sero-survey of equine infectious anemia, glanders and

Microsoft Word - Title PageSERO-SURVEY OF EQUINE INFECTIOUS ANEMIA, GLANDERS AND PIROPLASMOSIS IN FIVE
DRAUGHT EQUINE POPULATED URBAN AREAS OF PUNJAB
Thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
FAISALABAD, PAKISTAN
October 2011
To, The Controller of Examinations, University of Agriculture, Faisalabad We, the supervisory committee, certify that the content and form of thesis submitted
by Mr. Muhammad Hammad Hussain, Reg. No. 95-ag-1001 have been found satisfactory
and recommend that it be processed for evaluation by the External Examiner(s) for the award
of degree.
SUPERVISORY COMMITTEE
Member ___________________________ (Prof. Dr. Ijaz Javed Hassan)
Member ___________________________ (Prof. Dr. Muhammad Siddique)
DEDICATION
I would love to dedicate this manuscript to my loved ones: my
loving family, mentors, special friends and loves of my life. All
are truly the bright stars in my life.
i
ACKNOWLEDGEMENTS
I bend myself modestly in front of Almighty ALLAH the Lord of the worlds, the
Omnipotent, the Beneficent, the Merciful and the Gracious and thank Him for everything I have
been blessed with in my life. Peace and blessing of Allah be upon Holy Prophet Hazart
Muhammad (Peace be Upon Him), the Apostle of Allah, the greatest social reformer, and who is
the forever source of knowledge.
My deep sense of gratitude for my Supervisor, Prof. Dr. Ghulam Muhammad,
Department of Clinical Medicine & Surgery, for his dynamic supervision, auspicious guidance and
keen interest during my research work.
I express my profound sense of appreciation to Dr. Ijaz Javed Hassan, Professor
Department of Physiology & Pharmacology, for his technical guidance, constructive criticism and
needful help during the course of my study.
I am feeling dearth of words to express my gratitude and appreciation to Dr. Muhammad
Siddique (Late), Professor of Microbiology, for providing valuable suggestions, competent guidance
and boosting up my morale during the conduct of this study.
With deep emotions of benevolence and gratitude, I offer my appreciation to Prof. Dr.
Laeeq Akhtar Lodhi, (Dean FVS) for his skilful and ever inspiring intelligent guidance, absolute
friendly atmosphere during the completion of this project.
This work was supported in part by the University of Agriculture, Faisalabad through
provision of financial grant. I am extremely thankful to The Brooke Hospital for Animals,
Pakistan and especially Dr. M. Saleem, Leader, The Brooke Project, Faisalabad for provision of
support regarding collection of samples from Lahore, Gujranwala, Faisalabad and Multan.
I am sincerely and earnestly indebted to my parents, family members, beloved wife
daughter, son and friends who have always wished to see me glittering high on the skies of success.
(Muhammad Hammad Hussain)
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Table- 3.1
Sampling plan for the sero-survey of equine infectious anemia, galnders and piroplasmosis in 5 draught equine populated urban areas of Punjab 30
Table-1
Geographic and climatic characteristics of the 5 draught equine populated urban areas of Punjab selected for sero-survey of equine infectious anemia, glanders and piroplasmosis
44
Table-2 Reported equine population targeted for sero-survey of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
44
Table-3 Areas and equine type related distribution of samples (n=430) taken for sero-survey of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
48
Table-4 Sex related distribution of samples (n=430) taken for sero-survey of equine infectious anemia, glanders and piroplasmosis from 5 draught equine populated urban areas of Punjab
48
Table-5 Age related distribution of equines (n=430) sampled for sero-survey of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
49
Table-6 Housing pattern observed in equines (n=430) sampled for sero-survey of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
51
Table-7 Watering pattern used at work and home by the owners of equines (n=430) sampled for sero-survey of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
51
Table-8 Number of equine owners practicing tick, fly and mosquito control measures in 5 draught equine populated urban areas of Punjab sampled for sero-survey of equine infectious anemia, glanders and piroplasmosis.
52
Table-9 Hematological values recorded in equines (n=430) surveyed for seroprevalence of equine infectious anemia, glanders and piroplasmosis from 5 draught equine populated urban areas of Punjab
57
Table-10 Comparison of hematological values recorded in horses (n=65), mules (n=33) and donkeys (n=332) sampled for sero-prevalence of equine infectious anemia, glanders and piroplasmposis in 5 draught equine populated urban areas of Punjab
58
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S. No. Title of the Table Page No.
Table-11 Comparison of hematological values found in male (n=295) and female (n=135) equines sampled for seroprevalence of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
59
Table-12 Comparison of hematological values found in 3 different age groups of equines sampled for seroprevalence of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
59
Table-13 Comparative Seroprevalence of equine glanders in 5 draught equine populated urban areas of Punjab 65
Table-14 Frequency of clinical signs in RBT positive (n=34) equines of 5 draught equine populated urban areas of Punjab sampled for seroprevalence of equine infectious anemia, glanders and piroplasmosis
66
Table-15 Comparative age related seroprevalence of glanders in equines (n=430) of 5 draught equine populated urban areas of Punjab surveyed for prevalence of equine infectious anemia, glanders and piroplasmosis
67
Table-16 Sex related seroprevalence of glanders in equines (n=430) of 5 draught equine populated urban areas of Punjab surveyed for prevalence of equine infectious anemia, glanders and piroplasmosis
68
Table-17 Haematological values in equines found sero-positive for glanders (n=34) in 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
69
Table-18 Housing pattern observed in Rose Bengal Plate Agglutination Test (RBT) positive (n=34) and negative (n=396) equines surveyed for the seroprevalence of glanders in 5 draught equine populated urban areas of Punjab
70
Table-19 Prevalence of glanders in association with watering habits of owners (n=430) of 5 draught equine populated urban areas of Punjab surveyed for the seroprevalence of equine infectious anemia, glanders and piroplasmosis
70
Table-20 Bivariable analysis for predicting glanders in equines sampled from 5 draught equine populated urban areas of Punjab 72
Table-21 SPSS output of binary logistic regression analysis for predicting glanders in equines sampled from 5 draught equine populated urban areas of Punjab
73
Table-22 Seroprevalence of piroplasmosis in equines (n=430) of 5 draught equine
populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
77
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Table-23 Clinical findings in the equines (n=430) of 5 draught equine populated
urban areas of Punjab found sero-positive (n=226) for piroplasmosis (T. equi and B. caballi)
78
Table-24 Seroprevalence of Theileria equi in the equines surveyed for the seroprevalence of equine infectious anemia, glanders and piroplasmosis from 5 draught equine populated urban areas of Punjab
79
Table-25 Comparative age related seroprevalence of T. equi infection in equines of 5 draught equine populated urban areas of Punjab surveyed for prevalence of equine infectious anemia, glanders and piroplasmosis
80
Table-26 Comparative sex related dynamics of Theileria equi infection in 5 draught equine populated urban areas of Punjab surveyed for seroprevalence of equine infectious anemia, glanders and piroplasmosis
81
Table-27 Seroprevalence of Babesia caballi infection in 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
84
Table-28 Comparative age related seroprevalence of B. caballi infection in 5 draught equine populated urban areas of Punjab surveyed for prevalence of equine infectious anemia, glanders and piroplasmosis
84
Table-29 Comparative sex related dynamics of Babesia caballi infection in 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
85
Table-30 T. equi and B. caballi mixed infection in 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
88
Table-31 Seroprevalence of T. equi and B. caballi mixed infection in 5 draught equine populated urban areas of Punjab surveyed for prevalence of equine infectious anemia, glanders and piroplasmosis
88
Table-32 Age related seroprevalence of mixed infection with Theileria equi and Babesia caballi in equines of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
89
Table-33 Sex related prevalence of mixed infection with Theileria equi and Babesia caballi in 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
89
Table-34 Hematological values of horses found positive (n=53) for piroplasmosis in 5 draught equine populated urban areas of Punjab
94
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Table-35 Hematological values of donkeys found positive (n=191) for piroplasmosis in 5 draught equine populated urban areas of Punjab 95
Table-36 Hematological values of mules found positive (n=26) for piroplasmosis in 5 draught equine populated urban areas of Punjab 96
Table-37 Comparison of hematological values in equines (n=226) found positive for piroplasmosis in 5 draught equine populated urban areas of Punjab 97
Table-38 Comparison of housing pattern of equines (n=430) found positive (n=226)
and negative (n=204) for equine piroplasmosis in 5 draught equine populated urban areas of Punjab
102
Table-39 Seroprevalence of piroplasmosis with reference to presence of ticks on the cohorts in equines found positive (n=226) and negative (n=204) for equine piroplasmosis in 5 draught equine populated urban areas of Punjab
103
Table-40 Seroprevalence of piroplasmosis with reference to tick control practiced in equines found positive (n=226) and negative (n=204) for equine piroplasmosis in 5 draught equine populated urban areas of Punjab
104
Table-41 Preferred treatment protocol adopted by veterinarians and animal health workers (n=100) of 5 draught equine populated urban areas of Punjab in suspected cases of equine piroplasmosis
105
Table-42 Bivariable analysis for the seroprevalence of T. equi in equines (n=177) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
106
Table-43 SPSS output of binary logistic regression model for predicting T. equi infection in equines (n=177) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
107
Table-44 Bivariable analysis for the seroprevalence of B. caballi in equines (n=93) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
108
Table-45 SPSS output of binary logistic regression model for predicting B. caballi infection in equines (n=93) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
109
Table-46 Bivariable analysis for the seroprevalence of mix infection with T.equi and B. caballi in equines (n=44) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
110
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Table-47 SPSS output of binary logistic regression model for predicting mix infection with T.equi and B. caballi in equines (n=44) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
111
Table-48 Bivariable analysis for the seroprevalence of piroplasmosis in equines (n=226) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
112
Table-49 SPSS output of binary logistic regression model for predicting piroplasmosis in equines (n=44) of 5 draught equine populated urban areas of Punjab surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis
113
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Figure-1
Map showing global distribution of equine infectious anemia (EIA) during last six months (July – December) of 2008
12
Figure-2 Map showing global distribution of equine glanders during last six months (July – December) 2008
17
Figure-3 Map showing the global distribution of equine piroplasmosis during last six months (July – December) of year 2008
25
Figure-4 Google maps showing the 5 sampled draught equine populated urban areas of province, Punjab, Pakistan
31
S. No. Title of the Figures Page No.
Plate-I Photograph showing the commercial ELISA kit (VMRD, Inc., USA) used for the serodiagnosis of equine infectious anemia
35
Plate-II Photograph showing the 96 well microtitration plate after completing the assay ready for visual determination and interpretation through plate ELISA reader
35
Plate-III Photograph showing the strong positive results of rose Bengal plate agglutination test for equine glanders
37
Plate-IV Photograph showing the micro-titration plate of commercial cELISA (VMRD, Inc., USA) used for the serodiagnosis of equine piroplasmosis during the study: Visual Determination
40
Plate-V Photograph showing the communal water troughs used by the draught equine owners during working hours
71
Plate-VI Photograph showing the communal water troughs used by the draught equine owners in the communal equine housings
71
Plate-VII Photograph showing the communal housing pattern used by owners of draught donkeys in the study areas
114
Plate-VIII Photograph showing the roof type being used in the construction of the communal draught equine housing systems in the study area
115
Plate-IX Photograph of tick infestation on the medial aspect of the thigh in a horse sampled during the study
116
Appendix
Proforma for the recording of information regarding reo-survey of equine infectious anemia, glanders and piroplasmosis in 5 draught equine populated urban areas of Punjab
168
CONTENTS
4 RESULTS 42
5 DISCUSSION 117
6 SUMMARY 141
________________________________________________
reportable equine diseases viz., equine infectious anemia (EIA), glanders, and piroplasmosis
in 5 draught equine populated urban areas (Lahore, Gujranwala, Faisalabad, Multan and
Bahawalpur) of Punjab. Assuming the expected prevalence to be 50 percent (unknown status)
with confidence limits of 95% and a desired absolute precision of 5%, a total of 430 blood
and sera (comprising 332 donkeys, 65 horses and 33 mules) along with relevant information
were randomly collected. The samples were subjected to microscopic examination (thin and
thick Giemsa stained smears), hematological analysis and commercial enzyme linked
immunosorbant assay (ELISA). Sera were tested for EIA through ELISA (VMRD, Inc.,
USA), for glanders through rose Bengal plate agglutination test (RBT) and a commercial
competitive ELISA (cELISA, VMRD, Inc., USA) was used for piroplasmosis screening.
Positive and negative samples were identified on the basis of degree of agglutination (++ to
++++ considered positive) in case of RBT and as per instructions of the manufacturer
regarding ELISAs. Data thus generated was analyzed by using epidemiological software tools
(Epiinfo™, WINPEPI, Survey Toolbox and IBM SPSS). Results: Giemsa stained smears
were negative for blood parasite. None of the samples was positive for EIA, indicating
towards possible disease free status or failure of the disease to mount immune response in
sampled equine population. Seroprevalence of glanders was found to be 7.9% (n=34) with
highest prevalence in Lahore (11.6%) followed by Bahawalpur (10.3%), Multan (7.3%),
Faisalabad (6.7%) and Gujranwala (5.3%). Significantly different (P<0.05) seroprevalence
was recorded in horses (16.9%) than mules (12.1%) and donkeys (5.7%). Seroprevalence
recorded in young (< 5 years), adult (< 10 years) and old (> 10 years) equines was 7.9%,
9.2% and 5.2% respectively. Prevalence was significantly different (P<0.05) in males 5.7%
and female 12.59% equine. Multivariable analysis indicated that female equines (OR = 2.3,
95% CI= 1.16–4.77) and and those sharing water sources or access to communal water
troughs (OR =2.7, 95% CI=0.93-7.86) were more likely to be sero-positive for glanders.
Seroprevalence of piroplasmosis was found to be 52.5% (T. equi=41.2% and B.
caballi=21.6%; P<0.05) in the selected areas. Forty four (10.2%) sera were found positive for
both piroplasms (mixed infection). Significantly higher (P<0.05) seroprevalence was
recorded in Lahore (95.6%, n=66) than Faisalabad (64%, n=96) than Multan (52.9%, n=36),
Gujranwala (50.7%, n=38) and Bahawalpur (50%, n=34). Seroprevalence of piroplasmosis
was 69.2% in horses (T. equi=56.9%, n=37; B. caballi=24.6%, n=16) followed by 63.6% in
mules (T. equi 48.5%, n=16; B. caballi 30.3%, n=10) and 48.2% in donkeys (37.3%, n=124).
Adjusted analysis for possible confounding factors revealed that equines living alone or with
equine cohorts (OR=1.30, 95%CI=0.84–2.01), with ticks infested equine cohorts (OR=1.19,
95%CI=0.65-2.18) whose owners did not practice tick control (OR=1.43, 95%CI=0.93-2.21)
were more likely to be sero-positive for piroplasmosis. Hematological alterations observed in
RBT (glanders) positive equines regarding white blood cell counts, red blood cell counts,
hemoglobin concentrations and hematocrit in all there RBT positive equine species.
Macrocytic hyperchromic type of anemia was observed in all RBT positive horses, mules and
donkeys. Hematological analysis of piroplasmosis positive equines indicated decreased white
blood cell counts, red blood cell counts PCV and hemoglobin concentration values in
seropositive horses, donkeys and mules. Erythrocytic indices pointed towards microcytic
hyperchromic type of anemia in piroplasmosis sero-positive equines. In conclusion, the high
RBT based prevalence of equine glanders indicates towards possible endemic nature of this
disease in Pakistan. Potential role of communal water sources and faulty management in the
spread of equine glanders requires further investigations through carefully designed
experimental studies. Seroprevalence of piroplasmosis was alarmingly high in the selected
population which could be attributed to the faulty housing, lack of tick control practiced by
owners and lacuna regarding knowledge among animal health professionals about the correct
diagnosis and treatment of T. equi and B. caballi.
Chapter-I (Introduction)
1
CHAPTER-I
INRODUCTION
Equines have had deep, influential and enduring effects on human civilizations that started with
the domestication and then riding of the horse. And in an amazing way, they completely,
dramatically and almost instantaneously changed the face of the world. Among many purposes of
these animals, draught is the most beneficial use for mankind. A number of equine breeds are used
for draught purposes with the variation being largely geographic. Draught equines are versatile
breeds used today for a multitude of purposes, including draught equine showing, farming and
pneumatic cart pulling etc. These animals share the traits of strength, stamina, health, longevity,
patience and a docile temperament which made their presence vital to generations of pre-industrial
farmers.
The importance of equines in Pakistan is well known. According to Pakistan Livestock
Census (Anonymous, 2006) total equine population of Pakistan is about 4.77 millions. Major
proportion of equidae in Pakistan includes 4.2 million donkeys, 0.15 million mules and 0.34
million horses. About 33.33% donkey, 64.66% mule and 60.61% horse population is kept for non-
agricultural (mostly draught) purpose. These animals provide livelihood to the rural societies
living in arid, semi-arid and hilly regions through transport and draught. Whereas, a considerable
population of equines serves the purpose of transport as well as draught in the big industrial
metropolises, a small population of equines is used in army, police, racing industry and sports.
Equines like all other domestic species are susceptible to a variety of infectious diseases.
Chances of spread of infectious as well as vector born diseases are more in draught equine
population of urban areas where the contact among affected and healthy animals is frequent and
2
inevitable. Spread of infectious diseases could occur through common feeding and watering
utensils, insect vectors and use of contaminated instruments for health as well as managemental
purposes (Radostits et al., 2007). Epidemiological studies are of utmost importance in these
congested areas with high disease pressure to develop health and management packages for the
better exploitation of equine resources. Managemental practices contributing to the spread of
diseases can be identified through such studies.
As alluded earlier, like all other species, equines can become victim of many conditions
caused by various pathogens of viral, bacterial and parasitic origin. Among these diseases, equine
infectious anemia, glanders and piroplasmosis are placed in the List B of equine diseases by
World Animal Health Organization formerly known as ‘Office Internationale Des Epizooties’
(OIE) and transport/export of horses from the countries having pockets of these OIE reportable
diseases are strictly prohibited (OIE , 2004).
The equine infectious anemia (EIA) is a potentially fatal viral blood borne disease of
family Equidae i.e., horse and horse family (Ishii and Ishitani, 1975; Higgins and Wright, 1993;
Radostits et al., 2007). Disease is caused by EIA virus of subfamily Lentiviridae of family
Retroviridae and is a close relative to Human Immunodeficiency Virus (HIV) (Montaginer et al.,
1984; Nakajima and Sugiura, 1994).
After getting infected, equines may exhibit one of the three clinical states of the disease i.e.
acute, chronic and inapparent. Inapparent carries are the animals mostly found positive on the
basis of serological diagnosis and pose same threat as infected horses to equine community,
because environmental and managemental stresses and indiscriminate use of steroids are known to
induce recurrence of EIA (Issel et al., 1990). Concerning EIA, no treatment or vaccination is
3
available till now and stringent implementation of ‘test and destroy’ policy (OIE, 2004) is the only
control method available.
A scientifically validated diagnostic test is sine qua non for control and eradication of a
disease. For the serodiagnosis, many tests have been developed and implemented but among those
agar gel immuno diffusion (AGID) and enzyme liked immuno-sorbant assay (ELISA) were found
to be simple and reliable (OIE, 2004). Both the tests have their own pros and cons but use of
ELISA in prevalence studies is found to be more convenient and fruitful (Winston et al., 1987;
Pearson and Gipson, 1988; Cordes and Issel, 1996).
Equine infectious anemia has been reported from many countries of the world including
India (Uppal and Yadav, 1989; Singh et al., 1997). Although, vectors (horse fly and mosquito)
responsible for the transmission of EIA do occur in Pakistan, the occurrence of this disease has not
yet been reported. Documentation of presence or absence of EIA is important for equine health
planning as well as attaining disease free status to facilitate the export of these animals.
Glanders is another OIE list B respiratory disease of horse and akin thereto. It is caused by
gram negative bacteria Burkholderia mallei (Rotz et al., 2002). B. mallei is a host adapted parasite
(Waag and DeShazer, 2004) but it has also been reported in man (Srinivasan et al., 2001) and
other animal species (Smith et al., 1990). Glanders was a major cause of death in horses from
Middle Ages onward and as late as World War-I. It was found to be associated with fatal
secondary infections in humans (Wilkinson, 1981).
Glanders can occur in four different clinical forms viz. nasal, pulmonary, cutaneous
(Farcy) and pulmuno-cutaneous. Clinically, this disease is characterized by nasal discharge with or
without nasal septum ulcers, dyspnoea, nodules/ulcers on the body, and lymphadenitis /
lymphangitis. Transmission occurs through nasal or cutaneous discharge of clinically diseased
4
animals, by aerosol routes and through communal water trough system by contamination
(Radostits et al., 2007).
Field diagnosis of glanders is made on clinical grounds by observing the signs and mallein
test. Mallein testing has several pitfalls. Firstly, it gives false positive results. Secondly, it is
difficult to conduct under field conditions and thirdly it requires 48 hours for measuring outcome
of the test. Over the years, many diagnostic tests viz., complement fixation test (CFT), indirect
hemagglutination test (IHA), modified-counter immuno electrophoresis test (mCIET), Rose
Bengal plate agglutination test (RBT) and enzyme-linked immunosorbant assay (ELISA) have
been developed for the sero-diagnosis of glanders and used in many seroprevalence based studies
on the disease. Falling back on the gold standard test (i.e. isolation of B. mallei) is not possible in
non-clinical cases (Naureen et al., 2007).
Although, stringent implementation of ‘test and slaughter’ policy have successfully
eradicated glanders from most of the developed parts of the world like USA, Canada, UK, and
Australia (Derbyshire, 2002), reports indicate that the disease is still prevalent in many developing
countries (Al-Ani, et al., 1998; Mota et al., 2000; Sabirovic et al., 2005; Witting et al., 2006)
including Pakistan (Naureen et al., 2007). Even though the incidence of disease is thought to be
much higher, only a few prevalence studies based on clinical cases and covering only very small
area have been conducted in Punjab (Nasreen, 1977; Vaid, 1981; Bashir, 1984). Need of the hour
is to conduct some prevalence studies to estimate the current status of glanders in Pakistan
(Muhammad et al., 1998, Saqib et al., 2003; Naureen et al., 2007).
Equine piroplasmosis, caused by Theileria equi and Babesia caballi (Knowles, 1988;
Friedhoff et al., 1990; Radostits et al., 2007; Zinora et al., 2007) is an emerging equine disease
worldwide (Shkap et al., 1998; Uilenberg, 2006; Vial and Gorenflot, 2006). The disease can occur
5
in chronic to per acute forms, with T. equi causing more severe infections and signs as compared
to B. equi which usually exhibit chronic disease. Disease is transmitted through ticks of three
genera viz., Dermacentor, Rhipicephalus and Hyaloma (Yoshihara, 1997; Uilenberg, 2006; Vial
and Gorenflot, 2006).
Conventional diagnosis is based upon the demonstration of organisms in the Giemsa
stained blood smears under microscope (Knowles, 1988) or through transmission tests (Ali et al.,
1996). For the serological diagnosis, many tests are used viz. complement fixation test (Donnelly
et al., 1980; Weiland, 1986), indirect fluorescent antibody test (Zinora et al., 2007), enzyme linked
immunosorbant assay (Chahan et al., 2006), latex agglutination test (Xuan et al., 2001),
polymerized chain reaction (Alhassan et al., 2005), DNA Probes (Posnett and Ambrosio, 1989;
Posnett and Ambrosio, 1991) and loop-mediated isothermal amplification (LAMP) test (Alhassan
et al., 2007a; Alhassan et al., 2007b).
Throughout the world, disease is controlled through the therapeutic as well as prophylactic
use of imidocarb dipropinate and diminazine (Radostits et al., 2007; Vial and Gorenflot, 2006) and
implementation of tick (vector) control measures. To date, no vaccine is available for this disease
and for the proper eradication of disease stringent implementation of ‘test and slaughter policy’ is
advocated by Office International Des Epizooties, OIE (2004).
Not only the prevalence but also the presence of vector (ticks) of this important equine
disease is well established in our neighboring country India (Malhotra et al., 1978; Ali et al.,
1996; Ambawat et al., 1999). Although, we share almost similar climatic and geographic
conditions with India and prevalence of potential tick vectors is also reported from different areas
of Pakistan, our knowledge about the disease is limited to only a few studies based upon
conventional blood smear examination (Kokab, 1986; Khan et al., 1987). In view of the foregoing,
6
there is a need to conduct some well-planned epidemiological studies to know the prevalence of
this disease and review our current control methods.
Adequate resources and careful planning are major components for conducting meaningful
prevalence studies for a disease. In resource poor countries like Pakistan, where funds for
screening of animal populations for the presence of a single disease are usually scarce, planning of
such study will require proper allocation of resources, suitable and practical sampling frame and
use of recent and cost effective epidemiological documentation tools on which our future studies
can be built. To address the problem of finances in resource poor economies, it is advisable that
wherever possible multiple disease prevalence methods should be adopted to determine the
incidence / prevalence of different diseases by using a single sample taken from single species of
animal.
Keeping the aforesaid in view, and considering lacunae regarding equine infectious anemia,
glanders and piroplasmosis in Pakistan, the present study has been planned with the following
objectives:
I. Define the exposure of equines in the selected areas to EIA, glanders and piroplasmosis
II. Determination of hematological alterations (if any) associated with these diseases
III. Determine the association of such host determinants as the age, breed and sex (if any) with
the occurrence of these diseases
IV. Investigate the possibility of attaining disease free status regarding EIA for the areas under
study
V. Conceptualize the control measures for these disease by analyzing epidemiological data
generated by the survey
CHAPTER-II (Review of Literature)
I. Justification for the epidemiological surveys:
Draught horses, mules and donkeys not only pull the loads but also help sustain the livelihood of
their owners and families. Any disease or disability rendering these animals sick and unavailable
for work can have some serious effects on owner and his family. Draught work can also act as a
stress factor in these animals, reducing resistance to many diseases prevalent in the area
including infectious as well as many endoparasitic problems. Moreover, chances of spread of
infectious and vector borne diseases are more as these animals come in frequent contact with
each other through communal watering troughs, common feed mangers and at the rest stations.
In Pakistan, most of the draught equines survive in a sub-optimal productive state owing to stress
of diseases on one hand and poor nutrition on the other. That’s why any effort to increase their
productivity (draught power) under the situation can upset their physiological equilibrium and
lead to catastrophic results. Thus, exploitation of draught power from these animals is possible
only when diseases are brought under full control.
Among the two standard methods of disease management viz., control and eradication, disease
control is the only scientifically realistic or economically feasible method. This makes epidemic
disease control a desirable method for the developing world (Burridge, 1981). By performing
some meaningful epidemiological studies on epidemic diseases, most prevalent diseases can be
checked through some proven methods of control including identification and treatment of cases
and carriers, removal of reservoirs of infection, quarantine, sanitary measures and mass
immunization programs (Lawrence et al., 1993).
II.a. The disease and the organism
Equine infectious anemia (EIA) is a blood borne, chronic viral disease of the Equidae. Clinically,
the disease is manifested by persistent infection with recurrent viremia cycles and fever episodes,
anemia, edema and weight loss (Cheevers and McGuire, 1985; Sellon et al., 1994; Radostits et
al., 2007). EIA virus has great historic significance because this was the first retrovirus proven to
be transmitted through insects (Stein et al., 1942), the first persistent virus for which ‘antigenic
drift’ was defined (Kono, 1972) and the first retrovirus disease for which a diagnostic test viz.,
Coggins Test was recommended (Coggins et al., 1972).
The disease can manifest itself in three forms viz., acute, chronic and inapparent. In rarely seen
severe acute form, the clinical signs are non-specific with elevated rectal temperature and
affected horse may die within 2-3 weeks. Usually the clinical picture of disease is variable with
many haematological and biochemical alterations like anemia, thrombocytopenia,
hypergammaglobinemia and elevated liver enzymes (Spyrou et al., 2003; Leroux et al., 2004;
Cullinane et al., 2007; Radostits et al., 2007). The animals often recover and remain carriers
throughout their life. Recovered animals may suffer from recurring clinical (chronic) disease,
which manifests itself with the signs of fever (105-108 °F), petechial hemorrhages, depression,
weight loss, dependent edema and anemia. This form is caused by new mutant EIAV strains
(Kono, 1972; Payne et al., 1987) and affected equine is often termed as “swamper” (Clabough et
al., 1991). The disease is more detrimental in its inapparent form as it poses the real threat to
other healthy horses. These carriers show negligible or no clinical signs and suffer from constant
viremia and thus may spread infection to other horses through biting insects and hypodermic
9
needles etc., (Cordes and Issel, 1996). No seasonal, sex or age based association could be
ascertained on the basis of previous studies (Radostits et al., 2007).
II.b. Geographic distribution
Equine infectious anemia is worldwide in distribution (OIE, 2004) and since 2005 disease was
reported with/without clinical signs in 40 countries. According to World Animal Health
Database (Anonymous, 2008), during 2008, EIA has been reported at different levels in 20
countries (Canada, Chile, Costa Rica, Cuba, Denmark, Finland, Georgia, Greece, Iran, Ireland,
Israel, Lebanon, Mexico, Netherlands, Portugal, Russia, Spain, United Kingdom, United States
of America and Uruguay). During past few years, authors have reported the varying degree of
prevalence of EIA from different regions of world (Pare and Simard, 2004; Bicout et al., 2006;
Menzies and Patterson, 2006; Reynolds, 2006). A continuous surveillance is required to establish
and maintain the disease free status in a country as demonstrated by few Turkish workers (Turan,
et al., 2002; Ataseven and Arslan, 2005; Kirmizigl et al., 2009). It is feared that actual incidence
of EIA is more than our estimate, as many countries are still not conducting seroprevalence
studies to rule out or confirm the disease.
In Pakistan, to date, only one study regarding the investigations of EIA is available (Gill et al.,
2008), which indicates towards the possible disease free status of equines in Faisalabad
metropolis. In our neighbouring country (India), incidence of EIA is documented since the first
case was detected at Karnatika in 1987 (Uppal and Yadav, 1989). Since then the disease has been
reported from the states of Maharashtra, West Bengal, Haryana, Dehli and Indian Punjab as well
(Singh et al., 1997). Clinical disease has also been reported from neighbouring Islamic Republic
of Iran during recent years (Anonymous, 2008).
II. c. Diagnostic methods
Since the first description of diseases in 1843, many diagnostic techniques (precipitin test,
hemagglutination, immune adherence, indirect hemagglutination and serum protein and lipid
changes) were used for the diagnosis of EIA but the confirmed serological diagnosis remained a
big issue until the development of Coggins test. Coggins and co workers (1972) developed an
agar gel immunodiffusion (AGID) test for the diagnosis of EIA. This test detects the
precipitating antibodies against group reactive and antigenically stable core protein p26 (Payne et
al., 1984). This test has stood the test of time and the positive AGID test result is reliable and
confirms virus presence (Issel and Cook, 1993). Like all the serological tests, the AGID has its
own limitations. For instance, horses suffering from the disease first time and tested with AGID
can test negative for antibodies. This can also be seen in cases of foals acquiring colostral
passive immunity from EIA test positive dams. The results can be obtained within 24-48 hrs
(Issel and Cook, 1993). Since 1972, this test has been used as a gold standard in many
comparative test evaluation and prevalence studies on EIA (OIE, 2004).
Enzyme-linked immunosorbent assay (ELISA) has been used since late 1980s for faster and
more sensitive serodiagnosis (Issel and cook, 1993). A more sensitive competitive ELISA
(cELISA) has been developed to detect antibodies against core p26 protein and investigators
have found a good correlation between AGID and cELISA (Soutullo et al., 2001; Piza et al.,
2007). As compared to AGID, the ELISA test is rapid and results are easier to interpret (Uppal
and Yadav, 1992; Issel and Cook, 1993; Cordes et al., 2001; Cullinane et al., 2007; Piza et al.,
2007; Susan et al., 2008). The chances of getting false positive results are more with ELISA as
compared to AGID as it is less specific. For this reason, OIE recommends that all ELISA
positive samples should be confirmed by performing AGID test. However, the low levels of p26
11
antigen in the sample attained from horses suffering from initial bots of clinical disease can only
be detected through ELISA and often yield negative AGID test results (Cullinane et al., 2007;
Piza et al., 2007). Whenever rapid test results are needed, disease status is unknown and horses
are present at remote location, the ELISA becomes the test of choice (Issel and Cook, 1993;
Cullinane et al., 2007; Piza et al., 2007). Furthermore, ELISA also can be employed as an
international pre-movement screening test because of its sensitivity and rapidity (Cullinane et al.,
2007). Other techniques like immunoblot test and PCR have also been developed for the
diagnosis of EIA but their use is limited as a research tool only (Issel and Cook, 1993).
II.d. Treatment and control
To date no vaccine and cure is available for the EIA (OIE, 2004) and the control of disease
depends upon wide scale testing in population at risk and destruction / immobilization of infected
horses (Issel and cook, 1993; OIE, 2004; Issel et al., 2005). Disease has only been controlled in
developed countries through rigorous testing and destruction. For example, during the past two
and a half decades, the USA equine owners contributed about 600 million dollars for testing and
control of EIA which shows the amount of personal and financial efforts required to control the
disease. Vaccine development against EIA virus is still a great challenge (Issel et al., 2005).
As the disease has been present in neighbouring countries (India and most recently in Iran) to
whom we share porous borders and vectors (fly and mosquito) also exist in Pakistan, some
carefully planned prevalence studies are needed to confirm the disease status regarding EIA in
Pakistan.
Figure-1 Map showing global distribution of equine infectious anemia (EIA) during last six months (July – December) of 2008 (Anonymous, 2008)
III.a. The disease and the organism
Glanders is an anciently known disease of equids and has been described by Hippocrates
between 450 B.C. and 425 B.C. and a century later by Aristotle who named the infection
malleus, a Latin word for a malignant disease or epidemic (Minett, 1930). Apart from equines,
the organism can also cause disease in domestic and wild felids, dog, sheep & goat and camel
(Al-Ani and Roberson, 2007; Gregory and Waag, 2007). The disease is caused by Burkholderia
mallei (previously known as Loefflerella mallei, Pfeifferella mallei, Malleomyces mallei,
Actinobacillus mallei, Bacillus mallei and Pseudomonas mallei, (OIE, 2004). Burkholderia
mallei is Gram-negative, rods with rounded ends, 2-5 µ long and 0.5 µ wide, often stain
irregularly, and do not have capsules or form spores. B. mallei may have bipolar staining ‘safety
pin’ (Songer and Post, 2005). Capsular polysaccharide acts as an essential virulence factor for
this organism (Lopez et al., 2003). The organism produces toxins (extra-cellular enzymes) which
disrupt host cellular functions include pyocyanin, lecithenase, collagenase, and, lipase.
Pyocyanin interferes with terminal electron transfer, whereas lecithenase, collagenase, and lipase
are lytic in their action (Songer and Post, 2005). Recently, Russian workers (Narbutovich et al.,
2005) reported plasma coagulase (a thermostable enzyme) activity of natural and mutant strains
of B. mallei and B. pseudomallei. However, their exact role in pathogenesis is unknown.
There are three clinical forms of glanders viz., nasal, pulmonary and cutaneous (farcy) and the
course of disease may be acute, sub-acute or chronic (Hartwigk and Gerber, 1986). Furthermore,
an apparently carrier status (latent infection) is maintained by the recovered animals (Al-Ani and
Roberson, 2007; Gregory and Waag, 2007) that can result in to transmission of disease to
equines and humans. Spread of the organism can be due to direct contact through aerosol droplet
and indirectly though contaminated feed, water and grooming utensils (Neubauer et al., 2005;
Al-Ani and Roberson, 2007: Gregory and Waag, 2007). Pulmonary form is observed in
practically all cases of the disease (Al Ani et al., 1998). The acute form of glanders occurs most
frequently in donkeys and mules (Udall, 1939; Hayes, 1968) and is often fatal within a month or
so (Minett, 1959). Davies (1955) proposed that in mules the course of disease is sub-acute as the
susceptibility of the mules seems to be mid way that of the horse and donkey. Chronic form of
disease usually occurs in horses (Dungworth, 1993) and animal remains sick for months then
dies or becomes carrier (Hungerford, 1990).
Acute glanders begins with chill and high fever of 106 to 108 °F (Udall, 1939). It is clinically
characterized by attacks of coughing, mucoid nasal discharge from one or more rarely both
nostrils, inspiratory dyspnoea, pneumonia, rapidly developing ulcers on nasal mucosa and
enlargement of submaxillary lymph nodes (Cole, 1942; Dalling, 1966; Hartwigk and Gerber,
1986, OIE, 2004; Radostits et al., 2007). Death occurs within a few days due to septicaemia (Al-
Ani, et al., 1987; Radostits et al., 2007).
Chronic form of the disease develops as a sequel to either clinical infection or non-fatal acute
cases and is manifested by progressive loss of body weight, a ‘run down appearance’ and
unthrifty hair coat (Al-Ani et al., 1998; Al-Ani and Roberson, 2007) and these chronically
infected equines are known reservoirs of disease in nature (Neubauer et al., 2005). In chronic
infection, nasal and skin forms commonly occur together. When the localization is chiefly
pulmonary, there is chronic cough, frequent epistaxis, laboured breathing, and febrile episodes.
Inflammatory nodules and ulcers develop in the nasal passages and give rise to a sticky yellow
discharge from one or both nostrils, accompanied by enlarged firm submaxillary lymph nodes
(OIE, 2004; Al-Ani and Roberson, 2007; Radostits et al., 2007; Saqib et al., 2008). In skin form
15
(Farcy), lesions may occur in any part of body, but are more commonly present on areas exposed
to injury, such as hind legs (Dalling, 1966). In a collaborative study, a neurologic form of
glanders has been proposed in which equines had developed cross-stepping of hindquarters.
However, B. mallei could not be demonstrated in nervous tissue (Lopez et al., 2003).
The course of disease is usually acute in donkeys and horses usually suffer from chronic and
inapparent form of the disease, whereas the disease could manifest itself in both acute and
chronic forms in mules. (Al-Ani et al., 1998; Al-Ani and Roberson, 2007; Gregory and Waag,
2007). Risk of acquiring glanders found to be higher in old age animals and animals older than 2
years of age are more susceptible (Radostits et al., 2007; Al-Ani and Roberson, 2007). However,
some studies report that equines can get the disease at any age (Al-Ani et al., 1998; Saqib, 2003).
Hematological alterations associated with glanders are reported as marked increase in white
blood cell counts, anemia, below normal PCV and haemoglobin concentration (Saqib 2000;
Manso, 2003; Al Ani and Roberson, 2007; Saqib et al., 2008).
Overcrowding, unhygienic conditions, draught stress, contact with infected equines, sharing of
feed and water troughs and poor nutrition make the equine more susceptible to the glanders as
the chances of spread through infectious secretions are more from one animal to other under
these conditions (Henning, 1956, Al-Ani et al., 1987; Jerabek, 1994, Nagal et al., 1995;
Muhammad et al., 1998; Manso, 2003; Neubauer et al., 2005; Al-Ani and Roberson, 2007;
Gregory and Waag, 2007). Water can become contaminated with B. mallei through a diseased
equine and organism can remain there for the weeks to come (Gangulee et al., 1966; Al-Ani and
Roberson 2007; Gregory and Waag, 2007; Radostits, 2007). Glanders in equines can occur in
any season but mostly cases were reported in association with cold weather (Manso, 2003; Saqib
et al., 2003; Al-Ani and Roberson, 2007; Gregory and Waag, 2007).
Aggressive control measures have essentially eradicated glanders from most of the developed
countries such as USA, Canada, UK, and Australia (Derbyshire, 2002). However, the disease is
prevalent in developing countries like Pakistan (Muhammad et al., 1998; Saqib et al., 2003;
Naureen et al., 2007; Naureen et al., 2008), India (Verma et al., 1990; Pawaiya and Chauhan,
2008), Iraq (Al-Ani, et al., 1998), China (Ma et al., 1986), Iran (Bazargani et al., 1996), United
Arab Emirates (Sabirovic et al., 2005; Witting et al., 2006), Brazil (Mota et al., 2000; Manso,
2003) and Turkey (Arun et al., 1999). The disease has been reported from Brazil, Eriteria, India,
Iran, Mangolia, Pakistan, Russia and Philippines between 2006 and 2008 (Anonymous, 2008).
A recent outbreak in India after almost 2 decades started in 2006 with clinical disease was
reported from Maharashtra, Uttar Pradesh, Punjab and Uttarakhand provinces (Pawaiya and
Chauhan, 2008). Disease outbreak has also been reported in equines of Lahore Polo Club,
Pakistan (Naureen et al., 2008). This recrudescence of disease in India and Pakistan could be
attributed to meager pittance (about Rs. 50 per equine in Pakistan) given to the glanders positive
equine owners for killing their animals (Muhammad et al., 1998; Saqib et al., 2003; Pawaiya and
Chauhan, 2008). This has forced the poor equine owners to avoid reporting any signs of the
disease to veterinarians and authorities, hiding of clinically affected equids to avoid testing,
selling of suspected or chronically infected equines to un aware persons belonging to some other
community or cities to compensate their economic losses (Muhammad et al., 1998; Saqib et al.,
2003; Gregory and Waag, 2007; Pawaiya and Chauhan, 2008; Saqib et al., 2008).
17
Figure-2 Map showing global distribution of equine glanders during last six months (July – December) 2008
III.c. Diagnostic methods
The mallein test is a corner stone for the diagnosis of equine glanders. The mallein is purified
protein derivative (PPD), available commercially. It is a solution of water-soluble protein
fractions of heat-treated Burkholderia mallei cells (OIE, 2004). Mallein test can be performed by
following three different methods: (i). intradermo–palpebral mallein test (ii). Opthalmic test (iii).
Sub-cutaneous Mallein test (OIE, 2004).
Mallein test has been widely and effectively used for the diagnosis of glanders but has
limitations in terms of sensitivity, particularly in clinical and advanced cases of the disease (Jana
et al., 1982; Verma, 1981; Neubauer et al., 2005). Indian workers (Misra and Arora, 1990) have
found serological cross-reactions between Pseudomonas mallei (old name of B. mallei) and some
other bacteria, whereas Iraqi workers (Al-Ani et al., 1993) encountered false positive mallein
reaction, particularly between B. mallei and Streptococcus equi (Strangles) infection. Also, the
mallein testing of glanders negative equines have been reported to yield positive CFT test results
for these animals (Hagebock, et al., 1993; Neubauer et al., 2005; Gregory and Waag, 2007). The
serological tests used for diagnosis of glanders include complement fixation test (Higgins and
Wright, 1998), micro-complement fixation test (Verma, 1990), avidin-biotin dot enzyme-linked
immunosorbent assay (Verma et al., 1990), micro–enzyme–linked immunosorbent assay (Al–
Ani et al., 1993), arrayed immunoblotting mehod (Katz et al., 1999), competitive enzyme-linked
immuno-assay (Katz et al., 2000), agglutination and precipitin tests (Gillespie and Timoney,
1981), counter immunoelectrophoresis (Jana et al., 1982), haemagglutination test (Gangulee et
al., 1966), haeagglutination inhibition tests, indirect haemagglutination test (Zhang and Lu,
1983; Ferster et al. 1986), Rose Bengal plate agglutination test (Naureen et al., 2007) and latex
agglutination test (Saqib et al., 2008).
19
The complement fixation test (CFT) is reported to be 90 – 95 % sensitive but has its own
limitations especially regarding the sera from donkey, mule and pregnant mares having anti
complementary characteristics (Gregory and Waag, 2007). In the wake of limitations of CFT and
other serological tests, there is a need to develop tests using well characterized antigens to avoid
false positive results (Neubauer et al., 2005).
III.d. Treatment and control
Treatment of glanders is forbidden in countries where ‘Glanders and Farcy Act - 1899’ is
implemented. In Pakistan, legislation calls for detection and destruction of glanderous animals
(Farani, 1983). However, an incredibly low indemnity paid to the owners does not oblige or lure
them to let their animals be destroyed. As a salvage measure, equine owners sell their animals at
reduced price that leads to spread of disease in new areas (Muhammad et al., 1998). The
organism is sensitive to aminoglycosides, sulfonamides, tetracyclines and, quinolones,
nitrofurones and resistant to early ß-lactams and colistin, metronidazole, cephalexin (Darling and
Woods, 2004).
Experimental chemotherapy of glanders has been performed in equines, hamsters, guinea pigs,
and monkeys (Miller et al., 1948; Batmanove, 1991; Batmanove, 1993; Batmanove, 1994;
Iliukhin, et al., 1994; Manzeniuk et al., 1994; Muhammad et al., 1998; Russel et al., 2000;
Manzeniuk, 1995). Saqib and colleagues (2003) have reported an experimental cure of equine
glanders with a combination of enrofloxacin and trimethoprim + sulfadiazine with a 21-day
treatment protocol. In a recent case of laboratory-acquired glanders (Srinivasan et al., 2001), the
patient received imipenem and doxycycline intravenously for 1 month followed by oral
azithromycin and doxycycline for 6 months. This treatment regimen was successful and there
was no relapse of the disease. However, there is as yet no consensus recommendation for
treatment of human/animal glanders, although there is extensive literature on antibiotic
susceptibility of B. mallei (Al-Ani and Roberson, 2007; Gregory and Waag, 2007).
Lack of stern implementation of Glanders and Farcy Act (1899) had made glanders a major
concern in Pakistan because of high incidence in endemic areas and the disease is thought to be
quite prevalent in many areas of Pakistan. To date, only a few prevalence studies based on the
screening of clinical cases have been conducted in Punjab (Nasreen, 1977; Vaid, 1981; Bashir,
1984). Glanders is an emerging disease and as such the cause of a serious concern for animal
health monitoring authorities and there is a dire need to conduct a seroprevalence study to
establish the current prevalence of this disease in Pakistan (Muhammad et al., 1998, Saqib et al.,
2003, Naureen et al., 2007).
IV. Equine Piroplasmosis
IV.a. The disease and the organisms
Equine Piroplasmosis is a tick borne protozoan disease of horse family (Knowles, 1988;
Friedhoff et al., 1990; Radostits et al., 2007; Zinora et al., 2007). The disease is caused by
hemotropic protozoa viz. Theileria equi and Babesia caballi and regarded as an emerging equine
disease all over the world (Shkap et al., 1998; Vial and Gorenflot, 2006; Uilenberg, 2006). The
disease was previously known as ‘Babesiosis’ but due to the recent research developments some
basic differences (extra-erythrocytic shizogony in T. equi) were found between the life cycles of
parasites resulting in the change in status of B. equi as Theileria equi (Vial and Gorenflot, 2006;
Zinora et al., 2007).
Incubation period of the diseases varies from 12-19 days for T. equi and 10-30 days for B. cabali.
T. equi infected animals usually exhibit more severe and acute form of disease as compared to B.
cabali infections which usually follows the chronic course (Shkap et al., 1998; Vial and
21
Gorenflot, 2006; Radostits et al., 2007). Clinically, the disease can be categorized in four
different forms viz., peracute, acute, chronic and rare atypical forms. In peracute cases the
animals are usually found in moribund conditions or found dead. Fever, anorexia, depression,
icterus, hemoglobinuria, colic, regenerative hemolytic anemia and dissemminated intravascular
coagulopathies (DIC) are usually seen in acute form of piroplasmosis (Seifi et al., 2000;
Camacho et al., 2005).
Most common heamatological alterations associated with priplasmosis are Hemolytic anemia,
thrombocytopenia and decrease in hematocrit (PCV) values (De Waal et al., 1992; Taboada and
Merchant, 1991; Camacho et al., 2005). Significantly less hematocrit (PCV) was reported in non-
clinical seropositive horses as well by Camacho et al. (2005). Significantly lower RBC counts
and anemia was found to be more significantly associated with T. equi infected horses by
Camacho et al. (2005). T. equi is more virulent than B. caballi and results in more consistent
hemoglobinuria and death, while B. caballi causes persistent syndrome characterized by fever
and anemia (Henry, 1992; Camacho et al., 2005)
Chronic form of the disease usually follows the acute phase and observed clinical picture is
debility, loss of condition and exercise intolerance but most of the times these clinical signs
cannot be ascertained (Akkan et al., 2003) in case of serological studies. Sometimes a rare
atypical form is also seen with signs of gastro-enteritis, bronchopneumonia and abortions (Vial
and Gorenflot, 2006; Radostits et al., 2007).
Equines born and raised in the endemic areas enter into the carrier state of the disease which
compromises their draught potentials considerably (Abdelkebir et al., 2001). These carrier
animals are responsible for the maintenance of infection in endemic areas (Camacho et al.,
2005). Heavy draught stress, strenuous exercise and poor nutrition may results in to the
22
recrudescent infection and clinical disease in these animals (Hailat et al., 1997; Seifi et al., 2000;
Camacho et al., 2005). Spleen is responsible for the immune response against hemoparasites and
its removal results in recrudescence of latent hemoparasitic infections, therefore, splenectomy
results in acute and fatal disease in case of clinically normal carrier animals (Abdelkebir et al.,
2001).
Ticks of three genera (Dermacentor, Rhipicephalus and Hyaloma) are responsible for the spread
of piroplasmosis all over the world (Ali et al., 1996). In ticks sexual reproduction of these
organisms takes place and they can transmit the disease trans-stadially and transovarially.
Iatrogenic spread of the disease can also occur through contaminated blood transfusion,
injections and surgical instruments (Yoshihara, 1997; Uilenberg, 2006; Vial and Gorenflot,
2006). Clinically, piroplasmosis should be differentiated from equine infectious anemia, surra,
dourine, African horse sickness and plant poisoning (Ali et al., 1996; Vial and Gorenflot, 2006;
Radostits et al., 2007).
Horses are more susceptible for B. caballi infections as compared to mules and donkeys (Acici et
al., 2008) and susceptibility to disease is directly proportional to the age of animal (Abdelkebir et
al., 2001). The chances of contracting T. equi infections were found more in donkeys then horses
and mules by Abdelkebir et al. (2001) but a recent study in Turkey contradict this finding were
T. equi infection was found least prevalent in donkeys (Acici et al., 2008). Only a handful of
authors have suggested sex dependent prevalence of B. caballi and T. equi (Shkap et al., 1998;
Rüegg et al., 2007). However, no such difference is reported based upon the sex of the equines
by many authors (Olivera and Garcia, 2001; Asgarali, et al., 2007; Karatepe et al., 2009).
23
Various prevalence studies carried out to in the different regions of the world found that
prevalence of the T. equi increases with age of equines (Brüning, 1996; Oliver and Garcia, 2001;
Asgarali, et al., 2007; Rüegg et al., 2007; Karatepe et al., 2009). However, some authors did not
agree upon any difference between prevalence of T. equi and B. caballi according to age of
equines (Tenter et al., 1988; Shkap et al., 1998; Acici et al., 2008).
The higher prevalence of T. equi as compared with B. caballi, is mostly reported in the endemic
regions of the world (Barbosa et al., 1995; Ribeiro et al., 1999; Akkan et al., 2003; Boldbaatar et
al., 2005; Camacho et al., 2005; Asgarali, et al., 2007; Salim et al., 2008; Karatepe et al., 2009).
Which could be due to the fact the infections with T. equi are usually for life (Schien, 1988;
Rüegg et al., 2007; Rüegg et al., 2008) and B. caballi infections usually clear from the host in 1-
5 years (Rüegg et al., 2007; Rüegg et al., 2008). Prevalence of piroplasmosis can differ with
reference to locations as demonstrated by various studies based upon the differences found in the
climatic / geographic conditions, vector population, draught stress and equine keeping practices
(Heuchert et al., 1999; Skhap et al., 1998; Acici et al., 2008; Salim et al., 2008; Karatepe et al.,
2009). But selection of areas with similar geographic, climatic and equine management attributes
may lead to prevalence findings independent of locations (Abdelkebir et al., 2001; Chahan et al.,
2006).
Managemental conditions that favor the spread of T. equi includes congested and confined
housing resulting in the increased transtadial intrahost transfer among vectors and subsequently
transmission (Abdelkebir et al., 2001).
IV.b. Geographic distribution
About 90% of world equine population lives in the areas endemic for equine piroplasmosis
(Schein, 1988) and this is an emerging threat worldwide. Since 2006, the incidence of disease
has been reported from 31 countries of the world. During 2008, the incidence disease has been
reported in equines from Argentina, Belize, Bolivia, Brazil, Chile, Colombia, Costa Rica,
Greece, Israel, Jordan, Malta, Martinique (France), Mexico, Morocco, Myanmar, Peru, Portugal,
Qatar, Russia, South Africa, Spain, Suriname, Switzerland, United Kingdom, USA, Uruguay,
and Zimbabwe (Anonymous, 2008).
Piroplasmosis is endemic in china, Brazil, Central Mangolia (Xuan et al., 2001), Morocco
(Abdelkebir et al., 2001), Tranidad (Asgarali, et al., 2007), Iran (Seifi et al., 2000), Spain
(Camacho et al., 2005). Various prevalence studies indicated sero-reactivity in Mangolia (88-
84%), South Africa (61-40%), Colombia (94-90%), Sudan (91-86%), Brazil (81-90%), Morocco
(69%) and Israel (76-80%). Disease was also frequently reported from India (Malhotra et al.,
1978).
However, in high endemic areas the clinical form of disease is usually seen rarely (Pfeifer et al.,
1995). Japan is considered free from piroplasmosis because no clinical case has been reported to
date (Ikadai et al., 2002). In recent years, researchers all over the world are on the look for the
current prevalence of piroplasmosis in various regions (Akkan et al., 2003; Boldbaatar et al.,
2005; Camacho et al., 2005; Asgarali, et al., 2007; Acici et al., 2008; Karatepe et al., 2009;
Salim et al., 2008).
25
Figure-3 Map showing the global distribution of equine piroplasmosis during last six months (July – December) of year 2008
IV.c. Diagnosis
Diagnosis can be made by the demonstration of intra-erythrocytic parasites in Giemsa stained
blood smears which is rarely probing (Knowles, 1988; Weiland and Reiter, 1988; Shkap et al.,
1998; Akkan et al., 2003; Acici et al., 2008; Salim et al., 2008) in chronic forms of disease and
endemic regions where low prasitemia is usually observed (Calder et al., 1996; Salim et al.,
2008). Transmission test can be performed for the conformation by transfusing about 500 ml
anticoagulant mixed blood of suspected animal to disease free equines (Ali et al., 1996).
For the sero-diagnosis of piroplasmosis different tests have been developed over the years.
Complement fixation test (CFT) remained a gold standard for many years (Donnnelly et al.,
1980; Weiland, 1986) but due to its limitations regarding provision of false positive/negative
results, alytical ingredient production, anticomplementory activity of many sera and the inability
of IgG (T), the major immunoglobulin isotype of equids, to fix the complement forced scientific
community to develop new tests (Abdelkebir et al., 2001). A more sensitive and specific indirect
fluorescent antibody test (IFAT) was developed and used by many scientists (Amerault et al.,
1979; Callow et al., 1979; Donnelley et al., 1980; Ribeiro et al., 1999; Akkan et al., 2003;
Zinora et al., 2007) but the test is not efficient for use with a large number of sera (Abdelkebir et
al., 2001).
Keeping in view the requirements of surveillance and prevalence studies ELISA was developed
(Knowles et al., 1991; Shkap et al., 1998) which is more sensitive and specific (Abdelkebir et
al., 2001). Later on a much improved competitive ELISA was developed for the diagnosis and
reported to be best to use for epidemiological studies (Abdelkebir et al., 2001; Ikadai et al.,
2002; Damdinsuren et al., 2005; Chahan et al., 2006; Huang et al., 2006; Salim et al., 2008). On
the basis of successful demonstration of sensitivity and specificity in various studies (Knowles et
27
al., 1991; Shkap et al., 1998; Katz et al., 2000), ELISAs which detect the antibodies to
geographically conserved region of piroplasms are now one of the recommended tests for
piroplasmosis (OIE, 2004). Other tests developed for the confirmation includes PCR based
assays (Kappmeyer et al., 1993; Ali et al., 1996; Zintl et al., 2003; Alhassan et al., 2005; Salim
et al., 2008), DNA probes (Posnett and Ambrosio, 1989; Posnett and Ambrosio, 1991), latex
agglutination test (Xuan et al., 2001) and recently developed loop-mediated isothermal
amplification (LAMP) test (Alhassan et al., 2007a; Alhassan et al., 2007b). Latix agglutination
test developed by Japanese workers (Xuan et al., 2001) by using recombinant T. equi merozoite
antigen 1 (EMA-I) have shown identical comparable results with ELISA and can be trialed and
employed as an onsite screening tool for diagnosing T. equi infections.
IV.d. Treatment and control
Both T. equi and B. caballi respond to the babesiacidal drugs but T. equi is more refrectory to the
treatment than B. cabali (Vial and Gorenflot, 2006). Imidocarb can be used for therapeutic as
well as chemoprophylactic purposes and appears to be a drug of choice for eliminating the
carrier state. Recommended dose regimens for the T. equi is 4 mg/kg repeated four times at 72 hr
interval and two treatments of 2.2 mg/kg at 24 hr interval (Vial and Gorenflot, 2006; Radostits,
2007). However, T. equi infections can only be suppressed by treatment but complete elimination
of this parasite is not possible (de Waal, 1992; Knowles, 1996).
Although a few workers have tried to make a reliable vaccine (Kumar, et al., 2002), till today, no
vaccination is available for the piroplasmosis. So continuous disease surveillance, control of
vector (tick) population and implementation of test and slaughter policy forms the mainstay of
control programs (Radostits et al., 2007).
CHAPTER-III (Materials and Methods)
1. Study locales and settings
The study was conducted in 5 draught equine populated urban areas of Punjab, Pakistan.
Selection of the areas was made on the basis of reported district draught equine population
equine population in Livestock Census (Anonymous, 2006). For making the study more
meaningful and demographically diverse, 3 areas from Central (Faisalabad, Lahore and
Gujranwala Metropolises) and 2 areas (Multan and Bahawalpur Metropolises) from Southern
Punjab were selected. Wherever possible, the study area selection took into account the
presence of working areas of ‘Brooke Hospital for Animals’ in the selected cities to seek the
necessary cooperation by the draught equine owners and convenient sampling. The study
period spanned over 8 months from July 2007 to March 2008.
2. Sampling frame
As the prevalence of the diseases under study was unknown in Pakistan, the sample size was
calculated by considering the expected prevalence to be 50% with confidence limits of 95%
and a desired absolute precision of 5% to take maximum numbers of samples (Thrusfield,
2005). Samples size was calculated on the basis of following formula:
n = 1.962 Pexp(1 – Pexp) / d 2
Where:
29
The number of samples thus calculated was subjected to the following formula for the
estimation of required sample size (nadj):
nadj = (N X n) / (N + n)
Where:
n = calculated sample size through formula
Stratification of the sampling units was performed by proportional allocation of
sampling units to the draught equine population in the selected areas (Table-3.1). Random
selection methods were used for the selection of areas to be sampled within each city and the
simple random sampling was performed on-site.
3. Recording of information
history, treatment and clinical examination for the diseases under consideration was obtained
on a pre-designed proforma (Appendix-I).
4. Complete physical and clinical examination
Physical and clinical examination was performed and values regarding vital physiological
parameters and observations were entered in the proforma designed for the said purpose.
5. Samples collection
Samples were collected by using sterile vacutainers and labeled accordingly for
identification. For the serum, 10mL blood was drawn into a clot activator vacutainer without
anticoagulant. For the whole blood specimen, 4mL blood was collected into an anticoagulant
(Ethylenediaminetetraacetic acid (EDTA) @ 1 mg/ml) coated vacutainer. Thin and thick
smears on clean glass slides were made on-site and labeled accordingly.
Table-3.1 Sampling plan for the sero-survey of equine infectious anemia, galnders and piroplasmosis in 5 urban areas of Punjab
Urban Area
Total Draught
Faisalabad 6645 34.91 18 44144 37.20 123 3046 40.52 9
Lahore 4596 24.15 13 17386 14.65 50 721 9.59 6
Gujranwala 4612 24.23 14 18964 15.98 55 1841 24.49 6
Multan 1940 10.19 10 19076 16.07 52 954 12.69 6
Bahawalpur 1238 6.50 10 19076 16.07 52 954 12.69 6
19031 65 118646 332 7516 33
Total Samples Taken = 430
31
Figure-4: Google™ Maps showing the 5 sampled draught equine populated urban areas of province, Punjab, Pakistan
6. Samples transportation
These samples were transported to the postgraduate research laboratory of the
Department of Clinical Medicine & Surgery, University of Agriculture, Faisalabad, while
observing the critical requirement of temperature (4°C).
7. Sample processing
Hematological examination
Whole blood samples were subjected to hematological examination for red blood cell
(RBC) count, hemoglobin concentration (Hb), white blood cell (WBC) count packed cell
volume (PCV) and erythrocytic indices as per methods described by Coles (1986).
Erythrocyte indices (MCV, MCH and MCHC) were calculated to classify the type of
anemia (Coles, 1986, Thrall, 2004)
Serum collection
Serum was collected by following the recommendations of Benjamin (1978) and stored at
-40°C till further processing.
Thin and thick blood smear examination
Microscopic examination of Giemsa (Merk, Germany) stained thin and thick smears
(Henry, 1996) was conducted for any evidence of blood parasities (B. caballi, T. equi and
Trypanosoma)
Sero-diagnosis of equine infectious anemia
For this purpose commercial equine infectious anemia virus antibody test kit, ELISA
(VMRD, Inc., Pullman, USA) was used (Abdelkabir et al., 2001). For the Sample
processing and application on the ELISA kit, following methods described by the
manufacturer were followed:
33
Preparation
a. Warming up reagents: the serum samples, reagents and plate(s) were brought to the
room temperature before starting the test procedure.
b. Positioning controls and samples: Positive and negative controls in duplicate were
placed on different wells of the plate with each run. Samples were first placed in to non-
antigen coated plate and then transferred with multichannel micropipettor to the antigen-
coated plate. The control and serum sample IDs were recorded on the attached setup
record sheet. Plates were removed from the foil pouch and brought at room temperature.
d. Preparation of conjugate: 1X antigen-peroxidase conjugate (D) was prepared by
diluting 1 part of 100X D with 99 parts of conjugate diluting buffer (E).
e. Preparation of wash solution: 1X wash solution was prepared by adding one part of
the 10X wash solution concentrate (F) in 9 parts of deionized water.
f. Preparation of serum samples: undiluted serum samples were used for test.
Test Procedure
1. Loading controls and serum samples: 50 μl of controls and serum samples were
pipette into the antigen coated plate. Side of the plate was tapped several times for
ensuring coating of samples to the bottom of the wells. 10 minutes incubation was given
at room temperature (21-25°C).
2. Washing of wells: after incubation plate was washed once using ELISA washer
3. Addition of antigen-peroxidase conjugate: 50 μl of diluted antigen peroxidase
conjugate was added to each well and side of well tapped several times to ensure proper
coating. Plate was incubated again for 10 minutes at room temperature (21- 25°C).
34
4. Washing of wells: After incubation plate was washed four times with the automated
ELISA plate washer.
5. Addition of substrate solution: Substrate Solution (50μl) was added to each well and
side of plate tapped several times to make sure the proper coating. Plate was incubated at
room temperature (21-25°C) for 15 minutes.
6. Addition of stop solution: 50 μl of stop solution was pipette into each well.
Immediately after addition of stop solution, change in color from blue to yellow was
observed. Side of the loaded assay plate was tapped several times for proper mixing.
7. Reading and recording the test result: After the addition of stop solution, the plate
was observed visually against the positive control and then on ELISA plate reader at
optical density (O.D.) reading wavelength 450nm. For the visual determination, any of
the test wells yielding colour development equal to the positive control serum was
considered positive and the wells showing colour development equal to or less than the
negative control serum was considered negative. Samples having O.D. greater than or
equal to that of positive control on microplate reader interpretation were considered
positive (Issel and Cook, 1993).
35

Sero-diagnosis of glanders
Recently evaluated ‘Rose Bengal Plate Agglutination Test’ (RBT) was used for the
serodiagnosis of glanders. Antigen for the test was prepared as described by Naureen et
al. (2007). Briefly, 2 outbreak isolates present in the Department of Clinical Medicine &
Surgery, University of Agriculture, Faisalabad and China 5 strain were used for this
purpose. These were suspended in sterile phosphate buffer saline (PBH: pH 6.4) after
being evaluated for purity. Further these were used to seed the Roux flasks filled with
layers of glycerol-dextrose agar and incubated for one week at 37°C. Purity of the
growths were checked through Gram staining and harvesting was performed by adding
100 mL of phenol-saline (0.05% phenol in 0.85% sodium chloride solution). After gentle
agitation, the organisms were killed in water bath at 100°C by heating overnight. By
using the spectrophotometer, bacterial cell concentrations adjustment at 65 x 109 /
milliliter was performed and colored with Rose Bengal dye (1%). Centrifugation was
performed at 5,000 g for 20 minutes and re-suspension of sediment was performed in
NaOH lactic acid buffer (50mM, pH 3.5).
The test were performed as described by Naureen et al. (2007) and considered positive in
the presence of distinct agglutination with appearance of small or large rose-colored
flakes with 50 to 100% clarification of liquid (2–4 crosses) and negative if scored less
than 2 crosses.
37
Plate-III Photograph showing the strong positive (++++) results of rose Bengal plate agglutination test for equine glanders
Sero-diagnosis of equine piroplasmosis
Commercial Babesia caballi and Babesia equi antibody test kits, cELISA (VMRD, Inc.,
Pullman, USA) were used for this purpose (Abdelkabir et al., 2001). Sample processing,
application and interpretation were performed by following guidelines as provided by
manufacturer:
Preparation
a. Warming up reagents: Serum samples, reagesnts and plates were brought to the room
temperature (21-25°C)
b. Preparation of controls and samples: Serum samples, positive and negative controls
were diluted 1:2 with serum diluting buffer in non antigen coated transfer plates. Positive
and negative controls were run in duplicate and triplicate respectively on each plate.
Position of controls as well as serum samples was recorded on the setup record sheet.
d. Preparation of primary antibody: 1X primary antibody solution was made by
diluting 1 part of the 100X primary antibody with 99 parts of antibody diluting buffer.
e. Preparation of secondary antibody-peroxidase conjugate: 1X secondary antibody-
peroxidase conjugate was formed by diluting 1 part of the 100X secondary antibody-
peroxidase conjugate with 99 parts of antibody diluting buffer.
f. Preparation of washing solution: 1 part of the 10X wash solution concentrate was
diluted with 9 parts of distilled water to make 1X solution.
Test Procedure
1. Loading controls and serum samples: 50 μl of diluted controls and serum samples
were transferred to antigen coated plate through multichannel pipettor according to the
setup record. Loaded assay plate was tapped several times to ensure the proper coating of
39
solutions to the bottom of plate. Plate was then incubated for 30 minute at room
temperature (21-25°C).
2. Washing of wells: After incubation plate was washed 3 times by using an automatic
washer.
3. Adding primary antibody: Diluted 50 μl (1X) primary antibody was added to each
well and side of the loaded assay plate was tapped to ensure the proper coating of wells.
30 minutes incubation was provided at room temperature (21-25°C).
4. Washing of wells: Plate was washed 3 times after incubation in an automatic washer
5. Addition of secondary antibody-peroxidase conjugate: A 50 μl of diluted 1X
secondary antibody peroxidase conjugate was added to each well and side of well was
tapped to ensure proper coating. Plate was incubated at room temperature (21-25° C) for
30 minutes.
6. Washing of wells: Three times washing was performed after incubation
7. Addition of substrate solution: 50 μl of substrate solution was pipette in to each well
and side of well was tapped to make sure the proper coating of each well. Fifteen minutes
incubation was provided at room temperature (21-25°C)
8. Addition of stop solution: Stop solution (50 μl) was added to each well and side of
the well was tapped to ensure the mixing.
9. Reading and recording the test results: Plate reader was set at optical density (O.D.)
reading wavelength of 630 nm and plate was read.
10. Test Interpretation: Test was validated upon the points recommended by the
manufacturer and Percent inhibition (%I) was calculated by using the following formula:
Percent Inhibition (% I): = 100 - [(Sample O.D. x 100) ÷ (Mean Negative Control O.D.)]
Test sample producing ≥ 40% inhibition was declared positive and samples producing <
40% inhibition were considered negative.
40
Plate-IV Photograph showing the micro-titration plate of commercial cELISA (VMRD, Inc., USA) used for the serodiagnosis of equine piroplasmosis during the study: Visual Determination
Data analysis
Epidemiological data generated was analyzed by using the Epiinfo™ and other available
epidemiological softwares (WINPEPI; version 6.8 by J.H. Abramson; Survey Toolbox version
1.04 by Angus Cameron) to investigate different epidemiological attributes of these diseases.
Chi-square testing was performed to find out significant difference among sex, age and locations
based prevalence of the selected diseases. Univariate analysis was performed to calculate Odds
ratio (OR) for different determinants of disease. The associations between the outcome response
variables (sero-prevalence of EIA, glanders and piroplasmosis) and explainatory variables
(informations recorded through proforma) were estimated using binary logistic regression (IBM
SPSS Statistics 17.0 for Windows®, IBM Corporation, Route 100 Somers, New York, USA).
Individual animal was kept as unit of analysis for determining significance of association.
Outcome variables were dichotomized (0=negative and 1=positive) and response variables were
dichotomized or categorized wherever applicable. Bivariable screening was conducted and
variables yielding significant association at less than or equal 0.20 Wald P value were further
used in binary logistic regression model. A backward stepwise model was constructed. All
variables found significant in the initial screening model were kept at start and then based upon
likelihood ratio tests they were removed one by one. Hosmer-Lemeshow test, the Negelkerke R
square test and observed versus predicted values (Residual statistics) to identify outliers at 0.5
cut off point were used to assess the fit of the final models (Urdaz-Rodrihuez et al., 2009).
CHAPTER-IV (RESULTS)
CHAPTER-IV
RESULTS
For the current study 430 blood and serum samples were collected from 5 draught equine
populated urban areas (3 from central and 2 from southern Punjab) of Punjab to make the study
more diverse in terms of geography and climate. Geographic and climatic details of the studied
areas have been summarized in Table-1.
4.1. Geographic and Climatic Description of Selected Areas
Historical city of Lahore is the capital of province Punjab and the second largest city in Pakistan
with estimated population of 6.4 million people and population density of 3,566/km2. The city
lies 711 feet above the sea level with total land area of 404 km2 between 31°15′ and 31°45′ North
latitude and 74°01′ and 74°39′ East longitude. Weather of Lahore touches both extremes with
average maximum and minimum temperature in summer (May–July) can reach 36°C and 25°C
respectively, while in winter (December–February) temperature can drop to -1°C with average
maximum and minimum temperature of 19°C and 7°C respectively. The average annual rainfall
in Lahore is 452 mm.
With an estimated population of 2.7 million people the industrial city of Faisalabad is the third
largest city in Pakistan. Geographical coordinates of the city are 31°25′ North latitude and 73°05′
East longitude at an elevation of 605 feet above sea level. Climate of Faisalabad is extremely hot
in summer (mean maximum and minimum temperature, 39 and 27°C respectively) that may

reach up to 50°C and at times drop below 0°C in winter (mean maximum temperature and mean
minimum temperature, 21°C and 6°C respectively) with average annual rainfall of 346 mm.
Gujranwala is the fifth largest city of Pakistan (estimated population 1.4 millions) and is located
700 feet above sea level at 32.16° North latitude and 74.18° East longitude. Climatic conditions
are extreme both in summer and winter with annual mean maximum temperatures of 30.9°C and
18.8°C respectively. Gujranwala receives 325 mm average annual rainfall.
One of the oldest inhabited cities of the world, ‘Multan’ with estimated population of 1.4
million people is the largest city in southern Punjab and sixth largest metropolis of Pakistan. The
city is situated between 30.12° North latitude and 71.27° East longitude about 710 feet above the
sea level. The city has extreme climatic conditions and temperature in summer can sear up to
52°C (mean maximum temperature 40.3°C) and fall up to -2°C (mean minimum temperature
5.8°C) in winter. The average annual rainfall in Multan is 127 mm.
City of Bahawalpur (Capital of former Princely State of Bahawalpur) lies 370 feet above sea
level and is situated between 25.59° North latitude and 73.19° East longitude. Bahawalpur
receives average 187 mm of rainfall annually. Climate is extremely hot in summer (mean
maximum temperature 40C and mean minimum temperature 28C) and milder (mean maximum
temperature 22C and mean minimum temperature 8C) in winter.

Table-1 Geographic and climatic characteristics of the 5 draught equine populated urban areas of Punjab selected for sero-survey of equine infectious anemia, glanders and piroplasmosis
Metropolis Height from Sea Level (ft)
Geographic Coordinates Average Temperature
Av. Min.
Av. Max.
Av. Min.
Lahore 712 31.55 74.34 36 25 19 7
Faisalabad 600 31.40 73.04 39 27 21 6

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