The first occurrence of Trichinella murrelli inwild boar in Iran and a review of Iranian

trichinellosis

E.B. Kia1*, A.R. Meamar2, F. Zahabiun1 and H. Mirhendi1

1Department of Medical Parasitology and Mycology, School of Public Healthand Institute of Public Health Research, Tehran University of Medical

Sciences, Tehran, Iran: 2Department of Medical Parasitology and Mycology,School of Medicine, Iran University of Medical Sciences, Tehran, Iran

(Accepted 3 March 2009; First Published Online 17 June 2009)

Abstract

Trichinella larvae isolated from the thigh muscle of a wild boar, Sus scrofa,captured from Gilan Province, northern Iran, was processed for DNA analysis.Polymerase chain reaction (PCR) for amplification of the 5S rDNA fragmentdemonstrated a 700 bp band on agarose gel. Analysis of DNA sequencing byBLAST confirmed the isolate as T. murrelli. This report constitutes the firstrecorded occurrence of T. murrelli in Asia, and also the first occurrence in a wildboar host.

Introduction

Trichinellosis is a zoonosis, caused by ingestion of rawor undercooked meat containing larvae of the nematodegenus, Trichinella. Species of Trichinella can be foundworldwide in a variety of hosts, ranging from mammalsto birds and reptiles. Wildlife acts as a natural reservoirfor Trichinella. Links between the sylvatic and domesticcycle are well reported and, depending on humancultural food practices, the causative agent may enterthe human food chain, resulting in infection (Pozio &Murrell, 2006).

The genus Trichinella is divided into an encapsulatedclade and a non-encapsulated clade, referring to thecollagen capsule that surrounds the larvae of somespecies in the host muscle. Currently, the genus Trichinellais classified into eight species (T. spiralis, T. nativa,T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuaeand T. zimbabwensis) and three unclassified genotypes(Trichinella T6, Trichinella T8 and Trichinella T9) (Pozio &Zarlenga, 2005; Rodriguez et al., 2008). All knownTrichinella species are morphologically indistinguishableexcept for T. pseudospiralis, which is smaller in size thanother species. The geographical distribution of thedifferent species is related to ecology, climate and hostbehaviour (Pozio et al., 1992; Kapel, 1997; Muller, 2002).

The identification of sequence regions in the genomesof pathogens, which can be useful to distinguish amongspecies and genotypes, is of great importance forepidemiological, molecular and phylogenetic studies.The 5S ribosomal DNA intergenic spacer region has beenidentified as a good target to distinguish the eightTrichinella species and genotypes (Rombout et al., 2001;De Bruyne et al., 2005; van der Giessen et al., 2005).This method is simple (only one primer pair), sensitive(DNA from a single larva suffices) and inexpensive(low-cost sequencing is available). It can also beapplied directly to muscle biopsy specimens (Romboutet al., 2001).

In Iran, Trichinella has been reported in different wildreservoir hosts. In the northern part of Iran, it has beenreported in wild boars (Sus scrofa) (Afshar & Jahfarzadeh,1967; Mobedi et al., 1973), jungle cats (Felis chaus) (Mobediet al., 1973), brown bears (Ursus arctos) (Mobedi et al.,1973), golden jackals (Canis aureus) (Mobedi et al., 1973;Hamidi, 1977; Massoud & Mahdavi, 1987; Dalimi &Mobedi, 1992), red foxes (Vulpes vulpes) (Hamidi, 1977)and stray dogs (Hamidi, 1977); in Ardabil Province,north-western Iran, it has been identified in golden jackalsand red foxes (Zariffard, 1995); in Isfahan, central Iran, ingolden jackals, red foxes, stray dogs, hyena (Hyaenahyaena) and one species of rodent (Meriones persicus)(Sadighian et al., 1973); in Khuzestan Province, south-western Iran, it has been found in mongoose (Herpestesauropunctatus) (Mowlavi et al., 2000); in Bandar Abbas,

*Fax: þ98 21 88951392E-mail: keiaeshr@sina.tums.ac.ir

Journal of Helminthology (2009) 83, 399–402 doi:10.1017/S0022149X09990319q Cambridge University Press 2009

south of Iran, in golden jackals, red foxes, stray dogs(Hamidi & Mobedi, 1977) and recently in a leopard(Panthera pardus) (Mowlavi, personal communication).No infection has so far been reported in domestic animals.However, human infectivity has been documented twice,in both cases through consuming wild boar meat. The firstof these was a single case in 1966, which constituted thefirst report of trichinellosis in the country and wasdiagnosed based on clinical symptoms, history of thedisease and serology (Moin, 1966). There has also been asecond, more recent single-source outbreak (Kia et al.,2008). In spite of the occurrence of infection in variousanimal hosts from different parts of the country, nomolecular description of the species has been presentedprior to this work.

This study presents a molecular description ofTrichinella murrelli isolated from a wild boar in northernIran. This also constitutes the first recorded occurrence ofthis species in Asia.

Materials and methods

Source of sample

The source of the sample was part of a frozen thighmuscle of a wild boar hunted in Javaher Dasht forest,Siahkal, Gilan Province, northern Iran in April 2007.The sample was delivered to the helminthologicallaboratory of the School of Public Health, Tehran Universityof Medical Sciences in May 2007 by patients who hadcharacteristic symptoms of trichinellosis and were referredfor the diagnosis of their infection. The patients weresix household members living in Tehran who consumedsemi-cooked meat of the described muscle as kebab.

Parasitological examinations

Part of the muscle tissue was prepared for directmicroscopic examination and Trichinella encapsulatedlarvae were observed. Twenty gram of thigh muscle wasdigested by a pepsin–HCl method. The muscle wasminced by scissors and incubated in artificial gastric juicefor 24 h at 378C. After separation of larvae from thedigestion fluid by washing three times in distilled water,the size (length and width) of ten larvae was measured.In addition, a piece of muscle was preserved in 10%formalin and, following tissue processing by convention-al histological methods, 5mm sections were prepared andstained with haematoxylin and eosin.

DNA extraction

The parasite DNA was extracted using QIAamp DNAMini kit (Qiagen, Hilden, Germany) according to themanufacturer’s instructions for tissue extraction.

Polymerase chain reaction

A 700 bp region of the 5S ribosomal DNA gene wasamplified by polymerase chain reaction (PCR) with twodesigned primers, 5SF (50-GGA-ACA-CGC-AGT-GTC-GTA-GAC-30) and 5SR (50-TGG-TAT-GAT-CGT-AGA-CAT-TTG-C-30). Reactions were performed in a 50ml

reaction containing 1.5 mM MgCl2, 200mM of each dNTP,1.5 U of Taq polymerase (Roche, Mannheim, Germany),0.5mM of each primer and 5ml DNA sample. Reactionswere run in a Perkin-Elmer Thermocycler under thefollowing conditions: 958C for 6 min for one cycle; 948Cfor 30 s, 578C for 90 s and 728C for 45 s for 35 cycles;followed by a 5 min extension step at 728C. The ampliconwas electrophoresed on 1.5% agarose gel and visualizedunder an ultraviolet transilluminator after staining withethidium bromide.

Sequencing

The PCR product was sequenced by SEQLAB company(Germany) with an ABI 3100 genetic analyser. Thenucleotide sequence was aligned with those from theGenBank database, using BLAST software.

Results

Trichinella larvae were visible inside thick collagencapsules by direct microscopic examination of muscletissue. The average length and width of ten larvae afterpepsin–HCl digestion of the muscle was 1083.48mm and36.4mm respectively, with a worm burden of 7 larvae/g.In the histological sections of the muscle, encapsulatedTrichinella larvae were observed, surrounded by aninflammatory reaction.

The PCR reaction, using specific primers, yielded a700 bp band on agarose gel (fig. 1). After gel extraction ofthe PCR product and DNA sequencing, the sequence wasanalysed by BLAST and the isolate was identified asT. murrelli. The nucleotide sequence of T. murrelli wassubmitted to the Genebank/EMBL/DDBJ database underAccession Number AB426627.

Discussion

Trichinella circulates in a sylvatic cycle in various wildanimals (Afshar & Jahfarzadeh, 1967; Mobedi et al., 1973;Sadighian et al., 1973; Hamidi, 1977; Hamidi & Mobedi,

Fig. 1. Electrophoresis of PCR products of Trichinella murrellilarvae isolated from wild boar. Lanes: 1, molecular weight

marker; 2, Trichinella murrelli; and 3, negative control.

400 E.B. Kia et al.

1977; Massoud & Mahdavi, 1987; Dalimi & Mobedi, 1992;Zariffard, 1995; Mowlavi et al., 2000) in different parts ofIran. Despite this, human cases are very rare and there areonly two published reports of human infections (Moin,1966; Kia et al., 2008). This is mainly due to Muslim beliefsbased on not consuming pork and the meat of some otheranimal species. However, ethnic minorities and hunterswho practise consumption of raw or undercooked meat ofwild pork are at risk of trichinellosis. Nevertheless, priorto this work, no molecular description of the speciesinvolved in wild animal or human infectivity has beenpresented.

Shaikenov & Boev (1983) cross-bred four Trichinellaisolates from golden jackal from the north and south-westof Iran with standard strains. They reported the twoisolates from the north of the country as T. nelsoni andthose from the south as T. nelsoni and T. spiralis. Later, inKhuzestan Province, in two separate studies based onsensitivity of laboratory animals (white mouse and rat) toTrichinella, isolates from golden jackal (Massoud &Mahdavi, 1987) and mongoose (Mowlavi et al., 2000)were reported as T. nelsoni. Apart from the above-mentioned studies, no other studies reporting trichinel-losis in wildlife have attempted to characterize Trichinellaspecies.

This study is the first research on the molecularcharacterization of Trichinella in Iran. The method usedcombined PCR-based amplification, sequencing andcomparison with the published data, based on the 5SrDNA sequences of the best-known Trichinella species,previously determined by Rombout et al. (2001).

According to the results of the current study, the isolateof Trichinella detected from wild boar in the temperate areaof northern Iran was identified as T. murrelli, with only onenucleotide difference compared to T. murrelli fromNearctic America (Accession No. AY009947). This is thefirst report of T. murrelli in Asia and, to the best of ourknowledge, also the first report of occurrence of T. murrelliin wild boar in the world. Trichinella murrelli wasconsidered as a predominant aetiological agent of sylvatictrichinellosis exclusively in temperate regions of NorthAmerica (Pozio, 2001). It has been detected in red foxes,raccoons, coyotes, black bears, bobcats and in one horse, inConnecticut, Georgia, Illinois, Indiana, New Mexico,Pennsylvania and Texas, USA (Pozio, 2001), and recentlyin a domestic dog in Chase City, Virginia, USA (Dubey et al.,2006). Although, there was a horsemeat outbreak inFrance, caused by T. murrelli (Ancelle et al., 1988), itwas later concluded that the origin of the horse carcasswas from Connecticut, USA (Dupouy-Camet et al., 2001).

The newly studied T. murrelli isolate was theaetiological agent of a human outbreak with moderatepathogenicity, making it important for public health.Among the seven people who had consumed semi-cooked meat of the relevant boar, six people, all rangingfrom 24 to 27 years old, revealed symptoms characteristicof trichinellosis, including fever, myalgia, facial andperiorbital oedema, and cramps of the neck and legs,simultaneously. In three of the patients, for whom bloodsamples could be taken, the average eosinophil count was2091 mm3, and the average creatine phosphokinase andlactic dehydrogenase enzyme values were 890 U/l

(N ¼ 225–500) and 584 U/l (N ¼ 25–200), respectively(Kia et al., 2008).

In conclusion, T. murrelli is circulating in a sylvatic cyclein the temperate region of northern Iran. It was the causeof moderate trichinellosis in humans, even with a lowlarvae burden in consumed meat. Since Trichinella hasbeen detected in various wild animal hosts fromgeographic areas with different climates in the country,using DNA-based methods for identification of thespecies involved is necessary. Integration of the findingsfrom different human cases of T. murrelli from around theworld and comparison of its clinical patterns andpathogenicity in humans with the other Trichinella specieswill provide a better understanding of this zoonoticdisease and improve its diagnosis and treatment.

Acknowledgements

The authors would like to acknowledge the helpreceived from Mrs M. Sharbatkhori, School of PublicHealth, Tehran University of Medical Sciences andDr E. Razmjou, Faculty of Medicine, Iran University ofMedical Sciences.

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