CV1103 reference strains. All Arcobacter isolates exhibiting different rep-PCR patterns also revealed different PFGE patterns. Four pairs of Arcobacter isolates that had identical rep-PCR pattern (R5, R16, R17, and R21) also had indistinguishable PFGE pattern. However, two pairs of Arcobacter isolates that had the same rep-PCR pattern (R9 and R19) were distinguished by PFGE (P9, P10, P20, and P21) (Fig 1). The Simpson’s index of diversity of rep-PCR and PFGE was 0.989 and 0.992, respectively, indicating the high discriminatory

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power of these two techniques. The quantitative assessment of concordance between rep-PCR and PFGE was carried out by determining the Adjusted Rand and Wallace coefficients (Table 1). The Adjusted Rand coefficient was 0.798, which demonstrated the good congruence between rep-PCR and PFGE. The Wallace coefficient of PFGE to rep-PCR was 1.000, which indicated that if the isolates were identified to be of the same PFGE type, those isolates had 100% chances to be identified as the same rep-PCR type. On the other hand, the Wallace coefficient of rep-PCR to PFGE was 0.667, indicating that if the isolates were identified as the same rep-PCR type, those isolates

had 66.7% chances to be identified as the same PFGE type.

With the high discriminatory power and good correlation with the PFGE, the gold standard typing technique for bacteria in the family Campylobacteraceae, the rep-PCR technique as proposed in the present study can be used as a rapid and effective screening tool for studying genetic profiles of Arcobacter especially when large numbers of isolates are needed to be investigated. After the rep-PCR screening, an additional technique such as PFGE can be performed if a more thorough investigation of specific isolates is still required.

Figure 1 UPGMA dendrogram of Arcobacter isolates constructed based on the results of rep-PCR. The corresponding PFGE patterns

were shown for each isolate. The circle (●) in front of the strain ID indicates strains that shared the same rep-PCR pattern (R9 and R19), but had different PFGE patterns (P9, P10, P20, and P21). Arcobacter reference strains (A. cryaerophilus NCTC 11885, A. skirrowii CV1103, and A. butzleri NCTC 12481) are underlined.

Table 1 Adjusted Rand and Wallace coefficients

a Wallace coefficient of PFGE to rep-PCR b Wallace coefficient of rep-PCR to PFGE

Adjusted Rand coefficient (95% confidence interval) Wallace coefficient

(95% confidence interval) Typing method PFGE Rep-PCR PFGE Rep-PCR

PFGE 1.000 (1.000-1.000) 1.000

(1.000-1.000) 1.000a

(1.000-1.000)

Rep-PCR 0.798 (0.391-1.000)

1.000 (1.000-1.000) 0.667b

(0.431-0.902) 1.000

(1.000-1.000)

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Acknowledgments We thank Dr. Nicola J. Williams from the

University of Liverpool for providing the standard Arcobacter strains used in the present study. Ms. Panvipa Phasipol is a recipient of the Chulalongkorn University Graduate Scholarship to Commemorate the 72nd Anniversary of His Majesty King Bhumibol Adulyadej. This study was supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund).

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M 2002. Discrimination of Arcobacter butzleri isolates by polymerase chain reaction-mediated DNA fingerprinting. Lett Appl Microbiol. 35(2): 141-145.

Carrico JA, Silva-Costa C, Melo-Cristino J, Pinto FR, de Lencastre H, Almeida JS and Ramirez M 2006. Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes. J Clin Microbiol. 44(7): 2524-2532.

Chomczynski P and Rymaszewski M 2006. Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. Bio Techniques. 40(4): 454-458.

Collado L and Figueras MJ 2011. Taxonomy, epidemiology, and clinical relevance of the genus Arcobacter. Clin Microbiol Rev. 24(1): 174-192.

Gonzalez A, Ferrus MA, Gonzalez R and Hernandez J 2007. Molecular fingerprinting of Campylobacter and Arcobacter isolated from chicken and water. Int Microbiol. 10(2): 85-90.

Ho HT, Lipman LJ and Gaastra W 2006. Arcobacter, what is known and unknown about a potential foodborne zoonotic agent! Vet Microbiol. 115(1-3): 1-13.

Houf K, De Zutter L, Van Hoof J and Vandamme P 2002. Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods. Appl Environ Microbiol. 68(5): 2172-2178.

Hubert L and Arabie P 1985. Comparing partitions. J Classif. 2(1): 193-218.

Hunter PR and Gaston MA 1988. Numerical index of the discriminatory ability of typing systems: An application of Simpson's index of diversity. J Clin Microbiol. 26(11): 2465-2466.

Kabeya H, Maruyama S, Morita Y, Ohsuga T, Ozawa S, Kobayashi Y, Abe M, Katsube Y and Mikami T 2004. Prevalence of Arcobacter species in retail meats and antimicrobial susceptibility of the isolates in Japan. Int J Food Microbiol. 90(3): 303-308.

Majella M, Lahiff S, Morris D 2006. Molecular diagnostics and epidemiological typing methods for common foodborne. In: Molecular Diagnostics Current Technology and Application. JR Rao, CC Fleming, JE Moore (eds). Norfolk: Horizon BioScience: 93-122.

Miller WG, Wesley IV, On SL, Houf K, Megraud F, Wang G, Yee E, Srijan A and Mason CJ 2009. First multi-locus sequence typing scheme for Arcobacter spp. BMC Microbiol. 14(9): 196.

Ribot EM, Fitzgerald C, Kubota K, Swaminathan B and Barrett TJ 2001. Rapid pulsed-field gel electrophoresis protocol for subtyping of Campylobacter jejuni. J Clin Microbiol. 39(5): 1889-1894.

Rivas L, Fegan N and Vanderlinde P 2004. Isolation and characterisation of Arcobacter butzleri from meat. Int J Food Microbiol. 91(1): 31-41.

Son I, Englen MD, Berrang ME, Fedorka-Cray PJ and Harrison MA 2006. Genetic diversity of Arcobacter and Campylobacter on broiler carcasses during processing. J Food Prot. 69(5): 1028-1033.

Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH and Swaminathan B 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol. 33(9): 2233-2239.

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Susceptibility of Clostridium difficile Isolated from Healthy

Captive Asian Elephants to Metronidazole and Vancomycin

Nattawooti Sthitmatee1* Thanya Warinrak1 Waroot Wongkalasin2

Abstract

Susceptibility to metronidazole and vancomycin, drugs of choice for Clostridium difficile infection, of 15 C.

difficile isolates from 6 healthy Asian elephants was determined. All of the isolates belonged to only 1 ribotype pattern and carried both toxin A and B genes. The Minimal inhibitory concentration range of metronidazole and vancomycin, drugs of choice for treatment of C. difficile infection, was 0.125-4.0 µg/ml and 0.125-2.0 µg/ml, respectively. Moreover, MIC50 and MIC90 for metronidazole were 0.75 and 1.5 µg/ml while vancomycin was 1.0 and 2.0 µg/ml. There was no evidence of resistance to these antimicrobials. These results might be a preliminary data for further study of animal C. difficile.

Keywords: captive Asian elephant, Clostridium difficile, metronidazole, MIC, vancomycin 1Department of Veterinary Bioscience and Veterinary Public Health, 2Department of Companion Animal and Wild Life Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Muang, Chiang Mai 50100 *Correspondence author E-mail: fvmnatsthit@gmail.com

Short Communication

314 Sthitmatee N. et al. / Thai J Vet Med. 2013. 43(2): 313-316.

บทคดยอ

ความไวรบของเชอ Clostridium difficile ทคดแยกไดจากชางเลยงเอเชยตอยาเมทโทรนดาโซลและแวนโคมยซน

ณฐวฒ สถตเมธ 1* ธญญา วรนทรรกษ 1 วรตม วงศกาฬสนธ 2

การศกษานเปนการศกษาความไวรบของเชอ C. difficile จานวน 15 สายพนธ จากชางเลยงเอเชยจานวน 6 เชอกตอยาเมทโทรนดาโซลและแวนโคมยซน ซงเปนยาตวเลอกสาหรบโรคตดเชอ C. difficile จากการศกษาพบวาแบคทเรย C. difficile ทกสายพนธมรปแบบของไรโบทยปเดยวกน และทกสายพนธมทงยนของทอกซนเอและบตามลาดบ คาความเขมขนตาสดทสามารถยบยงการเจรญของเชอ (Minimal inhibitory concentration) ตอยาเมทโทรนดาโซลและแวนโคมยซนซงเปนยาตานจลชพทใชในการรกษาโรคตดเชอแบคทเรย C. difficile อยในชวง 0.125-4.0 ไมโครกรมตอมลลลตร และ 0.125-2.0 ไมโครกรมตอมลลลตร ตามลาดบ นอกจากนนคาความเขมขนตาสดทสามารถยบยงการเจรญของเชอรอยละ 50 และ 90 (MIC50 และ MIC90) ตอยาเมทโทรนดาโซลเทากบ 0.75 และ 1.5 ไมโครกรมตอมลลลตร สาหรบยาแวนโคมยซนเทากบ 1.0 และ 2.0 ไมโครกรมตอมลลลตร ทงนไมพบการดอตอยาตานจลชพทงสองชนดน ผลการทดลองทไดรบจะเปนขอมลเบองตนในการศกษาเชอ C. difficile จากสตวตอไป

คาสาคญ: คาความเขมขนตาสดทสามารถยบยงการเจรญของเชอ ชางเลยงเอเชย เมทโทรนดาโซล แวนโคมยซน Clostridium difficile 1ภาควชาชวศาสตรทางสตวแพทยและสตวแพทยสาธารณสข, 2ภาควชาคลนกสตวเลยงและสตวปา คณะสตวแพทยศาสตร มหาวทยาลยเชยงใหม อ.เมอง จ.เชยงใหม 50100 *ผรบผดชอบบทความ E-mail: fvmnatsthit@gmail.com

Introduction Clostridium difficile is a gram-positive anaerobic bacterium with sporulated bacilli which is recognized as a causative agent of a severe colon disease called pseudomembranous colitis in human. In addition, C. difficile is known to be the first cause of nosocomial diarrhea (Kelly and Lamont, 1998) or diarrhea associated with antimicrobial usage. Predominant virulent factors of this bacterium are two producing toxins A and B. Both are potent toxins and cause extensive colonic inflammation and epithelial tissue damage in the infected host (Carter et al., 2012). Diagnosis of C. difficile is generally performed by fecal culture with selective media in anaerobic condition and detection of its toxins (Delmée, 2001; Delmée et al., 2005). Bojesen et al. (2006) showed the first case report of elephant C. difficile infection in Denmark. Post-mortem necropsy and laboratory diagnosis confirmed overgrowing of C. difficile in gastrointestinal (GI) tract of elephant induced by Sulforaphane-like substance in broccoli even though the bacterium was recognized as a normal flora (Songer, 1996; Sthitmatee and Boonmar, 2011). Our previous study isolated 15 strains of C. difficile from 6 healthy elephants and concluded that C. difficile was a normal microbe in GI tract of healthy elephants (Sthitmatee and Boonmar, 2011). Moreover, the previous study in Austrian suggested that animal reservoirs were possible sources, via direct contact with food or meat products, of C. difficile infection in human (Indra et al., 2009). In Thailand, captive

elephants are for shows and forest sightseeing. Thus, there are many possibilities for human especially mahouts and tourists to direct expose to C. difficile. However, there is still scarce therapeutic information on animal C. difficile isolates, only in pigs (Post and Songer, 2004). Therefore, the objectives of the present study were (1) to obtain the therapeutic information on disease caused by C. difficile strains isolated from elephant source, determining the antimicrobial susceptibility of these 15 isolates to metronidazole (MTZ) and vancomycin (VAN), which are the drugs of choice for the treatment of C. difficile infection in human (Johnson, 2009; Leffler and Lamont, 2009) and (2) to characterize the isolates by detection of toxin genes and ribotyping. The advantage of this study is preliminary information for further investigation of this bacterium in animals.

Materials and Methods Bacterial culture: Bacteria were anaerobically cultured at 370C for 48 hours on cycloserine cefoxitin fructose agar (CCFA) supplemented with egg yolk (Oxoid, Basingstoke, UK), 500 µg/ml cycloserine (Oxoid) and 6 µg/ml cefoxitin (Oxoid) as described previously (Post and Songer, 2004). The anaerobic condition was composed of 5% Hydrogen, 5% Carbon dioxide and 90% Nitrogen, and was conducted by Bactron X-2 Anaerobic Chamber (Engineered Production System, Orange Country, CA). Positive colonies which appeared grayish, opaque and had typical horse manure odor were selected. Then, a

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single colony was picked and incubated in tryptic soy broth (TSB: Merck, Darmstadt, Germany) for DNA preparation.

DNA preparation and molecular characterization of the strains: Bacterial suspension was subjected to prepare chromosomal DNA with High Pure PCR template preparation Kit (Roche, Mannheim, Germany). Toxin gene typing was performed by multiplex PCR for identification of genes tpi, tcdA and tcdB as described previously (Lemee et al., 2004). The PCR ribotyping was employed by the protocol reported previously (Keel et al., 2007).

MIC: MIC was adapted and modified based on agar dilution method as described by a revised standard method of the Japanese Society of Antimicrobials for Animals in 2003. Briefly, metronidazole (MTZ: Sigma-Aldrich, St. Louis, MO, USA) and vancomycin (VAN: Sigma-Aldrich) were prepared with 2-fold dilution before adding into culture agar and pouring into sterile culture plates as described previously (Zheng et al., 2007). Agar medium used was 5% horse blood Brucella agar (Oxoid) supplemented with hemin (Acros Organics, Geel, Belgium) and vitamin K (Acros Organics). Subsequently, single colony was grown in TSB at 370C in anaerobic chamber until bacterial suspension reached McFarland Standard No. 1.5. Then, the plates were inoculated in anaerobic condition at 370C for 48 hours. The lowest concentration of an antimicrobial, which completely inhibited bacterial growth, was considered to be the endpoint and the concentration was regarded as the MIC. The resistance breakpoint values of VAN was ≥4 µg/ml and MTZ was >16 µg/ml, respectively (Zheng et al., 2007). Moreover, Bacteroides fragilis (ATCC 25285) and B. thetaiotaomicron (ATCC 29741) were included as controls.

Results and Discussion Six elephants were raised at 2 different

camps. There were four elephants in camp A, one male (21 years old) and three females (17, 22 and 25 years old) and, one male (18 years old) and one female (27 years old) in camp B. All captive elephants were fed on bananas, wild sugarcanes (Saccharum spontaneum) and additional commercial concentrate feeds during daytime. There were also abundance of water and plants such as grass, bamboo leaves, and wild sugarcanes in the area where the elephants were held. Moreover, elephant health management was under supervision of veterinarians. According to elephants’ individual health records, there was no evidence of antimicrobial usage six month prior to the experiment.

All the isolates carried both toxin A and B genes and the ribotyping analyses indicated that there was only 1 pattern among 15 isolates. MIC range of MTZ was 0.125-4.0 µg/ml while VAN was 0.125-2.0 µg/ml. Moreover, MIC50 and MIC90 for MTZ were 0.75 and 1.5 µg/ml while MIC50 and MIC90 for VAN were 1.0 and 2.0 µg/ml, respectively. In addition, there was no evidence of resistance to these antimicrobials.

Clostridium spp. is recognized as enteric pathogens in human, domestic animals and also wildlife (Songer, 1996; Magdesian et al., 2002). C. difficile caused pseudomembranous colitis in human, hemorrhagic necrotizing enterocolitis in foals (Magdesian et al., 2002), fatal enterocolitis in Asian elephants (Bojesen et al., 2006), and neonatal enteritis in piglet (Post and Songer, 2004; Avbersek et al., 2009). However, C. difficile could be isolated from the environment including healthy animal species (Medina-Torres et al., 2011) by control of the normal flora in GI tract. Disturbances to the normal flora in GI tract affect microbial growth and eventually cause disease in animals; for example, the over feeding broccoli in Danish elephants leading to overgrowth of C. difficile and resulting in fatal enterocolitis (Bojesen et al., 2006). C. difficile produces two types of exotoxins; A (TcdA: enterotoxin) and B (TcdB: cytotoxin) (Carter et al., 2012). These two toxins react to cell cytoskeleton by damaging cell tight junction and cause epithelium cell erosion. C. difficile infection in animals caused moderate to severe mesocolonic edema and pasty-to-watery yellowish colonic contents in 1-7 day-old piglets (Songer, 2004) and diarrhea calves (Hammitt et al., 2008). There is still no evidence of human infected with C. difficile genetically related to elephant C. difficile, however, mahouts and tourists have a higher risk of exposure to this bacterium. Moreover, the results of Indra et al. (2009) suggested that animal reservoirs are possible sources, via food, of human C. difficile infection. Therefore, molecular epidemiology among human strains and elephant strains need to be investigated.

MTZ and VAN are the drugs of choice for disease caused by C. difficile in human (Post and Songer, 2004). Ranges of MIC for MTZ and VAN have been reported (Wong et al., 1999; Aspevall et al., 2006). MIC of C. difficile strains to MTZ and VAN from Swedish University Hospital were 0.032-1.0 and 0.5-2.0 µg/ml (Aspevall et al., 2006) while in Hong Kong they ranged from 0.094-1.5 and 0.125-2.0 µg/ml, respectively (Wong et al., 1999). As our results, MIC range from elephant isolates were close to all human isolates. Moreover, MIC90 of MTZ and VAN in elephant isolates was similar to MIC90 from human isolates (Huang et al., 2009). However, resistance to antimicrobials in C. difficile varied between countries (Huang et al., 2009). Most isolates are still susceptible to VAN and MTZ but decrease in sensitivity is emerging. Huang et al. (2009) suggested that resistant mechanisms of C. difficile are similar to the Gram-positive bacterium but trends and mechanisms were still required for further study.

Acknowledgements This study was supported by Thailand

Research Fund and Commission of Higher Education, Ministry of Education, (grant No. MRG5280026).

316 Sthitmatee N. et al. / Thai J Vet Med. 2013. 43(2): 313-316.

References Aspevall O, Lundburg A, Burman LG, Akerlund T and

Svenungsson B 2006. Antimicrobial susceptibility pattern of Clostridium difficile and its relation to PCR ribotype in Swedish University Hospital. Antimicrob Agents Chemother. 50: 1890-1892.

Avbersek J, Janezic S, Pate M, Rupnik M, Zidaric V, Logar K, Vengust M, Zemljic M, Pirs T and Ocepek M 2009. Diversity of Clostridium difficile in pigs and other animals in Slovenia. Anaerobe 15: 252-255.

Bojesen AM, Olsen KEP and Bertelsen MF 2006. Fatal enterocolitis in Asian elephants (Elephas maximus) caused by Clostridium difficile. Vet Microbiol. 116: 329-335.

Carter GP, Rood JI and Lyras D 2012. The role of toxin A and toxin B in the virulence of Clostridium difficile. Trends Microbiol. 20: 21-29.

Delmée M 2001. Laboratory diagnosis of Clostridium difficile disease. Clin Microbiol Infect. 7: 411-416.

Delmée M, Van Broeck J, Simon A, Janssens M and Avesani V 2005. Laboratory diagnosis of Clostridium difficile-associated diarrhea: A plea for culture. J Med Microbiol. 54: 187-191.

Hammitt MC, Bueschel D, Keel MK, Glock R, Cuneo DP, DeYoung D, Reggiardo C, Trinh HT and Songer JG 2008. A possible role for Clostridium difficile in the etiology of calf enteritis. Vet Microbiol. 127: 343-352.

Huang H, Weintraub A, Fang H and Nord CE 2009. Antimicrobial resistance in Clostridium difficile. Int J Antimicrob Agents. 34: 516-522.

Indra H, Lassnig H, Baliko N, Much P, Fiedler A, Huhulescu S and Allerberger F 2009. Clostridium difficile: A new zoonotic agent?. Wien Klin Wochenschr. 121: 91-95.

Johnson S 2009. Recurrent Clostridium difficile infection: Causality and therapeutic approaches. Int J Antimicrob Agents. 33(S1): S33-S36.

Keel K, Brazier JS, Post KW, Weese S and Songer JG 2007. Prevalence of PCR ribotypes among Clostridium difficile isolates from pigs, calves, and other species. J Clin Microbiol. 45: 1963-1964.

Kelly CP and Lamont JT 1998. Clostridium difficile infection. Ann Rev Med. 49: 375-390.

Leffler DA and Lamont JT 2009. Treatment of Clostridium difficile-Associated Disease. Gastroenterology 136: 1899-1912.

Lemee L, Dhalluin A, Testelin S, Mattrat MA, Maillard K, Lemeland JF and Pons JL 2004. Multiplex PCR targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol. 42: 5710-5714.

Magdesian KG, Hirsh DC, Jang SS, Hansen LM and Madigan JE 2002. Characterization of Clostridium difficile isolates from foals with diarrhea: 28 cases (1993-1997). J Am Vet Med Assoc. 220: 67-73.

Medina-Torres CE, Weese JS and Staempfli HR 2011. Prevalence of Clostridium difficile in horses. Vet Microbiol. 152: 212-215.

Post KW and Songer JG 2004. Antimicrobial susceptibility of Clostridium difficile isolated from neonatal pigs with enteritis. Anaerobe 10: 47-50.

Songer JG 1996. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev. 9: 216-234.

Songer JG 2004. The emergence of Clostridium difficile as a pathogen of food animals. Anim Health Res Rev. 5: 321-326.

Sthitmatee N and Boonmar S 2011. Epidemiology of fermenter bacteria in intestine of captive Asian elephants (Elephas maximus). Chiang Mai: Chaing Mai University Press. 45 pp.

Stubbs SLJ, Brazier JS, O’neill GL and Duerden BI 1999. PCR targeted to the 16S-23SrRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J Clin Microbiol. 37: 461–463.

The determination method of minimum inhibitory concentration (MIC) of antimicrobials against bacteria isolated from animals (Revised standard method of the Japanese society of antimicrobials for animals in 2003). In: Proceeding of the Japanese society of antimicrobials for animals, 26: 64-74.

Wong SS, Woo PC, Luk W and Yuen K 1999. Susceptibility testing of Clostridium difficile against metronidazole and vancomycin by disk diffusion and Etest. Diagn Microbiol Infect Dis. 34: 1-6.

Zheng L, Citronb DM, Genheimera CW, Sigmona SF, Carmana RJ, Lyerlya DM and Goldstein EJC 2007. Molecular characterization and antimicrobial susceptibilities of extraintestinal Clostridium difficile isolates. Anaerobe 13: 114–120.

ECG Quiz

Chollada Buranakarl1 Suwarat Wadeerat 2

History The 5 years old mixed breed spayed female

weighing 15 kg was presented at the Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University with history of severe depression, anorexia and vomiting. Ten months ago, the dog was azotemia with creatinine of 4.5 mg/dl and anemia with PCV of 11%. Dog was given supportive therapy, doxycycline and immuno-suppressive drugs. Treatment and monitoring had performed for five months, after which BUN and creatinine were declined to 43 mg/dl and 3.2 mg/dl, respectively while PCV was increased up to 34%. Chronic kidney disease was maintained although she was still alert. The dog had never seen the veterinarian after since. He then returned to the veterinarian with clinical signs of azotemia. He had epistaxis, which was treated two days before presenting to the hospital.

From physical examination, the dog had pale mucus membrane with normal heart and lung sound.

At the hospital, dog had bloody diarrhea and anuria. Systolic blood pressure was 110 mmHg with bradycardia (heart rate was 45 beats/min). From blood results, the dog was positive for antibody against Ehrlichia canis using Snap 4Dx®. He had low red blood cell with PCV of 24%, 5.1 g/dl of total protein and leukocytosis (22,300 cell/mm3). Blood chemistries showed azotemia with increased BUN and creatinine up to 104 mg/dl and 9.4 mg/dl, respectively. The concentration of inorganic phosphate was increased up to 10.1 mg/dl. Plasma Na and K were 142 and 4.53 mEq/L. Plasma HCO3-

was 18.6 mmol/L with pH of 7.391. Acute kidney injury was diagnosed and the dog was treated with furosemide, dopamine and mannitol to increase urine volume. Supportive fluid therapy, anti-emetic and antibiotics were also given. The dog was still oliguric with urine output between 0.05-0.34 ml/kg/hr. The BUN and creatinine measured 5 days after admission were increased to 191 mg/dl and 11.4 mg/dl, respectively. Plasma K was 6.46 mEq/L by the time the ECG was performed (Figure 1).

Figure 1 Electrocardiographic recording on dogs with acute kidney injury

Please answer before turning to the next page.

1Department of Veterinary Physiology, 2Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University

Diagnostic Forum

318 Buranakarl C.& Wadeerat S./ Thai J Vet Med. 2013 43(2): 317-318.

Interpretation Sinus bradycardia without p-wave

The heart rate during resting was between

50-55 beats/minute. The shape of QRS complexes was similar to that originated from normal sinus node. However, the p-waves were very small or sometimes could not be detected suggesting that the conduction insight the atria may be slow. One supraventricular ectopic beat was found as shown at the 8th wave of the last panel of ECG tracing. Since the ECG tracing was performed by the telemetric ECG monitoring in the CCU unit, the conventional 10 leads of ECG should have been performed. The cause of bradycardia and loss of p-wave was due to electrolyte abnormalities especially potassium ion. Hyperkalemia in this case caused by oliguric kidney failure leads to bradycardia and many forms of ECG abnormalities. Besides p-wave abnormalities, QRS widening, fusion of QRS and T-wave, tall and tented T-wave are also seen when the hyperkalemia is more severe. Hyperkalemia

causes Na+ channel inactivation and speeds up membrane repolarization leading to tenting T-wave. Inactivation of Na channel causes slow conduction as shown by small or loss p-wave and QRS widening. Bradycardia is due to hyperpolarization by high potassium level, in which depolarization of phase 4 is prolonged. In severe hyperkalemia (K+ > 9 mmol/L), the electrocardiographic sine-wave pattern will be seen. When arrhythmia is detected with serum potassium higher than 6.5 mmol/L, treatment should be warranted. Calcium chloride or calcium carbonate should be given to stabilize cardiac membrane potential. Insulin along with glucose can be given to accelerate the K+ influx into the cell. Bicarbonate is another choice of drug that promotes K+ influx and correct acidosis. Finally, if urine output is low and K+ cannot be eliminated, hemodialysis or peritoneal dialysis should be considered.

Ophthalmology Snapshot

Nalinee Tuntivanich

History

A 2 year-old male Pekingese was referred to

the Ophthalmology Clinic, Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University for ocular surgery. He initially developed right ulcerative keratitis, which had shortly progressed to corneal rupture with iris protrusion. He had been topically on tobramycin.

Ophthalmic examinations revealed large

anterior staphyloma, which was 8 mm in diameter at the center of the cornea. Menace response was negative. STT 1 value was slightly high; intraocular pressure was in normal limit.

Corneal transplantation with double-layered

human amniotic membrane (HAM); combination of in lay and over lay techniques, was performed. Nictitan flap was considered as a secondary support after transplantation. Topical tobramycin was replaced with fluoroquinolone. Two weeks after flap removal; ocular surface was shown in Fig 1. Menace response returned.

Figure 1 Front view of the right eye of the Pekingese at two

weeks following HAM corneal transplantation. (For better quality, figures can be viewed in the TJVM website)

Question What was the lesion on the cornea?

Please turn to the next page for answers ……….

Ophthalmology Clinic, Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand

Diagnostic Forum

320 Tuntivanich N. / Thai J. Vet. Med. 2013 43(2): 319-320.

Answer

Formation of granulation tissue

Comments

Corneal damage that does not extend through basement membrane heals rapidly. Transient corneal epithelial hyperplasia may shortly be involved during healing process, before normalization. Deep or large corneal ulcer (with or without infection) however undergoes dissimilar healing process. Not only they heal with epithelial sliding and replication, stromal fibroplasia and angiogenesis also occur. As a result of budding of limbal venules, progressive ingrowth of new vascular tubes takes place from limbus to stroma. The ingrowth of vessels is accompanied by migration and activation of fibroblasts.

Injured endothelium undergoes fibroblastic metaplasia with high proliferative capability. Collagenases and proteases created from damaged cells and leukocytes result in dissolution of collagen and ground substances of the cornea. Wound healing of such a significant corneal injury, as in this case, is therefore characterized by severe inflammation during acute phase, followed by an intermediate phase of profound granulation tissue formation.

Transplantation of amniotic membrane (AM) is nowadays used in many ocular disorders. In this case, the bottom layer of AM was placed as an inlaid graft to fill in corneal defect. Healing process was created as the membrane subsequently integrated into host tissue. The top layer of AM was used as a temporary biological bandage or patch (overlaid graft) to suppress host inflammation and promote tissue healing with minimal scarring.

References Peiffer, Jr R.L., Wilcock, B.P., Dubielzig, R.R., Render,

J.A. and Whiteley, H.E. 1999. Fundamentals of veterinary ophthalmic pathology. In: Veterinary Ophthalmology. 3rd ed. Kirk N. Gelatt (ed). Maryland: British Lippincott Williams & Wilkins. 382-387.

Liu, J., Sheha, H., Fu, Y., Liang, L., Tseng, S.CG. 2010. Updates on amniotic membrane transplantation. Expert. Rev. Ophthalmol. 5(5): 645-661.

ULTRASOUND DIAGNOSIS

Phiwipha Kamonrat

History

A four-year-old, intact male, Saint

Bernard dog was presented at the Chulalongkorn University, Small Animal, Veterinary Teaching Hospital because of the chronic intermittent vomiting for five months. The dog had a mild depression and a less appetite. Four weeks ago, the dog went for surgical correction of gastric dilatation-volvulus by gastropexy. The dog had started to vomit on and off again during the last two weeks. A physical examination revealed pink mucous membranes, normal hydration status and abdominal distension. An abdominal tense was notified on palpation. Laboratory blood work showed a mild leukocytosis with neutrophilia (17,300 x 103 white blood cells/µl and 92% neutrophils). No blood parasite was found. Abdominal radiographs revealed a markedly gas and soft tissue-distended stomach displacing bowel loops caudally. There was no evidence of gastric rotation or splenic torsion. After a decompression of the dilated stomach by passing orogastric intubation, abdominal ultrasonography was performed to differentiate the gastric lesions especially a gastric outflow obstruction.

Ultrasonographic Findings Real-time, ultrasonographic images of the stomach were obtained using an 8 MHz microconvex, phased array transducer with the dog in dorsal recumbency. After a fast of 12 hours and a decompression of the stomach prior to ultrasonography, the stomach was still markedly dilated with luminal contents (Figure 1A and 2A). The alternating hyperechoic and hypoechoic layer definition of the gastric wall was preserved. Gastric wall measured 2 mm in thickness, which was within a normal range in dogs. Gastric contents consisted of a large amount of anechoic fluid mixed with echogenic food particles and hyperechoic gas (Figure 1B and 2B). Gas in the lumen created reverberation artifacts that masked the gastric far wall. In real-time scanning, there was continual gastric peristaltic movement, about 4-5 contractions per minute, without the movement of fluid and chyme into duodenum. Most of the contents still swirl in the lumen, however, there were no any morphologic changes of pyloric sphincter or duodenum detected. These findings might be related to a condition of a delayed gastric emptying time. Ultrasonography of other abdominal organs including the rest of the intestinal tract appeared normal in echotexture. Ultrasonographic Diagnosis Severe gastric dilatation without pyloric outflow obstruction

Department of Veterinary Surgery, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand

Diagnostic Forum

322 Kamonrat P. / Thai J Vet Med. 2013 43(2): 321-322.

Figure 1 Ultrasonographic images of the stomach of a four-year-old, intact male, Saint Bernard dog in dorsal

recumbency. A. After a fast of 12 hours, the stomach was still markedly dilated with luminal contents. The echogenicity and layering of the gastric wall were preserved. B. Gastric contents included anechoic fluid, echogenic food particles and hyperechoic gas with related reverberation artifacts.

Figure 2 Schematics of the relative positions of the structures scanned in figure 1. GW –gastric wall, Lu –gastric

lumen; R–reverberation artifact created by gas.

Comments

Ultrasonographic and radiographic studies compliment each other to confirm the presence and causes of gastric dilatation. The ultrasonography will add more information of the gastric wall thickness, layering and motility, peritoneal fluid and regional lymphadenopathy. In normal dogs, the gastric wall thickness ranges from 2 to 5 mm depending on distension of the stomach and measurements taken at the rugal or the interrugal level (Penninck, 2008). The five ultrasonographic layers of normal gastric wall are recognized as hyperechoic mucosal surface, hypoechoic mucosa, hyperechoic submucosa, hypoechoic muscularis and hyperechoic subserosa/serosa. Loss of layering is associated with pathologic changes but gas-related artifacts and limited resolution of ultrasound equipment may mask the visualization of the wall layer definition. Ultrasound is a valuable diagnostic tool for stomach motility evaluation. The normal gastric peristaltic movement ultrasonographically averages 4-5 contractions per minute. Gastric emptying time in dogs has been evaluated by serial measurement of antral area and volume after a test meal as developed by Bolondi et al.’s method (Choi et al., 2002). The identification of stomach distension with fluid, food

and gas contents, and the lack of passage of these contents through the pylorus in this dog may signify mechanical obstruction but ultrasonographic changes of pyloric sphincter or duodenum were not identified. Therefore endoscopy was employed for real time evaluation of the stomach and found only a mild hyperemia of the gastric mucosa without a mechanical obstruction of the pyloric sphincter or proximal duodenum. References Choi M., Seo M., Jung J., Lee K., Yoon J., Chang D.and

Park R.D. 2002. Evaluation of canine gastric motility with ultrasonography. J. Vet. Med. Sci. 64 (1):17-21.

Penninck D. 2008. Gastrointestinal tract. In: Atlas of Small Animal Ultrasonography. 1st ed. D. Penninck and M.A. Anjou (eds.) Ames: Blackwell Publishing. 281-318.

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