THEILERIOSIS IN SMALL RUMINANTS OF NORTHERN
BALOCHISTAN
REQUIREMENTS FOR THE DEGREE
LAHORE
2016
To
Lahore.
We, the Supervisory Committee, certify that the contents and form
of the thesis,
submitted by Mr. Mir Ahmad Khan Reg. No, 2005-VA-214, have been
found satisfactory and
recommend that it be processed for the evaluation of External
Examiner(s) for the award of the
degree.
=
Member:
Member:
Prof. Dr. Aftab Ahmad Anjum
In the name of Allah the most magnificent and the most
beneficent.
All praise for ALLAH All Mighty who has the control and
command of each and every thing.
It is He who has sent down to you, [O Muhammad], the Book; in it
are verses [that are]
precise - they are the foundation of the Book - and others
unspecific. As for those in whose
hearts is deviation [from truth], they will follow that of it which
is unspecific, seeking discord
and seeking an interpretation [suitable to them]. And no one knows
its [true] interpretation
except Allah. But those firms in knowledge say, "We believe in it.
All [of it] is from our Lord."
And no one will be reminded except those of understanding.
ii
ACKNOWLEDGEMENTS I am deeply grateful to my supervisor Prof. Dr.
Muhammad Arif Khan, Professor, and
Chairman of Department of Clinical Medicine and Surgery, University
of Veterinary and Animal
Sciences Lahore for providing me an opportunity to work with him.
His keen interest, analysis of
my work and rigorous critique not only improved the quality of this
dissertation but also my
overall understanding of the subject.
The enthusiasm towards science and your pursuit to do “good
science” helped to shape
my own scientific value. I am also thankful to my co-supervisor,
Prof. Dr. Muhammad Azam
Kakar, for his technical guidance and constructive criticism and
friendly attitude during the
course of this study from University of LUAWMS UTAL,
Balochistan.
I’ve been most fortunate to have the guidance of Prof. Dr. Muhammad
Sarwar Khan,
Director, Advanced studies, University of Veterinary and Animal
Sciences Lahore. I would like
to record my gratitude for his kind supervision as a member in my
supervisory committee,
sympathetic attitude, constructive advices, and scientific
discussions.
I wish to pay my gratitude to Prof. Dr. Aftab Ahmad Anjum,
Department of
Microbiology, University of Veterinary and Animal Sciences, Lahore,
as member in my
supervisory committee for his personal interest and valuable advice
in my research project.
I feel a momentous pleasure in transcribing my whole hearted thanks
to Dr Muhammad
Ijaz (Associate professor) Department of Clinical Medicine and
Surgery, UVAS Lahore, Dr
Imtiaz Ahmad Suduzai (Assistant professor) The University of Poonch
Rawalakot AJK.
I am greatly indebted to my great mother, wife, sisters, brothers,
teachers and colleagues,
for their support, understanding, prayers, love, patience and
sacrifices rendered to make this day
possible. Finally will pay love to my beloved kids Amir Hamza
Yaseenzai, Bibi Safia kakar and
Saleha Kakar, nephews Hafiz Muhammad Awais, Fahadullah, Rizwanullah
and Sher Baz Kakar.
At the end, as is customary all mistakes uncorrected are entirely
mine,
Mir Ahmad Khan
1 INTRODUCTION 1
4 RESULTS 58
5 DISCUSSION 90
6 SUMMARY 104
3.2. Determination of DNA concentration through Nanodrop of
Northern highland. 43
3.3. Determination of DNA concentration through Nanodrop of
Suleiman Mountain Region. 44
3.4. Sets of primers used to amplify Theileria rRNA gene sequences.
48
3.5. Quantities of final PCR reaction mixture. 49
4.1. Regional prevalence of Theileriosis in Sheep and Goats in
Northern Balochistan. 59
4.2. Union Council wise prevalence of Theileriosis in Sheep and
Goats. 62
4.3. Species wise prevalence of Theileriosis in Sheep and Goats in
Northern Balochistan. 65
4.4. Prevalence of Theileriosis in different breeds of Sheep in
Northern Balochistan. 68
4.5. Seasonal Prevalence of Theileriosis in Sheep and Goats in
Northern Balochistan. 71
4.6. Prevalence of Theileriosis in Sheep and Goats in different age
groups. 74
4.7. Sex wise Prevalence of Theileriosis in Sheep and Goats in
Balochistan. 77
4.8. Molecular identification of Theileria species causing
Theileriosis in Sheep and Goats in
Balochistan.
82
4.9. Analysis of Hematobiochemical parameters of Sheep affected
with theileriosis. 84
4.10. Analysis of Hematobiochemical parameters of Goats affected
with theileriosis. 85
4.11. Efficacy of three drugs against Theileria in blood of Sheep
and Goats affected with
theileriosis.
87
4.12. Comparison of efficacy of three drugs against Theileria in
blood of Sheep and Goats
affected with theileriosis.
No. 3.1. Sample collection Districts and their Union Councils.
28
3.2. Schematic diagram indicating detailed sampling strategy for
the study of Theileriosis in
sheep and goats in Northern Balochistan.
32
3.3. Clinical examination of animals for the presence of Ticks in
sheep farm. 36
3.4. Clinical examination of animals for the presence of Ticks in
sheep farm. 37
3.5. Clinical examination of ticks infested animal. 38
3.6. Quantification of genomic DNA of Theileria species. 46
3.7. Tagging of Animals for treatment trails. 52
3.8. Tagging of Animals for treatment trails. 53
3.9. Collection of leaves and flowers of Calotropis procera.
54
3.10. Calotropis procera leaves in powder form. 55
3. 11. Prepared Sachet of Calotropis procera leaves and flowers in
powder form. 56
4.1. Prevalence of Theileriosis in Sheep and Goats in two Regions
of Northern
Balochistan.
60
4.2. Union Council wise prevalence of Theileriosis in Sheep and
Goats in Northern
Balochistan.
63
4.3. Species wise prevalence of Theileriosis in Sheep and Goats in
Northern
Balochistan.
66
4.4. Prevalence of Theileriosis in 4 different breeds of Sheep and
Khurasani breed of
Goat in Northern Balochistan.
69
4.5. Seasonal Prevalence of Theileriosis in Sheep and Goats in
Northern Balochistan. 72
4.6. Prevalence of Theileriosis in Sheep and Goats in different age
groups in
Northern Balochistan.
75
4.7. Sex wise Prevalence of Theileriosis in Sheep and Goats in
Northern Balochistan. 78
4.8. Gel picture of T .ovis and T. lestoquardi from sheep and goats
in Balochistan 80
4.9. Gel picture of T .ovis from sheep and goats in Balochistan.
80
4.10. Gel picture of T. lestoquardi from sheep and goats in
Balochistan. 81
4.11. Graph showing efficacy of three drugs against Theileria in
blood from sheep and
goats affected with theileriosis.
INTRODUCTION
Balochistan province is located in the north west of Pakistan, it
shares borders
with Punjab and and Khyber Pakthoon Khwa (KPK) to the northeast,
the Arabian Sea to the
south, Iran to the west, Sind to the southeast and Afghanistan to
the north. Ecological distribution
of the Province has six zones, Balochistan Coastal Region, Suleiman
Mountainous Region,
Northern highlands, Central Brahvi Highlands, Kachhi Basin Region
and Chaghai Kharan desert.
The provincial economy is based on natural resources including
livestock. The Province has
unique culture, extremely dry desert climate, and the Suleiman
Mountains with monsoon tail
region. It covers an area of 347,190 square kilo meters
constituting 44% of the country. The
climate is unique having very cold winters in highlands and hot
summer in lowlands (Weiss et al.
2012).
The livelihood of the people of Balochistan depends on livestock
especially on small
ruminants having 12.80 million sheep and 11.784 million goats
(Livestock census 2006). The
Northern range lands constitute 38 % of the total area and are
thickly populated with sheep and
goats having better grazing because of heavy rainfall. In these
areas sheep and goats are reared
on large scales under range management system (Kakar, 2009).
Livestock diseases are crucial in
the life of farmers which lower the production and weaken the
economy. Mortality and
morbidity resulting from diseases deprives the farmer earnings by
short term and long term
production losses. The diseases of livestock are mostly confined to
tropical and subtropical
countries like Pakistan, where climatic conditions are suitable for
growth and development of
many vectors as ticks. Theileriosis belongs to this complex and
affects both large and small
ruminants with high morbidity and mortality rates resulting in high
economical losses and thus
poses a serious risk to livestock production (Schnittger et al.
2000). Recently, interest has been
focused in the world on the sheep and goats infecting Theileria
species which include T.
lestoquardi and China 1 which are reported highly pathogenic. The
disease is characterized with
fever, weakness, anorexia, conjunctival petechae, swollen lymph
nodes, anemia, and cough. The
later stages of the disease are associated with diarrhea, dysentery
and recumbency (Schnittger et
al. 2000; Ahmed et al. 2002).
Malignant Ovine Theileriosis caused by T. lestoquardi infection is
now considered one of
the major constraints for sheep production in many areas of the
world. It has been reported that it
infects lymphocytes and little is known about the mechanisms
involved in the pathogenesis. The
high mortality probably linked to the ability of T. lestoquardi to
stimulate proliferation of the
infected leukocyte. As a result severe tissue destruction and
pulmonary edema occurs leading to
respiratory failure (El-Imam et al. 2015). The disease was reported
from different parts of the
world affecting large and small ruminants. In China the disease was
found more serious in lambs
and exotic animals than native animals (Jianxun and Hong, 1997).
24.6% prevalence of
theileriosis was reported in sheep from Greece (Papadopoulos et al.
1996). The disease was also
found a serious threat to sheep and goats in Kingdoms of Saudi
Arabia (El-Azazy et al. 2001),
Sudan (Salih et al. 2003), Iran (Razmi et al. 2006) and Turkey
(Sayin et al.2009). In Pakistan
Nausheen et al. (2010) reported 43.37% prevalence of ticks in sheep
and 41.53% in goats which
transmit different protozoal infections including theileriosis.
Rhipicephalus was found to be the
most abundant tick spp infesting both sheep and goats.
(Zia-ur-Rehman et al. 2010) reported
16.5% prevalence in sheep in the Okara district, Pakistan of which
65.1% were showed schizont
of thieleria in lymphocytes. The disease was also reported a
serious concern in sheep and goats
from Lahore, Pakistan (Durrani et al. 2011).
INTRODUCTION
3
Tick-borne diseases can seriously affect the well-being of farmers.
Impact on livestock, is
felt more in the tropical and subtropical parts of the world.
Tick-borne diseases cause heavy
economic losses by decreasing milk production, weight loss and an
estimated annual loss of
US$7,000 million was reported with significant population of cattle
i.e. above 80 % at risk of
tick borne infections (Willadsen. 2005). Ticks also involve in
increasing risk for bacterial, viral,
and fungal infections and are considered to be the second important
vectors after mosquitoes of
human infectious diseases in the world (Jabbar et al.
2007,Hashemi-Fesharki, 1988).
The disease is now considered a serious threat to different species
of animals in Pakistan
needs prompt action for in time diagnosis and tactical control
measures (Zia et al. 2010). The
conventional method of diagnosis depends on Giemsa stained blood
smears examination. The
presence of parasite in red blood cells along with the clinical
examination was the method used
for so many years in the world which may lead to false
morphological diagnosis and needs
special diagnostic skills. It is difficult to differentiate the
species on the basis of morphology of
piroplasm and schizont stages, especially in mixed infections. An
exact differentiation between
hemo-parasites is crucial for diagnosis and understanding the
epidemiology of the disease.
Alternatively serological and molecular techniques were used in the
world for accurate diagnosis
and better understanding of the epidemiological pattern of the
disease. Amplification of parasite
DNA using Polymerase Chain Reaction (PCR) has advantages over the
conventional methods of
detecting animals infected with Theileria and Babesia species. It
is more sensitive and specific
than parasite detection by thin blood smears (Almeria et al. 2001;
Aktas et al. 2004 and Aktas et
al. 2005). PCR and sequencing results showed that sheep were
infected with T .lestoquardi, and
T ovis (Spitalska et al. 2004 Altay et al. 2007). T. luwenshuni and
T. uilenbergi are reported
highly pathogenic for sheep and goats in China. As they are
morphologically indistinguishable
INTRODUCTION
4
and poorly characterized, there is no easily applicable method
available to differentiate between
these species. The specific PCR primers were found useful tools for
the detection and
differentiation of different species in mixed infection in small
ruminants (Yin, 2008). The advent
of molecular techniques, accurate identification of tick-borne
pathogens and precise diagnosis of
disease is now available in the world (Ahantarig et al. 2008). PCR
was reported more sensitive
and reliable detection tool for accurate diagnosis of theileriosis
in sheep and goats as compared
to conventional diagnostic methods (Durrani et al. 2012).
To study the effect of the disease on hematological parameters
different tests were made
in different animals like haemoglobin percentage (Hb%), packed cell
volume (PCV), differential
leucocytic count (DLC), total leucocytic count (TLC) and total
erythrocytic count (TEC), which
were found significantly decreased in bovine babesiosis (Sharma et
al. 2000) might be equally
important in case of theileriosis. Comparison of these values with
normal values in buffaloes
showed marked decrease in the PCV, TEC and Hb% suffering from
theileriosis Durrani et al.
(2006) which are likely to be equally important in case of sheep
and goats. The anemia is
considered as major manifestation of Theileriosis infection in
animals (Glass, 2001; Preston et al.
2002), complemented by some factors such as corpuscular oxidation
(Saleh et al. 2011) and
development of autoantibody IgG dependent phagocytosis (Shiono et
al. 2004). Diagnosis of the
disease mainly depends upon clinical signs expressed during
Theileriosis accompanied by
microscopic examination of Giemsa-stained blood smear examination
(Cicek et al. 2009).With
the advent of the PCR the diagnosis has become more accurate for
the detection of various
pathogens including Theileria species in livestock (Aktas et al.
2006; Galuppi et al. 2012; Li et
al. 2010).
Theileriosis was treated with different allopathic medicines
especially in cattle and
buffaloes. Anti Theilerial drugs such as buparvaquone have been
used effectively for many years
in the treatment of tropical theileriosis in the field and
considered as a drug of choice (Qayyum et
al. 2010). However, data are lacking regarding its effectiveness in
case of sheep and goats.
Efficacy of various antiprotozoal drugs like buparvaquone,
imidocarb dipropionate and
Diminazene acceturate was proven in cattle and buffaloes with
different concentrations (Akhter
et al., 2010) should be thoroughly investigated its effectiveness
in small ruminants. Herbal plant
Calotropis procera, Family Asclepiadaceae (local name Spalmi or
Aak) in the tropical countries
of Asia and Africa is a well-known wild plant with multifactor
medicinal characteristics (Bharti
et al. 2010). Plant growth is reported throughout the year in arid
and dry zones of Indian sub-
continent (Singh et al. 2005).
Calotropis procera, was also used as an alternative medicine in
Pakistan by researchers in a
range of different animal diseases as an anti-inflammatory, anti
bacterial, anthelmintic and
antiprotozoal in cattle and buffaloes (Zafar and Durrani et al.
2005). Use of medicinal plants and
ethno-veterinary practices are diminishing because of technological
advancement. Affordability
of the modern allopathic drugs in Pakistan is one the important
virtue of ethno-veterinary system.
Prices of these drugs and resistance support such practices.
6
Prevalence of Theileriosis and its Vector in Sheep and Goats
Prevalence of hemo-protozoan of sheep and goats in North Western
part of Tamil Nadu,
India was reported by Velusamy et al. (2015). The prevalence
recorded was 11% in sheep and
3% in goats. Statistically significant difference was observed
between sheep and goats. Theileria
and Anaplasma specie of hemo-parasites were diagnosed in both
species of animals. The
seasonal prevalence was found maximum in summer followed by winter
and minimum in rainy
season. Yas et al. (2014) collected 500 blood samples of sheep male
and female suspected for
theileriosis. 22.8% animals were found infected with Theileria and
reported that most of the
cases were in chronic form of the disease. There was no significant
difference among male and
female animals. It was noted that prevalence was significantly
higher in summer and old age
groups. Razmi and Yaghfoori (2013) investigated prevalence of
theileriosis in sheep and tick
vectors in the south of Khorasan Razavi Province, Iran. Blood
samples were collected from 30
small and large flocks and ticks were sampled. Blood smears were
stained with Giemsa stain and
examined microscopically. 18.6% blood smears were found positive
for Theileria species. The
most prevalent ticks were Rhipicephalus turanicus.
Climate change and variability in different regions can change the
parasites fauna and
their vectors. So the regular surveillance can be very useful for
control strategies. Sheep with
clinical sign like fever, jaundice and anemia were tested for
presence of Theileria and Babesia
using Giemsa staining method in Iran. Blood smears examination
revealed Theileria was the
only parasite seen with 2% prevalence rate (Tahamtan et al.
2013).
REVIEW OF LITERATURE
7
Prevalence of Theileria hirci and Babesia motasi among goats in
Duhok Governorate
were assessed by Zangana et al. (2011) from April to September
2010. A total 500 local black
breed were randomly selected from thirty five flocks. Animals were
clinically examined and
blood smear were prepared. The result showed that T. hirci was
20.8% and B. motasi was 4% in
goats. The prevalence Theileria and Babesia infection was
statistically significant between male
and female and different age groups of goats. Hematological
findings revealed a significant
decrease in hemoglobin concentration, red blood cell count and
packed cell volume. Macrocytic
and normochromic type of anemia was also observed. Ovine
theileriosis were assessed in five
different regions in eastern half of Iran. A total of 220 Blood
samples from sheep were examined
through nested-PCR and microscopically. Theileria spp were positive
60% by nested-PCR and
22.27% by microscopic examination, out of 132 positive blood
samples, 55.3% were positive for
T. lestoquardi and 44.7% for T. ovis Heidarpour et al.
(2010).
A survey was conducted by Irshad et al. (2010) on prevalence of
tick infestation and
possible disease transmission of theileriosis in sheep and goats
maintained at National
Agricultural Research Centre Islamabad, Pakistan and Barani
Livestock Production Research
Institute Kherimurat, Attock, Pakistan. A total of 662 animals were
screened 43.37% sheep and
41.53% goats were found infested. Prevalence of theileriosis in
sheep was 7.36% and in goats
3.8%. A theileriosis of sheep and goats in China has been reported
by (Yin et al. 2007) to be due
to T. lestoquardi. They reported additional ovine and caprine
piroplasm species, designated T.
luwenshuni and T. uilenbergi. A study was carried out in Turkey by
Sayin et al. (2009) to detect
Theileria infection in sheep and goats on the basis of different
geographical and environmental
regions such as Central Anatolia, Eastern Anatolia and Southern
Anatolia. A total of 687 sheep
and 89 goats suspected to have Theileria infection were examined.
They reported 64.19 %
REVIEW OF LITERATURE
8
prevalence of infection with T. ovis in sheep and 12.36 % in goats.
The sero prevalence was
found 60.26 % in sheep and 8.99 % in goats by Indirect Fluorescent
Antibody Test. T.
lestoquardi was not found in any of the blood samples of sheep or
goats. Durrani et al (2008)
recorded the prevalence in buffaloes through blood smear
examination and PCR. The results
showed 39.9% prevalence through blood smear examination and 53.3%
through PCR hence
stated that PCR is more reliable and sensitive technique for
diagnosis of the disease. Ghosh et al.
(2007) were of the view that globally ticks transmit a variety of
pathogenic microorganisms and
are among the most important vectors of diseases in livestock,
human and companion animals.
Tick-borne diseases affect 80% of the world animal population and
are widely distributed
particularly in tropical and subtropical countries including
Pakistan. In subcontinent the livestock
sector is suffering from a number of disease problems. The damage
caused by Tick-borne
diseases is considered very high. (Altay et al.(2007) carried out a
prevalence survey to determine
the incidence of Theileria T. ovis and T. lestoquardi in small
ruminants through blood smear
examination and PCR in the Anatolia. Whole blood samples 677 sheep
and 142 goats were
collected from Malatya. Thin blood smears of 656 sheep and 139
goats were examined.
Theileria were detected in 18.29% sheep and 2.88% goats. T. ovis
was detected in 11.27% of
goats and 58.79% of sheep. T. lestoquardi was not detected in the
animals. The prevalence of
theileriosis was studied in sheep in the South Khorasan province in
Iran by Ramzi et al. (2006).
A total of 840 sheep from 34 flocks were examined and investigated
through blood smears
examination. 11.9% sheep were found infected with Theileria spp.
Difference in the infection
rates was significant among different areas. The difference of
prevalence in males and females
and age groups were not found significant. The prevalence was
highest in June (26.3%),
gradually decreased in July and August.
REVIEW OF LITERATURE
9
A study was conducted by Nagore et al. (2004) to investigate the
prevalence of
Theileriosis and babesiosis in sheep population. Sheep 320 located
in the Basque County Spain
were analyzed. They reported 64.7% prevalence of subclinical
infections in sheep. Three
Theileria and two Babesia genotypes were also identified in the
study. They also reported 78.7%
prevalence of piroplasmosis in another investigation. They also
found that these piroplasm
species were significantly involved in abortion in the study area.
Ramzi et al. (2003) conducted
a study to determine the population of ticks in sheep in Iran.
Suspected cases of Theileriosis from
28 flocks of sheep were clinically examined and reported 36.17%
prevalence of the disease in
sheep. Seasonally the prevalence was highest in June and July
whereas lowest in September and
August. Sex wise prevalence of hemo-parasitism was generally higher
in female animals. The
lower prevalence in young animals compared to adults can be
attributed to the restricted grazing
of young animals which tends to reduce their chance of contact with
the vectors of these
diseases. However, the parasitism effect on the mean PCV was
deleterious in younger as
compared to adult animals Enwezor et al. (2009). The age groups and
gender was not found
significantly affected. Guo et al. (2002) studied sheep
Theileriosis in Gannan Tibet. The disease
was found endemic in Gannan. Seasonal prevalence was found higher
in March to May and
lower in September and October. The incidence rate was 60.8% and
mortality 81.5% in young
animals as compared to 17.12% and 65.78% in adult sheep. The mean
incidence values were
40% and mortality 85.71%. The incidence rate for goats was 8.06%
and the mortality was
73.33%. El-Azazy et al. (2001) stated that data are lacking about
tick-borne diseases in sheep and
goats. They conducted a survey of vectors of malignant theileriosis
of sheep in Saudi Arabia.
Samples were collected from animals at the Veterinary Diagnostic
Laboratory in Jeddah,
Makkah and Bureida, Al-Qasim. Blood and lymph node smears were made
and examined.
REVIEW OF LITERATURE
10
Disease was found 2.8% in Jeddah and 24% in Bureida. The infected
sheep were infested with
Hylomma Ticks.
A study was carried out by Papadopoulos et al. (1996) on piroplasms
of small ruminants
in the Macedonia region of Greece. A total of 721 serum samples
were collected from sheep and
487 from goats. The prevalence of positive IFA titers for sheep and
goats sera were 24.6% and
0.6% for T. ovis, 52.1% and 36.4% for B. ovis, 10.5% and 4.2% for
B. motasi, 12.6% and 6.6%
for B. brassa and blood smears revealed the presence of T. ovis and
B. ovis in sheep and
Anaplasma ovis in a goats.
The role of vector in the spread of Theileriosis was reviewed by
Jongejan and Uilenberg
(1994). They discussed seven important genera of ticks Boophilus,
Dermacentor,
Haemaphysalis, Hyalomma, Ixodes, Rhipicephalus and Amblyomma. They
identified 840 tick
species involved in disease transmission in their hosts. Ixodids
also called hard ticks comprised
80% of the total tick species population. Staining techniques were
used to detect the parasites
infection in ticks for many years but today PCR is the more
sensitive, quick and reliable
technique even to differentiate the protozoan spp. along with the
prevalence and severity of
infection. Tick transmitted piroplasmosis was the important disease
affecting cattle in the South
mountain pasture of Hunan province. In their study ticks were
collected from cattle and
identified three genera and four species: Haemaphysalis
vietuamensis, Haemaphysalis
longicornis, Ixodes sinensis and Boophilus microplus). The tick
species were found potential
vector of T. sergenti. The disease affected calves from May to
August reaching a peak in June or
July. In cattle, the disease occurred from June to September with a
peak in September. The
pathological lesions observed were; subcutaneous hemorrhages in
tissues and serosa, intestinal
REVIEW OF LITERATURE
11
mucosa and most prominently mesenteric lymph nodes, enlarged liver,
gall bladder filled with
tawny colored bile, enlarged spleen and heart with numerous
hemorrhagic foci in the auricle.
A survey was conducted in Layyah & Muzaffargarh districts of
Punjab by Sohail et al.
(2008). They found that tick infestation is still a great threat to
livestock and dairy industry of
Pakistan. The prevalence was calculated in 3500 cattle and
buffaloes and found highest in cattle,
1076 (72.9%) whereas in buffaloes the prevalence rate of tick
infestation was recorded as 957
(47.3%). Hyalomma anatolicum anatolicum was found more prevalent
among 3500 animals
followed by Rhipicephalus sanguineus. The seasonal prevalence was
noted high in the month of
July with no prevalence in January, February, March November,
December. The importance of
ticks in disease transmission cannot be neglected in the world
because they cause heavy
economic losses In Australia in 1974 losses caused by cattle tick
(Boophilus microplus) were
calculated as USD 62 million Rajput et al. (2006) The importance of
piroplasmosis was reviewed
by Bock et al. (2004) causing heavy losses to cattle industry. The
studies shown economic value
by estimating 1.2 billion cattle exposed to babesia
infection.
Theileriosis due to T. hirci and babesiosis due to B. ovis and B.
motasi were the most
pathogenic protozoa of sheep. The major tick genera found were
Hyalomma, Rhipicephalus,
Haemaphysalis, Ixodes and rarely Dermacentor distributed in all
part of Iran. Yin et al (2002),
investegeted that the Hyalomma ticks transmit Theileria species in
Gannan area of China. These
results shown that the ticks were infected with two species of
Theileria. The species infective for
sheep could be related to the newly recognized, but not yet named,
pathogen recently reported in
small ruminants in China. In another study Razmi et al. (2003)
assessed the prevalence of
different genera of ticks in sheep causing ovine Theileriosis in an
endemic area of Iran.188 sheep
were clinically examined from 28 flocks and reported 36.17%
prevalence infected with T.
REVIEW OF LITERATURE
lestoquardi. 61.1% of the animals harbored Hyalomma a. anatolicum,
33.42% Rhipicephalus
sanguineus and 0.05% Hyalomma marginatum marginatum. The
examination of 345 ticks
showed that 15% of salivary glands of H. anatolicum. anatolicum and
4% of R. sanguineus
contained Feulgen positive bodies. Seasonally, the prevalence of
Theileria infection and ticks
was highest in July (62.5%) and June (23.6%), while a decrease was
observed in September
(24.5%) and August (17.39%). Ticks were not only involved in
disease transmission but also
allow development of the infectious agent inside. Singh and
Girschick (2003). Ticks-borne
diseases assuming more importance as they cause major economic
problems Makala et al.
(2003). A total of 109 species of ticks were identified by Yin and
Luo (2007) in China. 45
species were found infest small ruminants and five involved in the
transmission of tick-borne
diseases. Pathogenic microorganisms like protozoa, rickettsiae,
spirochaets, and viruses were
transmitted to the animals by ticks and most important vectors of
diseases affecting 80%
livestock in tropical and subtropical countries Ghosh et al.
(2007). T. uilenbergi (China 1) the
most pathogenic heamo-parasite could be transmitted to small
ruminants by at least two species
of ticks namely Haemaphysalis longicornis and Haemaphysalis
qinghaiensis Youquan Li et al.
(2010). Ovine theileriosis is an important tick-borne disease of
sheep in the tropical and
subtropical regions of the world Zia-ur-Rehman et al. (2010) caused
by T. hirci (T. lestoquardi).
The disease is economically important in sheep and goats, causing
clinical illness and mortalities
in Mediterranean Basin, west Asia, Middle East, Indian
subcontinent, and parts of Africa.
A study was conducted by Hashemi and Goodarzai, (2016) in Lorestan
province of Iran
to identify the vector of theileriosis in sheep through PCR. From
April to July (2012), a total
number of 265 herd ticks were collected from different predilection
sites of Theileria infected
sheep using a species specific primers for T .ovis and their result
revealed that Rhipicephalus
13
sanguineus were positive for T.ovis as well as 21 (30.88) percent
female ticks and 16 ( 19.04)
percent male ticks with a maximum numbers of R. sanguineus ticks
(57.35) percent and least
number(3/01) percent of Haemaphysalis punctuat ticks were recorded
in the study area in view
of that R. sanguineus is the primary vector of theileriosis in
sheep in Lorestan Iran.
A survey was conducted by Razmi et al. (2011) to investigate the
frequency of hard ticks
on sheep in Khorasan Razvi province of Iran. They collected and
identified 812 ticks from sheep.
Five different species Rhipicephalus turanicus (59.23%),
Hyalomma.marginatum turanicum
(25.73%), Hyalomma excavatum (14.8%), Hyalomma anatolicum (8.3%),
and Dermacentor
niveus (4.8%) were reported. R. turanicuss and H. m. turanicum were
predominant ticks in the
province. Hasson and Al-Zubaidi, (2012) conducted a study to
investigate the prevalence of ticks
infestation in sheep and goats. 366 animals were examined, out of
these 69 (18.85 %) were found
infested and infestation was more prevalent in sheep than goats.
Ali et al. (2013) conducted a
survey to estimate the prevalence of ticks infestation in grazing
sheep in Iran. The frequency
was Hyalomma 46.88%, Rhipicephalus 42.32 % and Haemaphysalis
10.78%.
Molecular Identification of Theileria in Sheep and Goats.
Theileria are intracellular apicomplexan protozoan infecting a wide
range of animals. In
the Caribbean Zhang et al. (2015) used genus-specific pan-Theileria
FRET-qPCR to identify
infected animals. They found Theileria spp. in 9% of the samples of
animals. Donkeys revealed
20.0% followed by sheep 17.4%, cattle 6.8%, goats 5.0% and horses
5.0%. They also
identified Theileria spp. OT3 in sheep and goats, T. equi in
donkeys, cattle, goats, and sheep,
NG-2013 in cattle, YW-2014 in donkey and B15a in goats. Saeed et
al. (2015) carried out a study
in Kohat and Peshawar districts of Pakistan to determine the
prevalence of T. lestoquardi in
sheep and goats. They collected 165 blood samples were from sheep
(n= 44) and goats (n= 121).
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14
Data on the characteristics of animals and the herds five samples
(3%) were found positive and
generated 730 bp product through PCR amplification of 18S SSU rRNA
gene, specific for T.
lestoquardi. None of the samples from Peshawar district were found
positive for this parasite. It
was observed that presence of tick on the domestic animals and dogs
along with mixed flocks
were highly significant risk factor for the spread of ovine
theileriosis. They concluded that PCR
is more sensitive and reliable diagnostic tool for detection of T.
lestoquardi in blood samples of
sheep and goats.
A molecular study were conducted in China by Yang et al. (2014) and
reported that
Theileria spp. can infect large and small ruminants causing
diseases and heavy economic losses.
Diagnosis of infection on the species level is often challenging
because identical parasites are
difficult to identify microscopically. It can be made easier while
using PCR assays which can
identify Theileria spp. In their study they detected all recognized
species of Theileria
successfully in whole blood samples from animals in China. DNA of
Theileria was detected in
53.2% of the sheep, 44.4% of the goats and 30.8% of the cattle.
Sequencing of some of the PCR
products showed T. ovis and T. luwenshuni were found in sheep and
T. luwenshuni in goats.
Shahzad et al. (2013) used primers specific for Theileria spp to
amplify small subunit ribosomal
RNA from Theileria species genomic DNA from blood samples of the
sheep. A species specific
primer pair for T. ovis was also used in the study to amplify 520
bp product from T. ovis genomic
DNA. 37% samples were found positive for T. ovis. All positive
samples were successfully
amplified using both primer pairs. PCR was found more sensitive and
specific for disease
diagnosis and species identification of Theileria.
A molecular study was conducted by Ramzi and Yaghfoori (2013) in
Iran to detect T.
ovis, T. lestoquardi in sheep and tick vectors. Blood samples were
taken from sheep and salivary
REVIEW OF LITERATURE
15
glands of ticks were isolated and analyzed by PCR. PCR-RFLP was
used to differentiate
Theileria species from PCR-positive samples. Microscopically
detected 18.6% of blood smears
samples, 58.6% samples were identified as T. ovis and 6.6% as T.
lestoquardi. In total, 169
Ixodids ticks were collected from different areas of the province.
The most prevalent ticks
were Rhipicephalus turanicus (n = 155); followed by Hyalomma
anatolicum anatolicum
and Hyalomma marginatum turanicum. 33 ticks were found positive for
Theileria species by
semi-nested PCR.
In an experiment in Turkey Aydin et al. (2013) investigated the
presence and distribution
of Theileria and Babesia species in sheep and goats in Turkey. The
genomic DNAs were
extracted from blood samples of animals. 360–430-bp fragment 18S
SSU ribosomal RNA gene
of Theileria and Babesia species was amplified using the genomic
DNAs. 3.37 % animals
including 34 sheep and 4 goats were found positive for Theileria
spp. Infection rates were
34.64 % in sheep and 10.04 % in goats, A Theileria sp. OT3 was also
detected in 2.04 % of
animals; besides Theileria sp. Durrani et al. (2011) collected 200
whole blood samples along
with 100 samples of ticks from 20 different flocks of sheep in
Lahore, Pakistan. The history of
fever and anemia were taken into consideration for screening of the
animals. On microscopic
examination 22% samples were found positive for Theileria, while
35% blood samples were
found positive for Theileria species by PCR. T. ovis was found
prevalent in 79% of the cases and
T. lestoquardi in 21% animals. In an experiment 250 sheep blood
samples were taken from
during tick season. Microscopic examination revealed that 9.2%
sheep were infected with
Theileria spp. Parasitemia was observed ranged 0.011% to 0.015%.
The results of nested PCR
assessment of DNA samples, 32.8% sheep were found positive. The
evaluation of positive
samples revealed, 54.8% and 40.2% T. lestoquardi and T. ovis
respectively. Mixed infections
REVIEW OF LITERATURE
16
were found in 4.8% cases. Based on their PCR product digestion
pattern they claimed that
mixture of T. annulata and T. lestoquardi was observed in some
cases. The presence of T.
annulata was unreported by sequence analysis Zaeemi et al. (
2011).
Theileria was reported very common and two species, T. lestoquardi
and T. ovis, are
suspected in case of ovine theileriosis in Iran. The
epidemiological aspects of the disease are
poorly understood and further investigations are required with more
sensitive and precise
techniques. Theileria species were determined in sheep from five
regions of Iran blood samples
were collected in EDTA and data revealed 60% infection rate by
nested-PCR compared with
22.27% by microscopic examination. 55.3% samples were positive for
T. lestoquardi and 44.7%
for T. ovis. Bami et al. (2010). Theileria species involved in
sheep theileriosis were determined
in eastern half of Iran. Blood samples from 220 sheep obtained from
five different regions. 60%
were found positive for Theileria spp. by nested-PCR compared with
22.27% by microscopic
examination. Two types of Theileria spp. T. lestoquardi (55.3%) and
T. ovis (44.7%) were found
involved in the disease Heidarpour et al.2010). T. luwenshuni and
T. uilenbergi were found
highly pathogenic for sheep and goats in China. The researcher
stated that these two species of
Theileria are morphologically indistinguishable and poorly
characterized. They used specific
PCR primers and successfully amplified the genomic DNA of the two
species and declared that
there method was useful for the detection and differentiation of
both species of Theileria in
mixed infection occurs in small ruminants Yin et al. (2008).
With the advent of molecular techniques, identification of
tick-borne pathogens at species
level and the accurate diagnosis of the disease well in time are
available in the world. Tick
transmitted diseases in animals like theileriosis were reported
from different parts of the world.
Two types of Theileria species T. ovis and T. lestoquardi were
found in ovine disease. Ahantarig
REVIEW OF LITERATURE
17
et al. (2008). In Italy and Iran PCR amplification of the target
18S rRNA gene, showed 78.7%
and 76.0% prevalence of the disease in sheep. The sequencing showed
that they were two
species involved, T. ovis and T. lestoquardi Olivier et al. (2006).
In Turkey a total of 218 blood
samples were collected from sheep. Theileria piroplasm DNAs,
extracted from sheep blood, was
subjected to the polymerase chain reaction (PCR) procedures, using
specific primers for
Theileria species and T. lestoquardi. Blood smears were examined
for Theileria piroplasms by
microscopic observation. In PCR analysis, the expected amplicons
were obtained from 41.2%
blood samples with a molecular size of 1098 bp for Theileria spp.
whereas none were amplified
by T. lestoquardi-specific primers. Piroplasms of Theileria species
were detected in 15.5% of the
blood samples Aktas et al. (2005). Specificity of molecular
surveillance of tick-borne diseases
based on polymerase chain reaction showed 76.0% and 59.5% of blood
and tick samples
respectively. 93.1% and 82.6% of the bacteria-positive blood and
tick samples were also positive
for Piroplasmida. Sequencing results showed that sheep were
infected with T. lestoquardi
Spitalska et al. (2004).
A nested PCR for the detection of T. ovis in sheep was used.
Sensitivity of the nested
PCR for T. ovis was assessed that one infected cell in 107 sheep
erythrocytes, equivalent to a
blood Parasitemia of 0.00001%, could be detected. This is more
sensitive than examining 200
fields under light microscopic examination (ME). In addition, of
the 124 field samples obtained
from sheep in eastern Turkey tested, 19.35% were positive for the
presence of Theileria spp. by
microscopic examination compared to 54.03% positive for T. ovis by
nested PCR Altay et al.
(2004). PCR detection of Theileria parasites in ticks was compared
with conventional staining of
dissected salivary glands using methyl green pyronin and its
comparative advantages are
discussed. Molecular epizootiology of piroplasmids i.e., Babesia
spp and Theileria spp. was
REVIEW OF LITERATURE
18
studied in sheep and goats from Spain, Portugal and France by
Criado-Fornelio et al. (2003).
Blood samples were taken from nine sheep and one goat. Piroplasmids
detected in Sheep and
goat was B. ovis, B. bovis, which shows a relatively lower degree
of homology (94%) with
regard to other B. bovis isolates from several countries. The same
is true for B. ovis that showed
a 94% identity with regard to Babesia sp. from South African cow
and a 92% with report to B.
bovis from Portugal.
Hematology.
A study was performed to assess the hematological parameters in
sheep infected with
Theileria spp. after conformation of the infection by microscopic
examination and PCR.
analysis. T. ovis was conformed in 88% of the inspected sheep by
PCR, and 67.8%
microscopically. Hematological analysis showed that mean RBC, PCV,
Hb, and MCHC were
significantly lower, whereas RDW and MCV were found higher compared
to the uninfected
sheep Khaki et al. (2015). Ayesha et al. (2013 investigated
hematological and biochemical
parameters in ten Sudanese desert ewes and compared with 10
clinically healthy ewes. Their
results showed a significant decrease in Hb%, PCV and WBC counts
compared to the control
group. The decrease in Hb% and PCV was observed 7-10 weeks after
tick application. It was
noted that there is marked decrease in WBCs occurred at week 5 and
week 6. In another
experiment Rashid et al. (2010) evaluated the changes in blood
parameters in animals affected
with piroplasms. Their investigations revealed a significant
decrease in Hb% and Hematocrit
values for positive animals compared to healthy controls.
A study were conducted by Khan et al. (2011) and studied blood
hematological and
biochemical parameters in cross bred cattle naturally infected with
theileriosis in semi arid zone
of Pakistan. Study comprised of 50 cross bred cattle ranging from
2-5 years in age. The clinical
REVIEW OF LITERATURE
19
signs observed were rise in body temperature, swollen lymph nodes,
mucosal hemorrhages and
conjunctivitis. A statistically significant decrease in TEC, PCV,
hemoglobin concentration,
serum total proteins, albumin, globulins, glucose, calcium,
phosphorus, cholesterol and
triglycerides concentrations was recorded in affected cattle while
significant increase was
observed in levels of serum bilirubin and ALT of affected cattle
compared with healthy cattle.
Similar results were reported by Shahnawaz et al. (2011) in cattle
and buffaloes affected with T.
annulata in Punjab, Pakistan. Biochemical investigation revealed a
significant decrease in values
of hemoglobin and glucose in infected cattle. Concentrations of
serum ALT, AST, LDH and
cholesterol in infected cattle was higher as compared to that of
non-infected cattle. Sulaiman et
al. (2010) examined 175 goats, 27 were found infected with Babesia
spp. When hematology was
performed, statistically significant decrease in TEC, Hb%, PCV and
platelets counts were noted.
A significant increase in ESR and WBCs was recorded along with
significant increase in
lymphocytes and neutrophils. Zia-ur-Rehman et al. (2010) studied
the impact of ovine
theileriosis on certain blood values. A total of 400 sheep were
examined for the presence of
Theileria spp in thin blood smears. A mild to severe anemia in the
infected animal was observed.
There was a marked decrease in total leukocyte count and hemoglobin
percentage noted along
with significant increase in total leukocyte count.
A study of hematological parameters in buffaloes suffering from
theileriosis in different
areas of Punjab, Pakistan Durrani et al. (2006) reported mean
values of PCV, TEC and
hemoglobin concentration (Hb%). The overall mean values of these
three hematological
parameters were 0.15 l/l, 3 X10 12
and 64.5 gm/l respectively. Comparison of recorded values
from diseased animals with normal values in buffalo showed a marked
decrease in the PCV, total
REVIEW OF LITERATURE
animal blood parameter values were found to be statistically
significant.
In an experimental study Singh et al. (2001) evaluated blood
samples collected from
experimentally infected calves with an average age of 53 days. The
percentage paracitemia was
71.7%± 3.3% recorded after 20 days of inoculation. Giemsa stained
thin blood smear
examination revealed macroschizonts in monocytes & lymphocyte.
A significant decrease in the
Hb% and PCV was observed after 20 days. Biochemical analysis showed
a marked decrease
(P<0.05) in the concentrations of calcium, and triglycerides
along with an increase (P<0.05) in
the concentration of blood urea nitrogen as compared to day 0
values. Statistically significant
decreases were measured in the total serum proteins and albumin.
Increased osmotic fragility of
erythrocytes during terminal stages of disease was observed along
with morphological alterations
in erythrocytes.
Researcher Friedhoff (1997) findings displayed general anemia along
with steady
decrease in total erythrocyte count (RBC) and hemoglobin (Hb)
concentration. (Dhar et al. 1986;
Mishra et al. 1994) findings of hematology analysis revealed
significant decrease in total
erythrocyte count, hemoglobin and total leukocyte count in
experimentally infected calves,
Similar findings Durrani and Kamal (2008) reported significant
decrease in Hb, WBC and total
leucocytes count in both affected Cross bred and Sahiwal cattle.
Goddeeris, (2000) also
described that there are significant difference in total leukocyte
count, hemoglobin and red blood
cell count concentration. Similar findings have also been described
by Kumar and Malik (1999)
who reported that heamoglobin, total leukocyte count and packed
cell volume decreased post-
infection. Osman and Al-Gaabary (2007) reported significant
decrease in hemoglobin, total
leukocyte count and erythrocytes count in buffaloes infected with
Theileria compared to control.
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21
A decrease in leukocytes count was noticed as a result of
devastation of lymphocytes in
lymphoid organs as well as dissemination of infiltration of these
into different organs cells
(Beniwal et al. 2000; Omer et al. 2002). Hematocrit values and TEC
were found decreased in
Theileria infected animals as compare to healthy animals Hasanpour
et al. (2008). In Egypt
Mahmmod et al. (2011) investigated the hematological findings
besides clinical disease
manifestations in the crossbreed cattle and water buffaloes
naturally infected with T. annulata.
Their hematological findings showed significant decreased in blood
parameter including PCV %,
RBCs count, WBCs count and hemoglobin amount in diseased animals as
compared to control
ones.
Serum AST and ALT concentrations are the indicators of hepatic
function as described
by Forsyth et al. (1999). Some authors Sandhu et al. (1998)
attributed the abnormally high level
of Creatinine kinase Aspartate Aminotransferase,
Gamma-glutamyltransferase and Alanine
aminotransferase and in the measurements of blood urea nitrogen,
bilirubin and uric acid in
Theileria infected calves. In accordance to them abnormally high
level of AST and ALT in
animals infected with Theileria infection was reported by
(Shahnawaz et al. 2011; Yadav and
Sharma, 1986). They reported alteration in these biochemical
reactions occurred due to indirect
damage of kidney and liver tissues. Similar findings were described
by (Hasanpour et al., 2008;
Singh et al., 2001b).
Therapeutic Trails against Theileriosis
An outbreak of theileriosis was investigated and treated in exotic
cows in India. Theileria
positive animals were treated with Butalex (Ranbaxy, India)
intramuscularly at the dose of 2.5
mg/kg body weight. Healthy cows were also given Butalex as
prophylactic measures. Since the
temperature and humidity in the months of July, August and
September was very high, the cows
REVIEW OF LITERATURE
22
were kept in an air-conditioned room. Second injection of
buparvaquone given after 48 hours of
first treatment. After some recovery supportive therapy with B
complex and iron sorbitaol plus
Folic acid and Hydroxycoblamin was given intramuscularly. At the
end 4 animals, 4 calves died
in spite of the treatment Kohli et al. (2014), Osman and Al-Gaabary
(2007) reported that
excellent result of buparvaquone was observed in early treatment of
30 naturally infected water
buffalos recovered soon from clinical state whereas in later stages
of disease low efficiency of
buparvaquone recorded.
Efficacy of drugs was measured by Ijaz et al. (2013) and they
revealed that the treatment
of 80 animals sheep (n=40) goats (n=40) positive for babesiosis.
The diseased animals were
divided into four groups A, B, C and D equally. The group A were
treated with imidocarb
dipropionate (Imizol® ICI, Pakistan) @ 2 mg/kg body weight
intramuscularly and
oxytetracycline @ 10 mg/kg body weight IM, group B was treated with
imidocarb dipropionate
alone, group C was given Diminazene aceturate (Fa-try.banil; Prix
Pharma, Pakistan) @ 3.5
mg/kg body weight intramuscularly and oxytetracycline @ 10 mg/kg
body weight. While group
D was treated with Diminazene aceturate alone. Efficacy of drugs
was measured on the basis of
disappearance of clinical signs and negative blood smear
examination at day 2, 4, 6 and 10 post
treatments. Combine treatment of imidocarb dipropionate and
oxytetracycline was proven the
most effective treatment in sheep and goats followed by combination
of Diminazene aceturate
with oxytetracycline.
A therapeutic study was conducted by Basheir et al. (2012) and they
recorded 32 vector
borne animal diseases prevalent in South Kordofan State Sudan.
Traditional treatment was
found to be a common practice particularly treatment with herbal
medicines and plants. Their
study revealed that infection with blood parasites was common. They
found 70% of blood
REVIEW OF LITERATURE
23
smears were positive for the blood parasites. In sheep and goats
Theileria spp. were found
common infections causing economic losses. In India the calves
infected with theileriosis were
treated with buparvaquone at a dose rate of 2.5 mg/kg body weight
along with Haemoferron 2 ml
IM and Artizone 2 ml IM, injected one week of treatment. They used
buparvaquone IM for the
successful treatment Naik et al. (2010). Clinically infected cattle
(n=35) with theileriosis were
subjected for the clinical therapeutic trail by Al-Hosary et al.
(2010). Different age groups of the
infected animals were used in the trails. Buparvaquone Injection
used for the treatment which
gave 91.4 % accuracy rate. They further stated that the treatment
accuracy may reach 100% if
given at the start of infection. In case of respiratory
complications or secondary bacterial
infections antibiotics should be incorpoarated in the treatment.
Their trails showed excellent
results when combine treatment of buparvaquone and Marbofloxacine
was given.
Efficacy of various antiprotozoal drugs on bovine theileriosis was
investigated in 38
buffaloes. Group-A was treated with a single dose of imidocarb
dipropionate (3 mg / kg body
weight intramuscular. In Group A-I one animal recovered completely,
02 moderately while
remaining 02 animals did not respond. The later 02 animals
recovered completely after
additional 02 doses of 4 mg/kg body weight. In case of group A-II
complete recovery was
recorded in one animal, moderate recovery in three animals while
one animal showed no
response. Group B was treated with Diminazene acceturate (3.5 mg /
kg body weight IM,
administered as single dose. Six animals recovered completely, 02
moderately while 02 animals
showed no response. Group C was treated with a single dose of
Butalex (2.5 mg / kg body
weight) IM, complete recovery was recorded in 4 animals, 2 animals
showed no response, while
2 animals with severe infection died after 24 hours. (Akhter et
al., 2010). A total of 310 blood
samples of sheep were collected for treatment by Rashid et al
(2010) from Livestock Experiment
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Station, Qadirabad, Sahiwal district, Pakistan. Microscopically
9.67% samples were positive for
piroplasm (babesia). The diseased animals were divided into two
groups for chemotherapy. One
group was treated with Diminazene diaceturate while other with
imidocarb dipropionate.
Negative blood smear determined drug efficacy. Diminazene
diaceturate proven effective (80%)
against babesiosis compared to imidocarb dipropionate was 100%.
Hematological studies
revealed a significant decrease in Hb% and Hematocrit values. An
experiment was conducted by
Sing et al. (1993) in which bovine theileriosis was treated with
buparvaquone (n=86) and
oxytetracycline (n=23) in India. The diseased animals ranged 6 days
to 3 years of age.
Buparvaquone cured 98.8% animals while oxytetracycline cured 30.4%
of the animals treated.
Treated animals remained free of infection for 12 to 18 months
following recovery. In another
study long-acting Oxytetracycline injected intramuscularly at
20m/kg by Singh et al. (1993). The
drug was not found effective on day 8, 10 and 12 post-infection.
Their findings showed that
Halofuginone lactate was effective. Buparvaquone at dose rate of 20
mg/kg body weight
intramuscularly on day 11 post-infection showed significant effect
when compared to
parvaquone. Their findings suggested single treatment with
buparvaquone, either at 5 mg/ kg or
2.5 mg/ kg intramuscularly, rapidly eliminated schizont and
piroplasms of T annulata. At 5 mg/
kg, it resulted in rapid recovery of all the treated calves.
A study carried out in Kenya by Muraguri et al. (1999) in which
buparvaquone drug was
used. According to their findings in naturally infected animals
efficacy of the buparvaquone and
parvaquone was 90 percent and 80 percent respectively.
Calotropis procera is a wild growing plant of family
Asclepiadaceae. The medicinal
activity of this plant is well known. Different parts of the plant
have been reported as an
excellent anti-inflammatory, analgesic, and antioxidant properties.
The antimicrobial activity of
REVIEW OF LITERATURE
25
Calotropis procera admitted in different reports Verma et al.
(2010). In another study Durrani et
al. (2009) carried out therapeutic trials of C. procera and
buparvaquone (Butalex) in cross bred
cattle after experimental infection with Theileria annulata. The
results of the trials indicated that
C. procera was effective in the treatment of theileriosis in
cattle. Liver and kidney function tests
after treatment with C. procera showed no toxicity at the dose rate
of 0.3 mg/Kg orally. The
efficacy of C. procera was noted higher than buparvaquone on 21 day
post treatment. C. procera
is a wild flowery plant all around the world in Tropical areas. In
Pakistan, C .procera was
successfully used as an anthelmintic in small ruminants in crude
powder mixed with jiggery. The
plant was also used for treatment of cattle infected with Theileria
Farah et al. (2014).
The antimicrobial effect of leaf and latex of Calotropis procera
(ethanol, aqueous and
chloroform extracts) were studied. The results of the study
revealed that ethanol was the best
extractive solvent. The extracts of C. procera latex gave the
widest zone of inhibition against E-
coli. Similar results were obtained in case of fungi. The MIC
(minimum inhibitory
concentration) for the ethanol extract was 5.0 and 20.0 mg/ml for
fungi. This study revealed that
the C. procera latex had a strong inhibitory effect on the test
organisms Kareem et al. (2008). A
trail was conducted by Mirzaiedehaghi, (2006) in naturally infected
sheep Theileriosis with
chloroform extract of Pegnum harmala for 5 days at dose rate of
5mg/day 65% of the cases of
sheep were recovered. In another study in Pakistan the cows
affected with theileriosis was
treated with Buparvaquone (Butalex) injection @ 5 ml/100 kg body
weight IM. The drug showed
100 percent effectiveness Zahid et al. (2005).
Anti-ticks activity of the different aqueous extracts of the
medicinal plants (Azadirachta
indica leaves, Nicotiana tabacum leaves, Calotropis procera flowers
and Trachyspermum ammi
seeds) was evaluated in Pakistan. At a dose rate of 50mg/ml of the
extract affected egg laying,
REVIEW OF LITERATURE
26
hatchability and total larval motility of the ticks. The suspension
of the plants reduced the tick
infestation intensity on the calves topically. The response against
the Rhipicephalus microplus
(Boophilus) of the plant extract was both dose dependent and time
dependent Zaman et al.
(2012). In Nigeria, Trypnocidal activity of the hydro ethanol
extract of the Calotropis procera
was studied by Hassan et al. (2013) in albino rats having the
infection of Trypnosoma brucei
brucei. The rats were treated with the dose rate of 200mg of
extract for seven days. The results
showed that the root extract of the Calotropis procera was 96.43%
effective as compared to the
Diminazene aceturate (barenil). Significant changes were also
observed on PCV in animals
which were treated with the Calotropis procera and Diminazene, The
WBCs and DLC were not
decreased by the Calotropis procera root extract and berenil. The
leaf extract of the Calotropis
procera showed the opposite effect of the above.
C. procera displayed the presence of cardiac glycosides, sterols,
alkaloids, flavonoids and
triterpenes. Saponins, cardenolides and flavonoids after the
phytochemical study of C. procera
Moustafa et al. (2010), Kumar et al. (2001) investigated C. procera
dry latex effect on rodents
and reported significant decreased in severity of diarrhea.
Previous studies Ahmed et al. (2004)
showed that C. procera orally administration at dose of 300 mg/kg
body weight to rats protect
significantly from myocardial damage induced by isoproterenol.
Several authors (Alencar et al.
2004; Basu and Chaudhuri, 1991; Kumar and Basu, 1994) reported that
C. procera have a
potentially anti-inflammatory activity Larhsini et al. (2002)
reported antipyretic effect of C.
procera comparable to the standard drugs such as acetylsalicylic
acid. Antioxidant activity of C.
procera was also reported by Olalye and Rocha, (2007).
REVIEW OF LITERATURE
Statement of the problem
Theileriosis affects both large and small ruminants with high
morbidity and mortality
rates resulting in high economical losses and thus poses a serious
risk to livestock production
(Schnittger et al. 2000). Recently, interest has been focused in
the world on the sheep and goats
infecting Theileria species which include T. lestoquardi and China
1 which are reported highly
pathogenic. Ovine Theileriosis caused by T. lestoquardi infection
is now considered one of the
major constraints for sheep production in many areas of the world.
The disease was also found a
serious threat to sheep and goats in Kingdoms of Saudi Arabia
El-Azazy et al. (2001), Sudan
Salih et al. (2003), Iran Razmi et al. (2006) and Turkey Sayin et
al. (2009). Caprine and ovine
theileriosis is an important tick-borne disease caused by Theileria
hirci (T. lestoquardi) causing
clinical illness and mortalities. The disease is now considered a
serious threat to these species of
animals in the Pakistan needs prompt action for in time diagnosis
and tactical control measures
Zia et al. (2010). The advent of molecular techniques, accurate
identification of tick-borne
pathogens and precise diagnosis of disease is now available in the
world Ahantarig et al. (2008).
Keeping in view the importance of this disease and population of
small ruminants in Balochistan
this study was designed with following objectives;
1- Prevalence of Theileriosis in sheep and goats and associated
risk factors in Balochistan.
2- Molecular identification of Theileria species in study
area.
3- Effect of disease on different hematological parameters.
4- Therapeutic trails against Theileriosis in sheep and
goats.
28
MATERIALS AND METHODS
The present study was accomplished in a series of four components
for assessment of
Theileriosis intensity, diagnostic options, health effects and
control in small ruminants at two
ecological zones (northern highland and suleiman mountainous
region) of northern Balochistan,
Pakistan.
Part-1. Prevalence of Theileriosis in sheep and goats: associated
risk factors in Balochistan.
Part-2. Molecular identification of Theileria species in study
area.
Part-3. Effect of disease on different hematological
parameters.
Part-4. Therapeutic trails against Theileriosis in sheep and
goats
3.1. Study Area
MATERIALS AND METHODS
The Balochistan province of Pakistan comprises of six
agro-ecological zones
(Balochistan Coastal Region, Suleiman Mountainous Region, Northern
highlands, Central
Brahvi Highlands, Kachhi Basin Region and Chaghai Kharan Desert)
and six divisions namely
Kalat, Makran, Naseerabad, Quetta, Sibi and Zhob. Among these, two
divisions like Quetta
(northern highland) and Zhob (suleiman mountainous region) were
included in present study. On
the basis of animal population, suitable environment for vectors of
Theileria, three Union
Councils where the facility of veterinary hospital was available
from district Quetta (Quetta
Division) and three from district Loralai (Zhob Division) were
marked for sample collection
(Fig.3.1)
latitude and 67.0252 o longitude.
The geographical character of this district is mountainous and the
climate is dry and cold.
Temperature ranges between -7 to15 o C in winter and relatively
mild 32 to 35
o C in summer. The
average annual rainfall is 226mm. The heaviest rainfall and
snowfall occurs in January and
February. The total geographical area of the district is about 2653
Km 2 which is generally
covered with different mountain i.e. Murdar, Chiltan and Zarghoon.
The elevation of hills may
vary from about 1254-3500 Meters. Because of its natural beauty the
people used to call it little
Paris/small London. and the famous lake in the district Quetta is
Hanna Jheel.
The district Loralai of Suleiman mountainous region lies between
30.3852° latitude and
68.6000° longitude and an altitude of 3873 to 4350 feet. The
climate varies with elevation, at
higher altitudes cold and dry with occasional snowfall. The
district lies outside the range of
monsoon. However, along the eastern belt on Suleiman range, weather
is dominantly influenced
by the monsoon season during the months of July and August
respectively. (Anonymous, 1997a
and Anonymous, 1997b).
MATERIALS AND METHODS
30
PART-I. Prevalence of Theileriosis in sheep and goats and
associated risk factors in
Northern Balochistan.
In this part of the study, prevalence of Theileriosis in sheep and
goats and its associated
risk factors from three Union Councils (Table 3.1) of the two
districts were studied. The
selection of the Union Councils was made on the basis of animal
population, availability of
veterinary hospital and resources.
Selection of Animals
A total of 2870 small ruminants were randomly included in the study
from northern
highland and suleiman mountainous region of northern Balochistan.
Four sheep breeds among
them two local (Bibrik and Harnai), and two exotic (Karakul, and
Shinwari (Rastha)) along with
one goat breed (Khurasani) of either sex from their home tract
districts in the target area were
studied. These animals were divided into three age groups that
were, Less than one year old, one
to two years old and above two years old.
Selection of herds
In the prevalence of theileriosis in sheep and goats of targeted
area a total of 200
farmer`s herds belonging to twenty villages of district Quetta and
twenty villages of district
Loralai zones of northern upland of Balochistan were randomly
selected. It was kept in mind
during the selection of sheep and goat herds that the herds might
contain major figure of breeds
of study interest. Herd selection was also based on the animal`s
age group, sheep and goat herds
with maximum number of predetermined age groups were preferably
selected for study.
Farmers/related persons were interviewed according to already
developed questionnaire.
Conducted different meetings with local farmers, livestock traders
and villagers at different
places of the targeted villages of district Quetta and district
Loralai regarding theileriosis in
sheep and goat in that order during the study period.
MATERIALS AND METHODS
31
Prevalence
Occurrence of disease in respective area for a period of one year
was referred as
prevalence, without differentiation in between old and new cases
were calculated as per formula
described by Thrusfield, (2002).
No. of individual having a disease at a particular point in
time
P =
______________________________________________________x100
No. of individuals in the population at risk at that point in
time
Table 3.1. Sample collection Districts and their Union
Councils.
Region Name of
Northern Highland (NHL) Quetta Quetta Kuchlak, Hanna Urak,
Aghberg
Suleiman Mountainous
Region (SMR)
Sinjavi
Animals suffering from theileriosis showing clinical signs as
pyrexia, anorexia, swollen lymph
nodes, weight loss, weakness, conjunctival petechial, anemia, cough
and presence of ticks on the
body were selected for blood collection. The blood was analyzed for
both hematological
examination and molecular identification of different Theileria
species responsible for the
disease in the targeted area of northern Balochistan
province.
MATERIALS AND METHODS
Figure 3.2. Schematic diagram indicating detailed sampling strategy
for the study of
Theileriosis in sheep and goats in Northern Balochistan.
TOTAL
SAMPLES
3.1.2. Collection of Samples
Samples were taken from the target population of sheep and goats
from three union
councils each of the two districts. The sampling was performed from
veterinary hospitals,
dispensaries and during clinical examination of the animals in
sheep goat farms during the year
March 2012 to February 2013. During four seasons, Blood samples
were taken from sheep and
goats marked with number date of collection and other related
information according to
especially designed questioners (Annexure 1). The samples were
taken from sheep and goats
with the following methods.
Ear tip puncture collection procedure
a) The hairs on area of ear tip were trimmed and sterilized with
methylated spirit swab then
pressed the area of ear tip between thumb and index finger of one
hand.
b) The ear tip was punctured by a sterilized needle.
c) Drop of blood from punctured tip was taken on glass slide for
thin blood smear
preparation.
Jugular vein protocol
A) The jugular vein was raised and the area of collection was
disinfected.
B) 5 ml of blood was drawn gently into the sterile syringe.
C) To stop further bleeding area was plugged with cotton.
D) Then the blood was immediately transferred to the 15 ml Falcon
tubes containing 200 µl
anticoagulant i.e. Ethylene diamine tetra-acetic acid (0.5 M EDTA),
for hematological
examination, PCR experiments and Giemsa staining analysis.
E) The temperature of the collected blood samples tubes was
immediately maintained at 4°C
to preserve the collected samples during transportation.
MATERIALS AND METHODS
3.1.3. Preparation of blood smears
The thin blood smear was prepared and processed on clean glass
slides from jugular vein
blood samples and ear tip blood samples according to Benjamin
(1986). A small drop of blood
dropped on clean glass slid near one end of the slide. Then using
another glass slide the smear
was spread holding it firmly at an angle of 45° before the drop of
blood.
The spreader slide was pushed back gently to the drop of blood to
spreads along two third
of the width by capillary action to the far end with steady even
motion. The smear was dried in
air and fixed. The dried blood smears fixed in absolute methyl
alcohol for 1 minute.
3.1.4. Staining of fixed blood smear
I- One part of the stock solution of Giemsas stain was taken and
diluted with four parts of
distilled water.
II- The fixed blood smears were placed on the staining racks and
the diluted stain was
poured on them and stained for 4 minutes.
III- Smears were washed with distilled water gently and thoroughly
and dried.
3.1.5. Examination of stained blood smears
A small drop of cedar wood oil was placed on the thin end of the
smear and then
observed with the help of oil immersion lens (100X) to detect the
presence of Theileria in the
RBCs and identified according to Urquhart et al. (2002) and
prevalence of the disease was
calculated as described by Thrusfield (2002).
MATERIALS AND METHODS
A. Species
Two species of the target population were analyzed for prevalence
of the disease
a) Sheep
b) Goats
B. Season
Four seasons from the year 2012- 2013 were divided into
i) Spring (March-April)
ii) Summer (May-August)
C. Age
Samples were taken from three different age groups of sheep and
goats to analyze the
susceptibility of the age group of the animals. For this purpose,
the animals were divided
into following 3 age groups;
1. <1 year
D. Gender
Male and female groups of sheep and goats were analyzed for
susceptibility to
Theileriosis.
36
Figure 3.3. Clinical examination of animals for the presence of
Ticks in sheep farm
MATERIALS AND METHODS
37
Figure 3.4. Clinical examination of animals for the presence of
Ticks in sheep farm
MATERIALS AND METHODS
MATERIALS AND METHODS
Part-2: Molecular Diagnosis and identification of Theileria.
The blood samples collected from sheep and goats from various
geographical locations of
Northern Balochistan were processed by polymerase chine reaction
(PCR) for further
confirmation and identification of the Theileria species, T. ovis
and T. lestoquardi. To test the
molecular prevalence separately of two infectious agents, we need
to design specific primer set
for each species.
MATERIALS AND METHODS
Frozen DNA
Lysis buffer, Tris HCL 10mM, EDTA 0.2 mM, pH 8
TNE buffer, Tris HCL 10mM, NaCl, EDTA 0.2 mM pH 8
10% SDS
DEPC water
The following detailed steps were used to extracted the genomic DNA
of Theileria
species through organic Method described by Sambrook and Russel
(2001).
A. RBCs Lysis step
The frozen DNA samples were thawed and mixed well at room
temperature before the
DNA extraction.
Five ml blood for total genomic DNA extraction was transferred into
50 ml
polypropylene tube and add 40 ml TAE buffer, vertex for 10 seconds
and centrifuge for
4000 rpm at 4°Casss for 15 minutes.
The supernatant was discarded and pellet was broken by tapping and
vortex and again
mixed with 40 ml of TAE buffer, vortex well and centrifuge it as
mentioned in step 2.
Four and five steps repeated, until the pellet become whitish in
color. At the end the
supernatant was carefully discarded.
The following three chemicals were added into the washed
pellet
30 µl 10% SDS
15 µg proteinase K
5 ml TNE buffer
The mixture was mixed well and incubated at 50°C in water bath
overnight for complete
digestion of blood leukocytes.
C. Separation step
500 µl phenol: chloroform was added in the digested mixture and was
vortexed until the
emulsion formed.
The mixture was incubated at room temperature for 10 minutes
Centrifuged the mixture at 12,500 rpm for 15 minutes at 4°C. The
organic and aqueous
phases were separated well.
Transfer 500 µL upper phase (aqueous phase) carefully into new
polypropylene tube.
D. Precipitation step
Equal amount of isopropanol was added into the upper phase
containing DNA.
The mixture inverted gently until the threads of DNA visible and
incubated the mixture
for 15 minutes at room temperature.
The mixtures were centrifuged at 10,000 rpm for 15 minutes at
4°C.
The supernatant was discarded carefully and X pellet remained in
the tube.
MATERIALS AND METHODS
E. Washing step
Precipitated pellet was washed with 70% ethanol; 1ml diluted
ethanol was added and
mixed the pellet well and incubated at room temperature for 20
minutes.
Centrifuge the solution at 5000 rpm for 5 minutes at 4°C. The
diluted ethanol was
discarded carefully and let pellet dried well.
The dried pellet was dissolved with 100 µl DEPC water and checked
the confirmation
and concentration by following procedure and stored at -20°C until
the PCR was run.
3.2.2. Quantification of DNA
The extracted DNA was quantified by using Nanodrop
spectrophotometer (Thermo
Scientific) and further confirmed through 0.8% agarose gel. 1µl of
DNA was used for
Nanodrop and concentration was noted by ng/µL and purity by 260/280
nm ration (1:8). Few
positive DNA samples values acquired by Nanodrop are described
below in (Table 3.2 and
Table 3.3).
Table 3.2. Determination of DNA concentration through Nanodrop of
Northern highland.
S. No. Sample Lab No. Species Conc. ng/uL 260/280 ratio
1 NHQ/001 Sheep 541 1.71
2 NHQ/002 Sheep 678 1.74
3 NHQ/003 Sheep 492 1.45
4 NHQ/004 Sheep 533 1.63
5 NHQ/005 Sheep 327 1.25
6 NHQ/006 Sheep 412 1.35
7 NHQ/007 Sheep 396 1.39
8 NHQ/008 Sheep 432 1.40
9 NHQ/009 Sheep 510 1.62
10 NHQ/010 Sheep 576 1.75
11 NHQ/011 Sheep 365 1.37
12 NHQ/012 Sheep 424 1.38
13 NHQ/013 Sheep 534 1.67
14 NHQ/014 Goat 614 1.73
15 NHQ/015 Goat 601 1.70
16 NHQ/016 Goat 485 1.43
17 NHQ/017 Goat 356 1.36
18 NHQ/018 Goat 280 1.24
19 NHQ/019 Goat 335 1.33
20 NHQ/020 Goat 515 1.62
MATERIALS AND METHODS
Table 3.3. Determination of DNA concentration through Nanodrop of
Suleiman
Mountain Region.
21 SMRL/021 Sheep 680 1.75
22 SMRL/022 Sheep 494 1.45
23 SMRL/023 Sheep 536 1.68
24 SMRL/024 Sheep 437 1.39
25 SMRL/025 Sheep 417 1.32
26 SMRL/026 Sheep 400 1.30
27 SMRL/027 Sheep 432 1.41
28 SMRL/028 Sheep 518 1.63
29 SMRL/029 Sheep 578 1.76
30 SMRL/030 Sheep 362 1.33
31 SMRL/031 Sheep 429 1.39
32 SMRL/032 Sheep 536 1.68
33 SMRL/033 Sheep 620 1.74
34 SMRL/034 Sheep 600 1.70
35 SMRL/035 Sheep 488 1.42
36 SMRL/036 Sheep 359 1.35
37 SMRL/037 Sheep 286 1.26
38 SMRL/038 Goat 432 1.40
39 SMRL/039 Goat 489 1.47
40 SMRL/040 Goat 365 1.34
41 SMRL/041 Goat 299 1.27
42 SMRL/042 Goat 364 1.35
43 SMRL/043 Goat 500 1.62
44 SMRL/044 Goat 539 1.65
45 SMRL/045 Goat 422 1.26
46 SMRL/046 Goat 418 1.33
47 SMRL/047 Goat 398 1.39
48 SMRL/048 Goat 432 1.40
49 SMRL/049 Goat 552 1.73
50 SMRL/050 Goat 500 1.62
MATERIALS AND METHODS
45
The samples were further confirmed through 0.8% agarose gel.
Agarose gel was prepared
by boiling 0.8g agarose powder in 100 ml TAE 1X buffer (Tri base,
acetic acid and EDTA 0.5
M), when the temperature reduced to 60°C, 10 µL ethidium bromide
(conc. 15 mg/ml) was
added and mixed well before poured into casting tray. The agarose
gel was got solidified and
transferred into the electrophoresis tank; it was filled with 1X
TAE buffer. 3 µL of DNA was
mixed with 3 µL 3 X bromophenol blue dyes and run in their
respective wells. The DNA result
was seen under UV light in gel documentation system (BioRad, USA).
The image was taken and
saved it in the system (Fig.3.6).
MATERIALS AND METHODS
MATERIALS AND METHODS
A) Primer designing
The Theileria species were identified though 18S ribosomal RNA.
Strains specific
primers were designed for T.ovis and T. lestoquardi by using primer
3 software (Table 3.4). The
Gen Bank accession number GU7269901 was used for the primer
designing of T. ovis and
accession number GU7269902 was used for the primer designing of T.
lestoquardi. Both primer
sets were checked for self-complementary and hairpin structure
through Oligo calc. please add
some here
48
Table 3.4. Sets of primers used to amplify Theileria rRNA gene
sequences.
Species Primer Sequence TM
Forward CGGAGTTTCTTTGTCTGA 52
Reverse GAAGGAGTCGTAAAAACTGA 56
B) Both set of primers were optimized by gradient PCR using Bio-Rad
thermos cycler. Both
primer amplification was performed with final reaction volume of 25
µL, including template
DNA 5 µL, MgCl2(25mM/µL) 2.5 µL, 10X Ammonium sulphate buffer 2.5
µL, dNTPs 2.5µL
(2.5mM), Forward primer 1 µL (10mM), Reverse primer 1 µL (10mM),
Taq DNA polymerase
(5U/µL) 0.2 µL, and water 10.3 µL (Table 3.5).
MATERIALS AND METHODS
Reagents Quantity (µL) Concentration
MgCl2 2.5 25mM
Buffer 2.5 10X
Water 10.3 -
Total 25 -
*All the DNA samples were diluted and brought them to almost
50ng/µL
C) Both primers were optimized through gradient PCR at a range of
50-60°C. The PCR was
done for T. ovis by using the initial de naturation temperature
95°C for 5 minutes, and 35 cycles
were carried out, each comprised denaturation at 95°C for 30
seconds, annealing at 56°C for 30
seconds and extension at 72C for 1 minute and final extension at
72°C for 10 minutes and store
at 4°C. The PCR was done for T. lestoquardi was using the same
condition as revealed above;
however the annealing temperature was 55°C.
D) The PCR products were run on 1.5% agarose gel, 10 µL of PCR
product was mixed with
5 µL 3 X bromophenol blue dyes and the electrophoresis was done at
constant voltage 100V for
40-50 minutes in 1X TAE buffer. 50 bp or 1kb ladder was also run
along with PCR products to
confirm the species specific amplification.
MATERIALS AND METHODS
3.3.1. Haematological examination
Animals positive with theileriosis were confirm by making thin
blood smear microscopic
slide and was examined. The samples of the animals conformed for
the presence of Theileria in
RBCs was immediately transported in ice boxes to Provincial Disease
Investigation Laboratories
of Quetta, Bolan Medical Complex Hospital and Fatima Jinnah TB
Sanatorium for hematology.
Each sample was labeled as per determined information that is famer
name, animal species, sex,
breed and age. 5ml blood samples from each sheep (n= 60 infected,.
n= 18 healthy); and goat
(n= 36 infected, n= 9 healthy) were collected directly from the
jugular vein into sterilized falcon
tube with anticoagulant coated with EDTA @ 1 mg/ml of blood. Each
blood sample collected
was analyzed for different hematological factors RBC, Hb%, PCV,
Platelets, WBC, MCV and
MCHC by using hematological analyzer as per Hasanpour et al.
(2008). Changes in biochemical
values as AST, ALT, BUN, Bilirubin and Creatinine were also
analyzed.
MATERIALS AND METHODS
Part. 4. Therapeutic trails against Theileriosis in sheep and
goats
Therapeutic trials
A total of 80 animals (sheep=40) and (goats 40) declared diseased,
showing clinical signs
and microscopically positive for theileriosis, were divided into
four groups A. B. C and D. These
animals were diagnosed Theileria positive in the field conditions
and tagged there for the treatment
trails with the consent of the owners of the animals. The medicines
for trails were provided by the
researchers free of cost and the diseased animal kept as control
were discussed with the owner and
kept under observation for 10 days in the larger interest of the
area.
The animals of group A (sheep 10; goats 10) were treated with
Buparvaquone (Butalex®,
ICI, Pakistan) @ 2.5 mg/kg BW I/M. single dose.
The animals of group B (sheep 10; goats 10) were treated with
Imidocarb dipropionate
(Imizol®, ICI, Pakistan) @ 2 mg/kg BW I/M. single dose
The animals of group C (sheep 10; goats 10) were treated with
Calotropis procera,
(locally known as Spalmi, Akk) flowers and buds as a local
treatment in dried powder
form at the dose rate of 0.3mg/ kg BW, twice daily for 5 days
(Durrani et al. 2009).
The animals of group D (sheep 10; goats 10) were kept as positive
control.
Efficacy of all three drugs was measured on the basis of Theileria
clear blood smear examination
at day 10 post-medication.
MATERIALS AND METHODS
MATERIALS AND METHODS
Figure 3.9. Collection of leaves and flowers of Calotropis
procera
MATERIALS AND METHODS
MATERIALS AND METHODS
56
Figure 3. 11. Prepared Sachet of Calotropis procera leaves and
flowers in powder form.
MATERIALS AND METHODS
STATISTICAL ANALYSIS
The map of the study area was prepared using Quantum Geographical
Information
System (QGIS). For test of significance Chi square analysis was
used for epidemiological
factors. These factors include Region, Districts, Union Councils,
species, breeds, season, gender,
age, Chi square was used on Epi Info TM
7 (Centers for Disease Control, Atlanta, GA, USA). Two
districts from each region and three Union Councils from each
district along with two species
Sheep and Goats were divided into three age groups (A) less than
one year old, (B) one to two
year old, and (C) above two year old. Four seasons Spring, Summer,
Autumn and Winter along
with four breeds of sheep and one breed of goat were analyzed for
association with disease. The
strength of association of all factors with Theileriosis in sheep
and goats was estimated through
odds ratio and the corresponding 95% confidence intervals. P-value
less than 0.05 were taken as
significant. Mean along with standard error was calculated in
Microsoft excel (2007). T Test
was used to test the difference between the normal and infected
animals on SPSS 16 (Statistical
Package for Social Sciences 16). In order to calculate the efficacy
of drugs against Theileria
parasite and ticks (the outcome variables was binomial with
presence or absence of parasites in
microscopy and presence or absence of ticks post treatment at
different days post treatment) the
Chi Square for trends was used to test the significant difference
between medicines at different
post treatment exposure time and odds ratio were calculated against
each post treatment exposure
time on Epi Info TM
7 (Centers for Disease Control, Atlanta, GA, USA). P value less
than 0.05
was considered as significant.
RESULTS
The present study was conducted to investigate the prevalence of
Ovine and Caprine
Theileriosis at two different regions of Northern Balochistan,
namely Northern Highland (NHL)
and Suleiman Mountainous Region (SMR) in the year 2012-2013.
Samples were collected from
2870 animals Sheep (n= 2200) and Goats (n= 670) for screening of
the disease. The samples
were collected and processed in Regional Disease Investigation
Laboratory, Department of
Livestock and Dairy Development Balochistan, T.B. Sanatorium
Hospital Quetta and Medicine
Laboratory, Department of Clinical Medicine and Surgery, University
of Veterinary and Animal
Sciences, Lahore.
4.1.1. Regional Prevalence
Data on overall regional prevalence of theileriosis revealed 19.19%
in Northern
Highlands and 17.48% in Suleiman Mountain Region (Fig. 4.1).
Chi-square analysis showed
non-significant (p>0.05) difference in the prevalence of the
disease in two regions of Balochistan
(Table 4.1).
ANNEXURE
59
Table 4.1. Regional prevalence of Theileriosis in Sheep and Goats
in Northern Balochistan
Region District Total
1.12 0.93-1.36 1.38 0.24 Suleiman
Mountainous
Region
ANNEXURE
60
Figure 4.1. Prevalence of Theileriosis in Sheep and Goats in two
Regions of Northern
Balochistan
RESULTS
61
4.1.2. Union Council wise Prevalence.
Three Union Councils from each district were compared for the
prevalence of the disease
in sheep and goats. Statistical analysis of the data showed highest
prevalence (22.71%) in Union
Council Kuchlak of district Quetta (Table 4.2) followed by Aghberg
(18.42%) and Hanna Urak
(15.53%). Statistically the data for district Loralai showed
highest prevalence in Union Council
Zangiwal Jogezai (19.83%) followed by Kach Amaqzai (16.30%) and
Sinjavi (15.92%). Chi-
square analysis of the data showed significant difference
(p<0.05) in comparison of different
Union Councils.
ANNEXURE
62
Cable 4.2. Union Council wise prevalence of Theileriosis in Sheep
and Goats.
District Union Council Total
Hanna Urak 380 59 15.53 0.62 0.44-0.89 6.96 0.0083
Aghberg 380 70 18.42 0.77 0.55-1.07 2.36 0.12
Lorlai
Sinjavi 490 78 15.92 0.64 0.47-0.89