sero-survey of equine infectious anemia, glanders and

SERO-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

In

Clinical Medicine & Surgery

By

MUHAMMAD HAMMAD HUSSAIN D.V.M., M.Sc. (Hons.) CMS

FACULTY OF VETERINARY SCIENCES UNIVERSITY OF AGRICULTURE

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

Chairman ___________________________ (Prof. Dr. Ghulam Muhammad)

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)

Comment [D1]: He is dean now???

ii

List of Table

S. No. Title of the Table Page No.

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 sero-prevalence 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

iii


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

iv


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

v


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

vi


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

vii

List of Figures

S. No. Title of the Figures Page No.

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

List of Plates

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

Title Page No.

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

Acknowledgement ----------- i

List of Table ----------- ii

List of Figures ----------- vii

List of Plates ----------- vii

Appendix ----------- vii

CHAPTERS TITLE PAGE

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 7

3 MATERIALS AND METHODS 28

4 RESULTS 42

5 DISCUSSION 117

6 SUMMARY 141

LITERATURE CITED 145 APPENDIX 168

________________________________________________

ABSTRACT

A cross-sectional epidemiological survey was conducted to check the seroprevalence of 3

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)

7

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).


8

II. Equine infectious anemia

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; Kirmizigȕl 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).


10

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.


12

Figure-1 Map showing global distribution of equine infectious anemia (EIA) during last six months (July – December) of 2008 (Anonymous, 2008)


13

III. Glanders

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


14

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).


16

III.b. Geographic distribution

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


18

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


20

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).


24

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


26

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)

28

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) / d2

Where:

n = required sample size

Pexp = expected prevalence

d = desired absolute percision


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 = total population

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

Information regarding owner’s particulars, animal’s details, cohorts, management, problem

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.


30

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

Horse

% of total selected draught

horse population

Samples Taken

Total Draught Donkey

% of selected draught donkey

population

Samples Taken

Total Draught

Mule

% of selected draught

mule population

Samples Taken

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


32

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

Plate-I Photograph showing the commercial ELISA kit (VMRD, Inc., USA) used for the serodiagnosis of equine infectious anemia Plate-II Photograph showing the 96 well microtitration plate after completing the assay ready for visual determination and interpretation through plate ELISA reader


36

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


38

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


41

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)

42

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


43

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.


44

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

Latitude Longitude Summer °C Winter °C Av. Max.

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

Gujranwala 744 32.16 74.19 38 24 21 6

Multan 710 30.18 71.47 40.3 23 27 5.8

Bahawalpur 370 29.37 71.68 40 28 30 14

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 (Anonymous, 2006)

Area Sampled Horse Mule Donkey Total

Faisalabad 6645 3046 44144 53835

Lahore 4596 721 17386 22703

Gujranwala 4612 1841 18964 25417

Multan 1940 954 19076 21970

Bahawalpur 1238 954 19076 21268

Total 19031 7516 118646 145193


45

4.2. Equine Population of Selected Areas

The total reported equine population providing draught power to the selected cities was 145,193

heads (Table-2). This population was comprised of horses (19031 heads), mules (7516 heads)

and donkeys (118646 heads) (Anonymous, 2006). Highest number of these equines dwelled in

Faisalabad (53,835 heads), followed by Gujranwala (25,417 heads), Lahore (22,703 heads),

Multan (21,970 heads) and Bahawalpur (21,268 heads). Donkey was the chief animal draught

power source in these areas as reported in Pakistan Livestock Census (Anonymous, 2006),

whose total population engaged in draught work in these areas was 118646 heads with highest

number reported in Faisalabad (44144 heads), followed by Multan and Bahawalpur (19076 heads

each), Gujranwala (18964 heads) and Lahore (17386 heads). According to Census (Anonymous,

2006), reported draught horses population in the study areas was 19031 heads (Table-2) which

was highest in Faisalabad (6645 heads), followed by Gujranwala (4612 heads), Lahore (4596),

Multan (1940 heads) and Bahawalpur (1238 heads). Total reported draught mule population of

targeted metropolises was 7516 heads. Highest number of mules engaged in draught work was

reported in Faisalabad (3046), followed by Gujranwala (1841 heads), Multan and Bahawalpur

(954 heads each) and Lahore (721 heads).

4.3. Demographic Characteristics of the Sampled Equine Population

4.3a. Locale and Equine Type Related Distribution of Samples

A total of 430 blood samples were collected from draught equines of areas under study (Table-

3). Depending upon the population proportion of each equine type, 332 donkeys, 33 mules and

65 horses were randomly sampled. Depending upon reported equine population of these areas


46

(Anonymous, 2006), 150 equines (123 donkeys, 9 mules and 18 horses) from Faisalabad, 69 (50

donkeys, 6 mules and 13 horses) from Lahore, 75 (55 donkeys, 6 mules and 14 horses) from

Gujranwala, 68 (52 donkeys, 6 mules and 10 horse) each from Multan and Bahawalpur were

randomly selected and bled for blood and serum samples.

4.3b. Sex Related Distribution of Samples

Out of 430 equines sampled, 295 (68.6%) were male and 135 (31.4%) belonged to female sex

(Table-4). Blood and serum samples from 25 male horses (6 from Multan, 5 from Bahawalpur, 5

from Faisalabad, 4 from Lahore and 5 from Gujranwala) and 40 mares (4 from Multan, 5 from

Bahawalpur, 13 from Faisalabad and 9 each from Lahore and Gujranwala) were randomly

collected. Eighteen (18) male (3 from Multan, 2 from Bahawalpur, 6 from Faisalabad, 3 from

Lahore and 4 from Gujranwala,) and 15 female (2 from Gujranwala, 4 from Bahawalpur and 3

each from Faisalabad, Lahore and Multan) mules were bled for blood and serum samples. Blood

and serum samples from 332 donkeys having following sexual distribution: 252 males (37 from

Multan, 40 from Bahawalpur, 98 from Faisalabad, 34 from Lahore and 43 from Gujranwala) and

80 females (15 from Multan, 12 each from Gujranwala and Bahawalpur, 25 from Faisalabad and

16 from Lahore) were collected.

4.3c. Age Related Distribution of Samples

Equines sampled (n=430) for the current study were divided into three age groups (i) less than

equal to 5 years of age (n=138) (ii) 6-10 years of age (n=195) and (iii) more than 10 years of age

(n=97) to determine the possible association of diseases under study with age of animals. Out of

65 horses sampled for the study, 11 were less than equal to 5 years of age, 29 were 6-10 years of


47

age and 25 horses were of more than ten years of age (Table-5). Distribution of sampled mule

population (n=33) according to age was 8, 18 and 7 respectively. Age related distribution of

sampled donkey population (n=332) was 119, 148 and 65 respectively. Area related age

distribution of equine samples is presented in Table-5.

4.4. Housing and Management Related Distribution of Samples

4.4a. Housing Pattern Adopted by Equine Owners of the Selected Locales

Analysis of housing pattern of sampled equine population (n=430) revealed the 46.3% of equines

were kept alone (199 heads), followed by 28.6% animals kept with other equine cohorts (123

heads) and 25.1% in mix herds (108 heads) with cattle, buffalo, sheep, goat, dogs and other

animals (Table-6). However, the difference observed in these housing practices among sampled

areas was statistically not significant, 2 (4df) =1.91, p =0.984.

4.4b. Watering Habits adopted by Equine Owners of the Selected Locales

Survey indicated that 320 (74.4%) equine owners provided water to their animals through

communal water troughs and shared buckets while at work. This trend was highest among the

equine owners of Lahore (82.6%), followed by Bahawalpur (76.5%), Faisalabad (74%),

Gujranwala (72%) and Multan (67.7%). However, this difference was not significant, 2 (4df)

=4.464, p=0.347. This pattern reversed when watering habit of owners at home was analyzed to

25.4% (109 heads) (Table-7) because most of the equines were kept alone. Use of common water

trough at home was recorded as 27.9% in Multan and Bahawalpur each followed by Lahore

(27.5%), Gujranwala (25.3%) and Faisalabad (22%). This difference regarding watering

practices at home was not significant between selected areas, 2 (4df) =1.031, p=0.905.


48

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

Location Horse Mule Donkey Total

Multan 10 6 52 68

Bahawalpur 10 6 52 68

Faisalabad 18 9 123 150

Lahore 13 6 50 69

Gujranwala 14 6 55 75

Total 65 33 332 430

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

Area Sampled Horses Mules Donkeys

Male Female Male Female Male Female

Multan 6 4 3 3 37 15

Bahawalpur 5 5 2 4 40 12

Faisalabad 5 13 6 3 98 25

Lahore 4 9 3 3 34 16

Gujranwala 5 9 4 2 43 12

Total 25 40 18 15 252 80


49

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

Area

Sampled

Horses

(n=65)

Mules

(n=33)

Donkeys

(n=332)

< 5 Y 6-10 Y > 10Y < 5 Y 6-10 Y > 10Y < 5 Y 6-10 Y > 10 Y

Multan 0 8 2 2 3 1 21 26 5

Bahawalpur 1 7 2 2 4 0 12 23 17

Faisalabad 4 5 9 1 5 3 45 51 27

Lahore 5 4 4 1 4 1 24 20 6

Gujranwala 1 5 8 2 2 2 17 28 10

Total 11 29 25 8 18 7 119 148 65


50

4.4c. Tick, Mosquito and Fly Control Measures Adopted by Equine Owners

Data related to tick, mosquito and fly control measures (Table-8) adopted by owners of sampled

equine population (n=430) revealed that only 25.8% (n=111) owners used tick control measures

in their routine practice and 39.8% (n=171) practiced some sort of fly and mosquito control

methods (Table-8). Whereas 148 equine owners did not employ any tick, mosquito and fly

control measures during their routine practice. The trend of using tick control measures was

significantly different, 2 (4df) =17.085, p < 0.01 in the equine owners of Multan (39.7%) and

Bahawalpur (36.8%) than Gujranwala (22.7%), Faisalabad (20.7%) and Lahore (15.9%).

Similarly, significantly different (2, 4df =29.109, p < 0.01) number of equine owners practiced

fly and mosquito control measures in Multan (61.8%) and Bahawalpur (54.4%) than equine

owners of Faisalabad (32.7%), Lahore (31.9%) and Gujranwala (28%).

4.5. Equine Infectious Anemia

In current study 430 blood and serum samples from 65 horses, 33 mules and 332 donkeys were

subjected to enzyme linked immunosorbent assay (ELISA) testing for detecting antibodies

against equine infectious anemia virus in 5 draught equine populated urban areas of Punjab.

However, all equines were found sero-negative for antibodies against EIA virus.


51

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

Location

Housing Pattern

Kept with non equine cohorts

(%)

Kept Alone (%) Kept with Equines (%)

Faisalabada 39 (26.0) 68 (45.3) 43 (28.7)

Lahorea 20 (28.9) 29 (42.0) 20 (28.9)

Gujranwalaa 19 (25.3) 35 (46.7) 21 (28.0)

Multana 16 (23.5) 34 (50.0) 18 (26.5)

Bahawalpura 14 (20.6) 33 (48.5) 21 (30.9)

Total 108 (25.1) 199 (46.3) 123 (28.6)

Values with same superscript are not significantly different (P > 0.05), 2 (4 df) =1.91, p = 0.98

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

Values with same superscript are not significantly different (P > 0.05), 2(4 df)=4.46, p = 0.347

LOCATION

Water at Work Water at Home

Communal water trough

(%)

Separate Bucket (%)

Communal water trough

(%)

Separate Bucket (%)

Multana 46 (67.7) 22 (32.3) 19 (27.9) 49 (72.1)

Bahawalpura 52 (76.5) 16 (23.5) 19 (27.9) 49 (72.1)

Faisalabada 111 (74.0) 39 (26.0) 33 (22.0) 117 (78.0)

Lahorea 57 (82.6) 12 (17.4) 19 (27.5) 50 (72.5)

Gujranwalaa 54 (72.0) 21 (28.0) 19 (25.3) 56 (74.7)

Total 320 (74.4) 110 (25.6) 109 (25.4) 321 (74.7)


52

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.

Values with different superscript whole number differ significantly (P < 0.01)

Values with different superscript numbers differ significantly (P < 0.01)

Location Owners using tick Control methods (%)

Owners using fly & mosquito Control methods (%)

Multan 27 (39.7)a 42 (61.8)a

Bahawalpur 25 (36.8)a 37 (54.4)a

Faisalabad 31 (20.7)b 49 (32.7)b

Lahore 11 (15.9)b 22 (31.9)b

Gujranwala 17 (22.7)b 21 (28.0)b

Total 111 (25.8) 171(39.8)

Chi-Square 2 (4) 17.08 P = 0.002 2 (4) 29.11 P = 0.000


53

4.6. Comparison of hematological findings in equines of 5 different areas

Data regarding hematological values of equines from 5 different areas is presented in Table-9.

Mean white blood cell (WBC) count was significantly higher (P < 0.05) in equines of

Bahawalpur (7.48 + 1.85) and Multan (7.25 + 2.11) than Faisalabad (6.38 + 2.12), Gujranwala

(6.81 + 1.89) and Lahore (6.39 + 1.94). Significantly lower (P < 0.05) mean red blood cell

(RBC) count was observed in equines of Lahore (5.42 + 1.36) as compared to the Bahawalpur

(6.11 + 150), Multan (5.99 + 1.94), Gujranwala (5.89 + 1.77) and Faisalabad (5.71 + 1.47).

There was not a significant difference (P > 0.05) between the values of mean hemoglobin (Hb)

concentration among equines of Lahore (9.87 + 2.01), Gujranwala (9.56 + 1.92), Faisalabad

(9.31 + 1.90) and Multan (9.30 + 2.36). However, these values were significantly higher (P <

0.05) than mean Hb concentration value in equines of Bahawalpur (7.35 + 1.77).

Highest value for mean packed cell volume (PCV) was recorded in blood samples of equines

from Bahawalpur (32.45 + 6.26) that was significantly higher (P < 0.05) than mean PCV values

observed in equines of Gujranwala (29.42 + 4.41), Faisalabad (29.36 + 5.94) and Multan (28.11

+ 5.74). Significantly lower (P < 0.05) mean hematocrit value (22.77 + 5.07) was recorded for

the equines of Lahore. There was a not significant difference (P > 0.05) in the values of mean

corpuscular volume (MCV) among equines of Faisalabad (54.64 + 17.30), Lahore (58.55 +

18.56), Gujranwala (54.68 + 20.61) and Bahawalpur (55.70 + 16.16). However, these values

were significantly higher (P < 0.05) than the equines of Multan (50.76 + 15.86). Significantly

lower (P < 0.05) value regarding mean corpuscular hemoglobin (MCH) was recorded in equines

of Multan (16.74 + 5.71) as compared to equines of Lahore (19.21 + 5.85), Faisalabad (17.60 +


54

6.36) and Gujranwala (17.33 + 5.15). However, this MCH value (16.74 + 5.71) was significantly

higher (P < 0.05) than the equines of Bahawalpur (12.34 + 5.34). Mean corpuscular hemoglobin

concentrations (MCHC) were not significantly different (P > 0.05) among equines of Lahore

(34.16 + 9.40), Multan (33.52 + 7.86), Gujranwala (33.06 + 7.59) and Faisalabad (32.79 + 8.72)

but were significantly higher (P < 0.05) than equines of Bahawalpur (22.40 + 8.21).

4.6a. Comparison of hematological findings among horses, mules and donkeys

Analysis of data regarding the hematological findings in horses, mules and donkeys is presented

in Table-10. Mean white blood (WBC) counts were decreased in horses (6.65 + 2.16), mules

(6.82 + 2.12) and donkeys (6.77 + 1.91). These values were significantly different (P < 0.05)

from reference values for mules (9.40 + 2.89) and donkeys (9.75 + 3.29) but in horses this

difference was not significant (P > 0.05) from reference value (7.18 + 2.87). Mean red blood cell

(RBC) counts were not significantly different (P > 0.05) than respective reference values for

horses (5.28 + 1.18), mules (5.77 + 1.68) and donkeys (5.78 + 1.56). However, mean RBC

counts were significantly lower (P < 0.05) in horses than mules and donkeys (Table-10).

Significantly lower (P < 0.05) mean hemoglobin (Hb) concentration was observed in horses

(9.62 + 2.18) than reference value (10.93 + 1.81). Mean Hb concentrations were not significantly

lower (P > 0.05) in mules (9.24 + 2.05) than reference values (10.10 + 0.73). In donkeys mean

hemoglobin concentration (9.25 + 1.82) was not significantly higher (P > 0.05) than reference

value (9.01 + 1.13). There was not a significant difference (P > 0.05) among equines regarding

these values. Mean packet cell volume values for horses (28.59 + 5.30), mules (28.86 + 5.36)

and donkeys (29.22 + 5.48) were significantly lower (P < 0.05) than their respective reference


55

values (37.75 + 4.57, 34.37 + 4.13 and 32.42 + 4.38 respectively). However, difference among 3

equine species was statistically not significant (P > 0.05).

There was a not significant difference (P > 0.05) regarding mean corpuscular volume (MCV)

among horses (56.30 + 14.65), mules (53.48 + 16.34) and donkeys (54.30 + 18.37). These values

were not significantly (P > 0.05) lower than their respective reference values (Table-10). Values

regarding mean corpuscular hemoglobin (MCV) indicated a not significant difference (P > 0.05)

between reference and observed values for all 3 equine species. MCV was significantly higher (P

< 0.05) in horses (19.82 + 7.28) than donkeys (17.09 + 5.50) and mules (16.93 + 5.29). There

was not a significant difference (P > 0.05) among equines regarding values of mean corpuscular

hemoglobin concentration (MCHC). These values were not significantly higher (P > 0.05) than

reference values in horses (34.17 + 7.57), mules (32.67 + 7.80) and donkeys (30.56 + 8.62).

4.6b. Comparison of Hematological findings on the basis of sex of equines

Analysis of hematological data on the basis of sex of equines is presented in Table-11. There was

a not significant difference (P > 0.05) in mean white blood cell (WBC) counts between male

(6.75 + 2.03) and female (6.86 + 2.00) equines. Similarly mean red blood (RBC) counts were

numerically but statistically not significantly higher (P > 0.05) in males (5.71 + 1.59) as

compared to females (5.64 + 1.61). A significant difference (P < 0.05) was observed between

males (9.25 + 2.01) and females (9.76 + 2.14) regarding mean hemoglobin (Hb) concentration.

Numerically higher value of mean packed cell volume (PCV) was recorded in female equines

(29.27 + 4.91) than males (29.08 + 5.74). However, this difference was statistically not

significant (P > 0.05). There was not a significant difference (P > 0.05) between male and female

equines regarding values of mean corpuscular volume (MCV), mean corpuscular hemoglobin


56

(MCH) and mean corpuscular hemoglobin concentration (MCHC). In female equines MCV

(55.80 + 17.26), MCH (18.52 + 6.11) and MCHC (34.02 + 8.49) values were numerically higher

as compared to MCV (54.44 + 17.64), MCH (17.39 + 5.93) and MCHC (32.74 + 8.53) values in

males.

4.6c. Comparison of hematological findings on the basis of age of equines

Data regarding hematological examination in different age groups of equines is presented in

Table-12. Mean white blood cell (WBC) count was significantly higher (P < 0.05) in equines of

less than equal to 5 years (8.23 + 1.78) of age (group-I) followed by 6.65 + 1.43 and 4.94 + 1.68

in group-II (< 10 years) and III (> 10years) respectively. Similar pattern was observed regarding

mean red blood cell (RBC) counts with significantly higher (P < 0.05) count was found in

equines of group-I (6.38 + 1.65) followed by group-II (5.73 + 1.45) and III (4.74 + 1.26). Mean

hemoglobin concentration was significantly higher (P < 0.05) in group-I (9.66 + 1.98) and II

(9.43 + 1.91) as compared to group-III (8.67 + 1.99). There was not a significant difference (P >

0.05) among all three age groups regarding mean hematocrit (PCV) values with numerically

higher PCV was recorded in group-I (29.62 + 5.82) followed by group-II (29.07 + 5.63) and III

(28.58 + 5.29). There was a significant difference (P < 0.05) among values regarding mean

corpuscular volume (MCV) among equines of 3 groups with highest value recorded in group-III

(64.66 + 23.33) followed by group-II (53.20 + 14.14) and I (49.61 + 16.25). Similar pattern was

recorded regarding values of mean corpuscular hemoglobin (MCH) where significantly higher (P

< 0.05) MCH was recorded in group-III (19.89 + 7.12) followed by group-II (17.34 + 5.17) and I

(16.14 + 5.50). Mean corpuscular hemoglobin concentration was not significantly (P > 0.05)

different in group-I (33.70 + 9.06) and II (33.41 + 8.51) but these were significantly higher (P <

0.05) from group-III (30.90 + 7.17).


57

Table-9 Hematological values recorded in equines (n = 430) surveyed for sero-prevalence of equine infectious anemia, glanders and piroplasmosis from 5 draught equine populated urban areas of Punjab

Areas WBC/10³μL RBC/106 μL Hb (g/dL) HcT (%) MCV (fl) MCH (pg) MCHC (g/dL)

Faisalabad 6.83 + 2.12b 5.71 + 1.47a 9.31 + 1.90a 29.36 + 5.94b 54.64+ 17.30a 17.60 + 6.36a 32.79 + 8.72a

Lahore 6.39 + 1.94b 5.42 + 1.36b 9.87 + 2.01a 22.77 + 5.07c 58.55 + 18.56a 19.21 + 5.85a 34.16 + 9.40a

Gujranwala 6.81 + 1.89b 5.89 + 1.77a 9.56 + 1.92a 29.42 + 4.41b 54.68 + 20.61a 17.33 + 5.15a 33.06 + 7.59a

Multan 7.25 + 2.11a 5.99 + 1.94a 9.30 + 2.36a 28.11 + 5.74b 50.76 + 15.86b 16.74 + 5.71b 33.52 + 7.86a

Bahawalpur 7.48 + 1.85a 6.11 + 1.50a 7.35 + 1.77b 32.45 +6.26a 55.70 + 16.16a 12.34 + 5.34c 22.40 + 8.21b

WBC = White blood cell RBC = Red blood cell Hb = Hemoglobin HcT = Hematocrit

MCV = Mean Corpuscular Volume MCH = Mean Corpuscular Hemoglobin MCHC = Mean Corpuscular Hemoglobin Concentration

Values with different alphabetic superscript s are significantly (P < 0.05) different from each other


58

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 piroplasmosis in 5 draught equine populated urban areas of Punjab

Animal

WBC/10³μL RBC/106 μL Hb (g/dL) HcT (%) MCV (fl) MCH (pg) MCHC (g/dL)

Reference Value

7.18 + 2.87 5.93 + 2.81 10.93 + 1.81 37.75 + 4.57 63.66 18.43 28.95

Horse

6.65 + 2.16NSa 5.28 + 1.18NSb 9.62 + 2.18Sa 28.59 + 5.30Sa 56.30 + 14.65a 19.82 + 7.28a 34.17 + 7.57

Reference Value

9.40 + 2.89 5.74 + 2.62 10.10 + 0.73 34.37 + 4.13 59.88 17.59 29.38

Mule

6.82 + 2.12Sa 5.77 + 1.68NSa 9.24 + 2.05NSa 28.86 + 5.36Sa 53.48 + 16.34a 16.93 + 5.29b 32.67 + 7.80

Reference Value

9.75 + 3.29 5.88 + 2.65 9.01 + 1.13 32.42 + 4.38 55.13 15.32 27.79

Donkey

6.77+ 1.91Sa 5.78 + 1.56NSa 9.25 + 1.82NSa 29.22 + 5.48Sa 54.30 + 18.37a 17.09 + 5.50b 30.56 + 8.62



Values with different superscript alphabetic are significantly (P < 0.05) different from each other

Values with ‘NS’ superscript are not significantly (P > 0.05) different from their respective reference values

Values with ‘S’ superscript are significantly (P < 0.05) different from their respective reference values


59

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

Animal


Male 6.75 + 2.03a 5.71 + 1.59a 9.25 + 2.01b 29.08 + 5.74a 54.44 + 17.64a 17.39 + 5.93a 32.74 + 8.53a

Female 6.86 + 2.00a 5.64 + 1.61a 9.76 + 2.14a 29.27 + 4.91a 55.80 + 17.26a 18.52 + 6.11a 34.02 + 8.49a

Values with different superscript are significantly (P < 0.05) different from each other

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

Animal


< 5 years 8.23 + 1.78a 6.38 + 1.65a 9.66 + 1.98a 29.62 + 5.82a 49.61 + 16.25c 16.14 + 5.50c 33.70 + 9.06a

< 10 years

6.65 + 1.43b 5.73 + 1.45b 9.43 + 1.91a 29.07 + 5.63a 53.20 + 14.14b 17.34 + 5.17b 33.41 + 8.51a

> 10 years

4.94 + 1.68c 4.74 + 1.26c 8.67 + 1.99b 28.58 + 5.29a 64.66 + 23.33a 19.89 + 7.12a 30.90 + 7.17b

WBC = White blood cell RBC = Red blood cell Hb = Hemoglobin HcT = Hematocrit MCV = Mean Corpuscular Volume MCH = Mean Corpuscular Hemoglobin MCHC = Mean Corpuscular Hemoglobin Concentration Values with different superscript are significantly (P < 0.05) different from each other


60

4.7. Equine Glanders

Rose Bengal Plate Agglutination (RBT) test based screening of the serum samples (n = 430)

revealed that overall seroprevalence of glanders (Table-13) in equine population of studied areas

was 7.9% (n = 34).

4.7a. Area Related Seroprevalence of Equine Glanders

Numerically higher seroprevalence of equine glanders (Table-13) was found in draught equines

of Lahore 11.6% (n=8) followed by Bahawalpur (10.3%, n=7), Multan (7.3%, n=5), Faisalabad

(6.7%, n=10) and Gujranwala (5.3%, n=4). However, this difference in prevalence of glanders in

studied areas was not statistically significant, 2 (4df) =2.84, P=0.584.

4.7b. Clinical Signs Observed in RBT Positive Equines

Analysis of data regarding clinical signs found in RBT positive equines (Table-14) indicated that

pale and congested mucous membranes (58.8%) was the most frequent clinical finding followed

by loss of stamina (41.2%), fever (38.2%), anorexia (38.2%), cough (29.4%), lymph node

swelling (21.6%), nasal discharge (14.7%) and dyspnea (8.8%). In one male donkey orchitis

(2.9%) was also observed.

4.7c. Seroprevalence of Glanders in 3 Different Species of Equids

Species related analysis of data indicated (Table-13) that prevalence of glanders was

significantly different (P<0.01) in horses (16.92%, n = 11) and mules (12.12%, n = 4) than

donkeys (5.72%, n=19), 2 (2df) =10.23, P=0.006. Among horses of five draught populated


61

areas, prevalence of glanders was highest in horses of Lahore (23.1%) followed by Multan and

Bahawalpur (20% each), Faisalabad (16.7%) and Gujranwala (7.1%). Prevalence of glanders in

mules of Lahore, Bahawalpur and Multan was 16.7%. One mule serum from Faisalabad

(prevalence 11.1%) gave positive RBT reaction. No mule of Gujranwala was found positive for

glanders. Four donkeys (8.0%) from Lahore were found seropositive for glanders followed by 4

(7.7%) from Bahawalpur, 2 (3.8%) from Multan, 6 (4.9%) from Faisalabad and 3 (5.4%) from

Gujranwala. There was no significant difference (P>0.05) among horses, mules and donkeys

with in different metropolises regarding sero-prevalence of glanders.

4.7d. Age Related Seroprevalence of Equine Glanders

Numerically higher but statistically non-significant, 2 (2df) =1.47, P=0.477 seroprevalence of

glanders (Table-15) was found in equines belonging to group-2 (6-10Y) i.e. 9.2% (n=18),

followed by 11 equines (7.9%) in group-1 (less than equal to 5 Y) and 5.2% (n=5) in animals of

age group-3 (more than 10Y). Similar seroprevalence pattern was recorded in horses where 7

animals (24.1%) were found sero-reactive in group-2 followed by 2 young horses (18.2%) in

group-1 and 2 old horses (8.0%) in group-3. Higher seroprevalence of glanders was noted in

mules belonging to age group-3 i.e. 14.3% (n=1) followed by 12.5% in group-1 (n=1) and 11.1%

in group-2 (n=2). Prevalence of glanders was numerically higher (6.7%) in donkeys of group-1,

where 8 animals were found sero-reactive followed by 6.1% (n=9) in group-2 and 3.1% (n=2) in

age group-3.


62

4.7e. Sex Related seroprevalence of Equine Glanders

Significantly higher seroprevalence (12.6%) of equine glanders (Table-16) was found in female

equines (n=135) as compared to male (n=295) animals where it was 5.8%, 2 (1df)

=5.94, P=0.014. Out of 65 horses (25 male and 40 female), 20% (n=8) female and 12% (n=3)

male serum samples were positive for RBT. Seroprevalence of glanders in male mules (n=18)

was higher 16.7% (n=3) as compared to females (6.7%, n=1). Eleven out of 252 male donkeys

(4.4%) were tested RBT positive and prevalence of glanders in female donkeys was 10% (n=8).

However, these differences in seroprevalence of glanders regarding sex of equines within each

species were statistically not significant (P>0.05).

4.7f. Hematological analysis of the RBT positive equine blood samples

Hematological analysis of the equines found seropositive for glanders indicated that there was

not significant (P > 0.05) increase (8.15 + 2.77) in the mean leukocyte counts in horses (n = 11)

than their respected reference value (Table-17). Whereas in mules (n =4) a not significant (P >

0.05) decrease (8.27 + 3.23) than reference white blood cell (WBC) count (9.40 + 2.89) was

observed. Significantly lower (P < 0.05) mean WBC count (7.52 + 1.80) than normal reference

value was observed in Rose Bengal plate agglutination test (RBT) positive donkeys (n =19).

However, these values were not significantly (P > 0.05) different among horses, mules and

donkeys.

In RBT positive horses mean red blood cell (RBC) count (4.16 + 0.62) was significantly lower

(P < 0.05) than reference value (5.93 + 2.81). This difference was also observed in RBC counts

of RBT positive donkeys where mean RBC count (4.10 + 0.57) was significantly (P < 0.05)

lower than the reference value (5.88 + 2.65). Mean RBC count was numerically lower (4.76 +


63

1.38) in sero-reactive mules than reference value (5.74 + 2.62). There wasn’t a significant

difference (P > 0.05) among horses, mules and donkeys regarding these counts.

Mean hemoglobin (Hb) concentration was found numerically higher in horses (11.70 + 1.73),

mules (10.45 + 2.27) and donkeys (10.57 + 1.47) than their respective reference values (Table-

17). However, this difference in mean Hb concentration among horses, mules and donkeys was

statistically not significant (P > 0.05).

Mean packed cell volume (PCV) in RBT positive horses was statistically not significantly (P >

0.05) lower (29.29 + 4.57) than normal value (37.75 + 4.57). This was also reflected by the

mean hematocrit value of donkeys (31.51 + 4.68) and mules (30.28 + 3.77). There wasn’t a

significant difference (P > 0.05) in mean PCV values among different equines. On the basis of

erythrocytic indices (Table-17) macrocytic hyperchromic type of anemia was observed in RBT

positive horses (n = 11), mules (n =4) and donkeys (n =19).

4.7g. Prevalence of equine glanders with reference to housing pattern of equines

Data regarding housing pattern of equines (Table-18) sampled for seroprevalence of glanders

revealed 16 (47.1%) out of 34 seropositive animals were kept alone, while 9 (26.5%) equines

were kept with equine cohorts and 9 (26.5%) were managed with other animals (small and large

ruminants, dogs etc.). This prevalence was higher in equines kept in mix herds (9.1%) followed

by animals kept alone (8.7%) and managed with equine cohorts (7.9%). However, housing was

not significantly associated with seropositivity, 2 (2df) =0.11, P=0.94.

4.7h. Prevalence of glanders with reference to watering habits of the draught equine owners


64

Analysis of the watering methods adopted by owners (Table-19) of RBT positive equines (n=34)

revealed that 30 (88.2%) animals were given water through the communal water troughs either at

work or home. Only 4 (11.8%) positive equines were given water through separate buckets at

work and home. Equines drinking water through communal water toughs were more likely to be

seropositive for glanders (OR=2.71, 95% CI 0.94-7.83) as compared to equines given water

through separate buckets. However, this difference was not significant, 2 (1df) =3.6, P=0.058.

4.6i. Multivariable analysis for the equine glanders

In total 6 variables were used in initial bivariable screening and out of these only equine species

(P=0.006), sex (P=0.014) and watering methods adopted by equine owners (P=0.058) were

significantly associated with high prevalence of glanders (Wald P < 0.2). Metropolis (p=0.58),

age (0.477) and housing (0.774) were not significantly associated with sero-prevalence of

glanders (Wald P>0.2) (Table-20). All variables yielding Wald P < 0.2 were entered into a

multivariable regression model which resulted in no variable remaining significantly associated

with the sero-prevalence of glanders (P>0.05) except equine species and therefore, no

multivariable model was left to present. However, when equine species was removed from a

biological perspective and model was built by keeping sex and watering together, the results

showed being a female equine (OR=2.3, 95% CI=1.13-4.69) and drinking water at communal

water trough (OR=2.6, 95% CI=0.90-7.69) had more chances of contracting glanders (Table-21).

Based upon Hosmer-Lemeshow test and the Negelkerke R Square this seemed to be reasonably

good fit model to the data (Chi-square=4.244. df=2, P=0.120 and 0.051 respectively). However,

this assumption was not found to be strictly significant in statistical terms.


65

Table-13 Comparative Seroprevalence of equine glanders in 5 draught equine populated urban areas of Punjab

Location Samples done

Horses

(Prev. %)

Mules

(Prev. %)

Donkey

(Prev. %) Total (Prev. %)

Multana 68 (10H, 6M, 52D) 2 (20.0)a 1 (16.7)a 2 (3.8)a 5 (7.3)a

Bahawalpura 68 (10H, 6M, 52D) 2 (20.0)a 1 (16.7)a 4 (7.7)a 7 (10.3)a

Faisalabada 150 (18H, 9M, 123D) 3 (16.7)a 1 (11.1)a 6 (4.9)a 10 (6.7)a

Lahorea 69 (13H, 6M, 50D) 3 (23.1)a 1 (16.7)a 4 (8.0)a 8 (11.6)a

Gujranwalaa 75 (14H, 6M, 55D) 1 (7.1)a 0 (0.0)a 3 (5.5)a 4 (5.3)a

Totala 430 (65H, 33M, 332D) 11 (16.9)1 4 (12.1)1 19 (5.7)2 34 (7.9%)

H = Horse M = Mule D = Donkey

Values with similar superscript alphabets in a column are not significantly (P > 0.05) different

Values with different superscript whole number in a row differ significantly, 2 (2df) =10.23, P=0.006


66

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

Symptoms

Frequency of Clinical Sign

Percentage (%)

Fever 13 38.2

Mucous Membrane (Pale, Congested) 20 58.8

Anorexia 13 38.2

Respiratory Signs

Nasal Discharge 5 14.7

Cough 10 29.4

Dyspnea 3 8.8

Stamina Loss 14 41.2

Lymph Node Swelling 8 21.6

Others

Orchitis 1 2.9


67

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

Values with similar superscript alphabets in a row are not significantly (P > 0.05) different, 2(2, n=430) = 1.47, P= 0.477

Equine

Samples Taken

(age group distribution)

Age Group (Prev. %)

< 5 Y

(n= 141)

< 10 Y

(n = 193)

> 10Y

(n = 96)

Horses 65 (11,29,25) 2 (18.2)a 7 (24.1)a 2 (8.0)a

Mules 33 (8,18,7) 1 (12.5)a 2 (11.1)a 1 (14.3)a

Donkeys 332 (119,148,65) 8 (6.7)a 9 (6.1)a 2 (3.1)a

Total 430 (138, 195, 97) 11 (7.9)a 18 (9.2)a 5 (5.2)a


68

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

Area Sampled Samples Taken Horses (Prev.%) Mules (Prev.%) Donkeys (Prev.%)


Multan H (6♂, 4♀)

M (3♂, 3♀)

D (37♂, 15♀)

1 (16.7) 1 (25.0) 0 (0.0) 1 (33.3) 1 (2.7) 1 (6.7)

Bahawalpur H (5♂, 5♀)

M (2♂, 4♀)

D (40♂, 12♀)

1 (20.0) 1 (20.0) 1 (50.0) 0 (0.0) 3 (7.5) 1 (8.3)

Faisalabad H (5♂, 13♀)

M (6♂, 3♀)

D (98♂, 25♀)

1 (20.0) 2 (15.4) 1 (16.7) 0 (0.0) 3 (3.1) 3 (12.0)

Lahore H (4♂, 9♀)

M (3♂, 3♀)

D (34♂, 16♀)

0 (0.0) 3 (33.3) 1 (33.3) 0 (0.0) 3 (8.8) 1 (6.3)

Gujranwala H (5♂,9♀)

M (4♂, 2♀)

D (43♂, 12♀)

0 (0.0) 1 (11.1) 0 (0.0) 0 (0.0) 1 (2.3) 2 (16.7)

Total H (25♂, 40♀)

M (18♂, 15♀)

D (252♂, 80♀)

3 (12.0)a 8 (20.0)a 3 (16.7)a 1 (6.7)a 11 (4.4)a 8 (10.0)a

Values with similar superscript alphabets in a row are not significantly (P > 0.05) different

Overall prevalence between two sexes differ significantly (P<0.05), 2 (1, n=430) = 5.94, P=

0.014


69

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

Animal


Reference Value

7.18 + 2.87 5.93 + 2.81 10.93 + 1.81 37.75 + 4.57 63.66 18.43 28.95

Horse (n = 11)

8.15 + 2.77NSa 4.16 + 0.62sa 11.70 + 1.73NSa 29.29 + 4.75HSa 71.74 + 14.59NSa 28.42 + 4.39NSa 40.87 + 9.47NSa

Reference Value

9.40 + 2.89 5.74 + 2.62 10.10 + 0.73 34.37 + 4.13 59.88 17.59 29.38

Mule (n = 4)

8.27 + 3.23NSa 4.76 + 1.38NSa 10.45 + 2.27NSa 30.28 + 3.77NSa 68.83 + 26.33NSa 24.02 + 10.33NSa 34.82 + 8.53NSa

Reference Value

9.75 + 3.29 5.88 + 2.65 9.01 + 1.13 32.42 + 4.38 55.13 15.32 27.79

Donkey (n = 19)

7.52 + 1.80Sa 4.10 + 0.57Sa 10.57 + 1.47HSa 31.51 + 4.68NSa 77.79 + 13.52NSa 26.24 + 5.07NSa 34.03 + 5.36NSa

Total (n = 34)

7.81 + 2.27 4.20 + 0.71 10.92 + 1.69 30.65 + 4.60 74.78 + 15.49 26.68 + 5.62 36.33 + 7.72



Values with ‘NS’ superscript are non significantly (P > 0.05) different from the reference value Values with ‘S’ superscript are significantly (P < 0.05) different from the reference value Values with same alphabetic superscript are non significantly (P > 0.05) different from each other


70

Table-18 Housing pattern observed in Rose Bengal Plate Agglutination Test (RBT) positive (n=34) and negative (n=396) equines surveyed for the prevalence of glanders in 5 draught equine populated urban areas of Punjab

Region

RBT Positive Equines Housing Pattern RBT Negative Equine Housing Pattern

Alone With Equine Others Total Alone With Equine Others Total

Multan 1 2 2 5 33 16 14 63

Bahawalpur 5 1 1 7 28 20 13 61

Faisalabad 6 2 2 10 62 41 37 140

Lahore 2 3 3 8 27 17 17 61

Gujranwala 2 1 1 4 33 20 18 71

Total 16 9 9 34 183 114 99 396

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

Region

RBT Positive Equines RBT Negative equines

Communal Water Troughs

Separate Watering Total

Communal Water Troughs

Separate Watering Total

Multan 4 1 5 42 21 63


Faisalabad 9 1 10 102 38 140

Lahore 7 1 8 50 11 61


Total 30 4 34 290 106 396


71

Plate-V Photograph showing the communal water troughs used by the draught equine owners during working hours

Plate-VI Photograph showing the communal water troughs used by the draught equine owners in the communal equine housings


72

Table-20 Bivariable analysis for predicting glanders in equines sampled from 5 draught equine populated urban areas of Punjab

S. No. Exposure Variable Comparison Chi Square df P OR 95% CI Wald P

1 Metropolis 2.84 4 0.58 0.597

Multan Gujranwala 1.409 0.362 5.47 0.621

Bahawalpur Gujranwala 2.037 0.569 7.29 0.274

Faisalabad Gujranwala 1.268 0.384 4.18 0.697

Lahore Gujranwala 2.32 0.668 8.11 0.185

2 Species of equine 10.23 2 0.006 0.009

Horse Donkey 3.35 1.51 7.44 0.003

Mule Donkey 2.27 0.72 7.12 0.159

3 Sex

Female Male 5.94 1 0.014 2.35 1.163 4.77 0.017

4 Age groups 1.47 2 0.477 0.485

6 to 10 5 or less 1.59 0.535 4.74 0.402

more than 10 5 or less 1.87 0.673 5.2 0.23

5 Housing Pattern

Kept with non equine cohorts Kept alone or with equines

0.082 1 0.774 0.89 0.403 1.96 0.774

6 Watering Methods

Communal Separate 3.6 1 0.058 2.7 0.931 7.86 0.067


73

Table-21 SPSS output of binary logistic regression analysis for predicting glanders in equines sampled from 5 draught equine populated urban areas of Punjab

Variables in the Equation

B S.E. Wald df Sig. Exp(B)

95% C.I.for EXP(B)

Lower Upper

Step 1a Sex .838 .362 5.359 1 .021 2.311 1.137 4.696

Watering Methods .969 .546 3.144 1 .076 2.635 .903 7.690

Constant -3.578 .538 44.281 1 .000 .028

a. Variable(s) entered on step 1: Sex, Watering Methods.


74

4.8. Equine Piroplasmosis

Out of 430 serum samples collected from studied areas, 226 (52.5%) were found cELISA

positive for piroplasmosis (Theileria equi, Babesia caballi or mix infection) (Table-22). No

piroplasm could be seen in the Giemsa stained blood smears. Seroprevalence of T. equi was

significantly higher, 2 (1df) = 38.07, P= 0.00 i.e. 41.2% (n=177) as compared to B. caballi

(n=93, 21.6%). A significant difference regarding this seroprevalence was observed between the

equines of Lahore (95.6%) and others metropolises, 2 (4df) = 24.49, P<0.01. Seroprevalence of

piroplasmosis was found to be 64% in equines of Faisalabad followed by Multan (52.9%),

Gujranwala (50.7%) and Bahawalpur (50%). Difference regarding prevalence of piroplasmosis

in equines of Faisalabad, Multan, Gujranwala and Bahawalpur was not significant, 2 (3df) =

5.99, P= 0.112.

4.8a. Clinical Signs Found in Equines Tested Positive for Piroplasmosis

Analysis of clinical examination data (Table-23) indicated that anorexia (n=47) was mostly

observed non specific clinical finding followed by debility (n=18) in equines found seropositive

for piroplasmosis. More specific clinical findings in decreasing order were pale mucous

membranes (n=47), fever (n=31), congested mucous membranes (n=21), eyelid edema with

muco-purrulent discharge (n=20), edema of the distal limb (n=12), enlarged lymph nodes (n=10)

and patechial hemorrhages on the mucous membranes (n=5).


75

4.8b. Seroprevalence of Theileria equi Infection the Equines of Selected Areas

Out of 430 equine serum samples collected from studied areas, 177 (41.2%) were found sero-

positive on the basis of commercial competitive enzyme linked immunosorbent assay (VMRD,

USA).

4.8c. Area Related Seroprevalence of Theileria equi Infection

Highest seroprevalence of Theileria equi (Table-24) was recorded in the equines Lahore (66.7%)

followed by those of Faisalabad (42%), Multan (35.3%), Bahawalpur (33.8%) and Gujranwala

(28%). This difference in seroprevalence was significant between Lahore and other metropolises,

2 (4df) =26.41, P<0.01. However, seroprevalence of T. equi in Faisalabad, Multan, Bahawalpur

and Gujranwala was not significantly different, 2 (3df) =4.56, P=0.206.

4.8d. Equine Species Dependant Seroprevalence of Theileria equi Infection

Among three species of sampled draught equine population (n=430), significantly different

seroprevalence,2 (2df) =9.39, P=0.009 (Table-24) of T. equi was found in horses i.e. 56.9%

(n=37) than mules (48.5%, n=16) and donkeys (37.3%, n=124). Prevalence of T. equi was

recorded highest in horses of Lahore, where all the 13 (100%) animals were found sero-positive.

This prevalence was recorded in descending order as 70% (n=7), 50% (n=5), 38.9% (n=7) and

35.7% (n=5) in horses of Multan, Bahawalpur, Faisalabad and Gujranwala respectively.

Prevalence of T. equi infection in mules of Lahore was 100% (n=6), followed by 66.7% (n=4) in

mules of Multan. Third highest prevalence was recorded in the mules of Faisalabad i.e. 44.4%

(n=4) followed by 16.7% (n=1) each in Bahawalpur and Gujranwala respectively. Out of 124


76

cELISA positive donkey sera, highest prevalence (54%) was recorded in Lahore where 27

animals were found positive followed by 42.3% (n=52), 32.7% (n=17), 27.3% (n=15) and 25%

(n=13) in Faisalabad, Bahawalpur, Gujranwala and Multan respectively.

4.8c. Age Related Seroprevalence of Theileria equi Infection

Analysis of age related data of Theileria equi positive equine population (n=177) is presented in

Table-25 indicated towards higher prevalence of T. equi in horses of age group-I (5 Years or

less) i.e. 72.7% followed by 60% in group-III (above 10 Years) and 48.3% in group-II (6-10

Years). Relatively steady pattern of T. equi prevalence was observed in mules where prevalence

increased with age of animals i.e. 25%, 55.6% and 57.1% in group-I, II and III. Highest

prevalence of T. equi was observed in donkeys of age group-II (39.9%) followed by group-III

(36.9%) and group-I (34.4%). Overall prevalence of T. equi increased with age in equines i.e.

36.9, 42.6 and 44.3 percent in group-I, II and III respectively. However, there was no significant

difference found among the seroprevalence regarding 3 age groups, 2 (2df) =1.56, P=0.457.

4.8d. Sex Dependant Seroprevalence of Theileria equi Infection

Prevalence of Theileria equi was not significantly different, 2 (1df) =0.93, P=0.45 (42.7%,

n=126) in male equines (Table-26) as compared to females (37.8%, n=51). Out of 65 (25 male

and 40 female) horses, this prevalence was higher in male horses (64%) as compared to females

(52.5%). Analysis of serum samples from mules (n=33) revealed that out of 18 males 10 were

sero-positive (55.5%) and 6 (40%) out of 15 females were sero-reactive for T. equi. Out of 332

donkeys sera, 100 (39.7%) males and 24 (30%) females were found sero-positive for Theileria

equi.


77

Table-22 Seroprevalence of piroplasmosis in equines (n=430) of 5 draught equine populated urban areas of Punjab surveyed for prevalence of equine infectious anemia, glanders and piroplasmosis Metropolis (Samples Taken)

T. equi Positive (Prevalence)

B. caballi Positive (Prevalence)

Mix infection (Prevalence)

Total (Prevalence) (T. equi + B. caballi)

Multan

(n = 68) 24 (39.7) 12 (17.6) 5 (7.4) 36 (52.9)b

Bahawalpur

(n = 68) 23 (33.8) 11 (16.2) 4 (5.9) 34 (50.0)b

Faisalabad

(n = 150) 63 (42.0) 33 (22.0) 17 (11.3) 96 (64.0)b

Lahore

(n = 69) 46 (66.7) 20 (28.9) 12 (17.4) 66 (95.7)a

Gujranwala

(n = 75) 21 (28.0) 17 (22.7) 6 (8.0) 38 (50.7)b

Total 177 (41.2)a 93 (21.6)b 44 (10.2) 226 (52.6)

Values with different superscript alphabets in the column differ significantly, 2 (4df)=24.49, P<0.01)

Values with different superscript alphabets in the rows differ significantly, 2

(1df)=38.07, P<0.01)


78

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)

Clinical Finding T. equi + B. caballi + T. equi & B. caballi

Fever 23 8 9

anorexia 38 9 19

Pale Mucous Membranes 35 12 16

Congested Mucous Membranes 16 5 6

Patechial Hamorrhages on M. M. 4 1 4

Edema of the Distal Limb 11 1 5

Enlarged Lymph Nodes 5 5 7

Asthenia (Debility) 14 4 4

Eyelid edema with muco-purulent discharge 17 3 8

Polyuria 0 3 5

Colic 5 2 2

Small & dry feces 3 2 3


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Table-24 Seroprevalence of Theileria equi infection in the equines surveyed for the prevalence of equine infectious anemia, glanders and piroplasmosis from 5 draught equine populated urban areas of Punjab

Location Samples Examined

Horses

(Prevalence %)

Mules

(Prevalence %)

Donkey

(Prevalence %)

Total

(Prevalence %)

Multan 68 (10H, 6M, 52D) 7 (70.0) 4 (66.7) 13 (25.0) 24 (35.3)b

Bahawalpur 68 (10H, 6M, 52D) 5 (50.0) 1 (16.7) 17 (32.7) 23 (33.8)b

Lahore 69 (13H, 6M, 50D) 13 (100) 6 (100) 27 (54.0) 46 (66.7)a

Gujranwala 75 (14H, 6M, 55D) 5 (35.7) 1 (16.7) 15 (27.3) 21 (28.0)b

Faisalabad 150 (18H, 9M, 123D) 7 (38.9) 4 (44.4) 52 (42.3) 63 (42.0)b

Total 430 (65H, 33M, 332D) 37 (56.9)a 16 (48.5)b 124 (37.3)b 177 (41.2)

Values with different superscript alphabets in the column differ significantly, 2 (4df) =26.41, P<0.01

Values with different superscript whole number in a row differ significantly, 2 (2df) =9.39, P=0.009

H: Horse M: Mule D: Donkey


80

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

Type of

Equine

Samples Examined

(<5y, < 10y, > 10y)

No. of equines positive for T. equi in each

age group (Prevalence %)

< 5 Y < 10 Y > 10Y

Horses 65 (11,29,25) 8 (72.7) 14 (48.3) 15 (60)

Mules 33 (8,18,7) 2 (25) 10 (55.6) 4 (57.1)

Donkeys 332 (119,148,65) 41 (34.4) 59 (39.9) 24 (36.9)

Total 430 (138, 195, 97) 51 (36.9)a 83 (42.6)a 43 (44.3)a

Values with similar superscript alphabets are not significantly different, 2 (2df) =1.56, P=0.457


81

Table-26 Comparative sex related dynamics of Theileria equi infection in 5 draught equine populated urban areas of Punjab surveyed for the seroprevalence of equine infectious anemia, glanders and piroplasmosis

Area Sampled

Samples Examined

Horses

(Prevalence %)

Mules

(Prevalence %)

Donkeys

(Prevalence %)



M (3♂, 3♀)

D (37♂, 15♀)

4 (66.7) 3 (75) 3 (100) 1 (33.3) 11 (29.7) 2 (13.3)


M (2♂, 4♀)

D (40♂, 12♀)

3 (60) 2 (40) 1 (50) 0 (0.0) 16 (40) 1 (8.3)


M (6♂, 3♀)

D (98♂, 25♀)

3 (60) 4 (30.8) 3 (50) 1 (33.3) 43 (43.9) 9 (36)


M (3♂, 3♀)

D (34♂, 16♀)

4 (100) 9 (100) 3 (100) 3 (100) 19 (55.9) 8 (50)

Gujranwala H (5♂, 9♀)

M (4♂, 2♀)

D (43♂, 12♀)

2 (40) 3 (33.3) 0 (0.0) 1 (50) 11 (25.6) 4 (33.3)

Total H (25♂, 40♀)

M (18♂, 15♀)

D (252♂, 80♀)

16 (64)a

21 (52.5)a

10 (55.5)a

6 (40.0)a

100 (39.7)a

24 (30.0)a


H = Horse M = Mule D = Donkey


82

4.8e. Seroprevalence of Babesia caballi Infection

In the studied equine population (n=430) of 5 areas, 93 sera were found positive for antibodies

against B. caballi infection and seroprevalence was found to be 21.6% (Table-27).

4.8f. Area Related Seroprevalence of Babesia caballi Infection

Seroprevalence of B. caballi infection (Table-27) in descending order was found to be 28.9, 22.7,

22, 17.6 and 16.2 percent in Lahore (n=20), Gujranwala (n=17), Faisalabad (n=33), Multan

(n=12) and Bahawalpur (n=11) respectively. However, this prevalence difference was not

statistically significant, 2 (4df) =4.09, P=0.394.

4.8g. Equine Species Related Seroprevalence of Babesia caballi Infection

Highest prevalence of B. caballi (Table-27) was observed in mules (30.3%) followed by horses

(24.6%) and donkeys (20.2%). However, this difference was not statistically significant, 2 (2df)

=2.218, P=0.33. Out of 65 horse sera from 5 different areas, highest prevalence was observed in

Lahore i.e. 30.8% (n=4) followed by 30% (n=3) in Multan, 28.6% (n=4) in Gujranwala, 20%

(n=2) in Bahawalpur and 16.7% (n=3) in Faisalabad. Within the mule species highest

seroprevalence was recorded in Lahore (50%) followed by Gujranwala (33.3%), Faisalabad

(33.3%), Multan (16.7%) and Bahawalpur (16.7%). Babesia caballi seroprevalence was highest

in donkeys of Lahore where 13 (26%) animals were found cELISA positive followed by

Faisalabad (21.9%), Gujranwala (20%), Bahawalpur and Multan (15.4% each).


83

4.8h. Age Related Seroprevalence of Babesia caballi Infection

Age related dynamics of B. caballi are given in Table-28. Higher seroprevalence of B. caballi

was found in age group-III (more than 10 years) i.e. 27.8% (n=27) followed by group-II (6-10

years) 22.6% (n=44) and group-I (5 years or less) where it was found as 15.9% (n=22). The

difference in seroprevalence between group-III and group-I was found to be significant, 2 (1df)

=4.88, P=0.02. However, in general this seroprevalence was not significantly affected by

different age groups, 2 (2df) =4.938, P=0.085. Highest seroprevalence was observed in horses of

age group-I (27.3%) followed by group-II (24.1%) and III (24%). Mule sera from age group-III

reacted most (57.1%) to commercial B. caballi antibody test kit cELISA as compared to animals

in group-I (37.5%) and II (16.7%). Sero-prevalence of B. caballi increased with age in donkeys

with lowest prevalence was recorded in animals of group-I (13.4%) that increased to 22.9% and

26.1% in group-II and III respectively.

4.8i. Sex Related Seroprevalence of Babesia caballi Infection

There wasn’t a significant difference, 2 (1df) =0.002, P=0.96 regarding the sero-prevalence of

B. caballi in two sexes of equines (Table-29). Higher prevalence (21.7%, n=64) was recorded in

male as compared to female (21.5%, n=29) equines. Prevalence of B. caballi was higher 44% in

male horses (n=11) as compared to 12.5% in females (n=5). Relatively less difference in

prevalence was observed in male (33.3%, n=6) and female (26.7%, n=4) mules. Out of total 252

male donkeys, 47 (18.6%) were found sero-positive that was lower than the females (25%,

n=20).


84

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

Location Samples Examined Horses (Prev. %)

Mules (Prev. %)

Donkey (Prev. %)

Total (Prev. %)

Multan 68 (10H, 6M, 52D) 3 (30.0) 1 (16.7)

8 (15.4) 12 (17.6)a

Bahawalpur 68 (10H, 6M, 52D) 2 (20.0) 1 (16.7) 8 (15.4) 11 (16.2)a

Faisalabad 150 (18H, 9M, 123D) 3 (16.7) 3 (33.3) 27 (21.9) 33 (22.0)a

Lahore 69 (13H, 6M, 50D) 4 (30.8) 3 (50.0) 13 (26.0) 20 (28.9)a

Gujranwala 75 (14H, 6M, 55D) 4 (28.6) 2 (33.3) 11 (20.0) 17 (22.7)a

Total 430 (65H, 33M, 332D) 16 (24.6)1 10 (30.3)1 67 (20.2)1 93 (21.6)


Values with similar superscript whole number are not significantly different, 2 (2df) =2.21, P=0.33

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

Type of Equine

Samples Taken

(<5y, < 10y, > 10y)

No. of equines positive for B. caballi in each age group (Prevalence %)

< 5 Y < 10 Y > 10Y

Horses 65 (11,29,25) 3 (27.3)a 7 (24.1)a 6 (24)a

Mules 33 (8,18,7) 3 (37.5)a 3 (16.7)a 4 (57.1)a

Donkeys 332 (119,148,65) 16 (13.4)a 34 (22.9)a 17 (26.1)a

Total 430 (138, 195, 97) 22 (15.9)3 44 (22.5)1,3 27 (27.8)1

Values with similar superscript alphabet in a column are not significantly different (P > 0.05)

Values with different superscript whole number in the row are significantly different (P < 0.05)


85

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

Area Sampled Samples Taken Horses (Prevalence %)

Mules (Prevalence %)

Donkeys (Prevalence %)



M (3♂, 3♀)

D (37♂, 15♀)

2 (33.3) 1 (25.0) 0 (0.0) 1 (33.3) 6 (16.2) 2(13.3)


M (2♂, 4♀)

D (40♂, 12♀)

2 (40.0) 0 (0.0) 1 (50.0) 0 (0.0) 7 (17.5) 1(8.3)


M (6♂, 3♀)

D (98♂, 25♀)

2 (40.0) 1 (7.7) 2(33.3) 1(33.3) 19 (19.4) 8 (32.0)


M (3♂, 3♀)

D (34♂, 16♀)

2 (50.0) 2 (22.2) 1(33.3) 2 (66.7) 8 (23.5) 5(31.3)

Gujranwala H (5♂, 9♀)

M (4♂, 2♀)

D (43♂, 12♀)

3 (60.0) 1 (11.1) 2 (50.0) 0 (0.0) 7 (16.3) 4(33.3)

Total H (25♂, 40♀)

M (18♂, 15♀)

D (252♂, 80♀)

11 (44.0)a 5 (12.5)a 6 (33.3)a 4 (26.7)a 47 (18.6)a 20 (25.0)a

Values with similar superscript alphabet are not significantly different, 2 (1df) =0.002, P=0.96


86

4.8j. Seroprevalence of Mixed Infection of Theileria equi and Babesia caballi

A total of 44 (10.2%) out of 430 equines were found sero-positive for both T. equi and B. caballi

(Table-30).

4.8k. Area Related Seroprevalence of Mixed Infection

Analysis of data presented in Table-31 revealed that highest sero-prevalence of mix infection

was in Lahore (17.4%) followed by Faisalabad (11.3%), Gujranwala (8%), Multan (7.4%) and

Bahawalpur (5.9%). Chi square analysis revealed that this prevalence was not significantly

different, 2 (4df) =6.469, P=0.167 in equines of Lahore, Faisalabad, Gujranwala, Multan and

Bahawalpur.

4.8l. Equine Species Related Seroprevalence of Mix Infection

Data regarding equine species related mix infection seroprevalence of B. caballi and T. equi is

presented in Table-31. Analysis indicated that mix infection was higher in mules (15.1%) of

studied population followed by horses (12.3%) and donkeys (9.3%). Prevalence of mix infection

in mules, horses and donkeys was found to be statistically not significantly different, 2 (2df)

=1.464, P=0.481. Mix infection was higher in horses of Lahore (30.8%) followed by of

Faisalabad (11.1%), Multan (10%), Gujranwala (7.1%) and Bahawalpur (0%). In mules (n=33),

mix infection was highest in Lahore (50%) followed by Faisalabad (22.2%). No mule sera from

Gujranwala, Multan and Faisalabad were found sero-positive for mix infection. Highest

prevalence of mix infection was found in donkeys of Faisalabad (10.6%) followed by donkeys of

Lahore (10), Gujranwala (9.1%), Multan (7.7%) and Bahawalpur (7.7%).


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4.8m. Age Related Seroprevalence of Mixed Infection

Higher prevalence (14.4%) of mix infection was recorded in equines of age group-III (more than

10 years) where 14 equines were found sero-reactive to both B. caballi and T. equi commercial

cELISA test kits followed by 10.8% (n=21) and 6.5% (n=9) in group-II (6-10 years) and I (5

years or less) respectively (Table-32). However, this difference was statistically not significant,

2 (2df) =3.99, P=0.136. Computed age based sero-prevalence of mixed infection in horses was

18.2% in age group-I followed by 16% and 6.9% in group-III and II. In mules (n=33), highest

prevalence (28.6%) was observed in group-III followed by 12.5% in group I and 11.1% in group-

II. In donkeys (n=332), sero-prevalence of mix infection increased with age and lowest

prevalence (5.04%) was observed in animals of age group-I that increased to 11.5 and 12.3

percent in group-II and group-III. However, all these differences were found to be statistically

not significant on chi square analysis (P > 0.05).

4.8n. Sex Related Seroprevalence of Mixed Infection

Data regarding sex related seroprevalence of mixed infection revealed that 29 out of 295 male

and 15 out of 135 female equines were found sero-positive for both prioplasms (Table-33). This

seroprevalence of mix infection was higher in females (11.1%) than males (9.8%). However, this

difference was statistically not significant, 2 (1df) =0.165, P=0.684.


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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

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

Location Samples Examined

Horses (Prevalence %)

Mules (Prevalence %)

Donkeys (Prevalence %)

Total (Prevalence %)

Multan 68 (10H, 6M, 52D)

1 (10.0) 0 (0.0) 4 (7.7) 5 (7.4)a

Bahawalpur 68 (10H, 6M, 52D)

0 (0.0) 0 (0.0) 4 (7.7) 4 (5.9)a

Faisalabad 150 (18H, 9M, 123D)

2 (11.1) 2 (22.2) 13 (10.6) 17 (11.3)a

Lahore 69 (13H, 6M, 50D)

4 (30.8) 3 (50.0) 5 (10.0) 12 (17.4)a

Gujranwala 75 (14H, 6M, 55D)

1 (7.1) 0 (0.0) 5 (9.1) 6 (8.0)a

Total 430 (65H, 33M,332D)

8 (12.3)1 5 (15.1)1 31 (9.3)1 44 (10.2)

Values with similar superscript alphabet in column are not significantly different, 2 (4df) =6.469, P=0.167

Values with similar superscript whole number in a row are not significantly different, 2 (2df) =1.464, P=0.481

T. equi + T. equi - Total

B. caballi + 44 (10.2%) 49 (11.4%) 93 (21.6%)

B. caballi - 133 (30.9%) 204 (47.4%) 337 (78.4%)

177 (41.2%) 253 (58.8%) 430 (100%)


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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

Equine Species Samples Taken Number of equines positive for B. caballi & T. equi in each age group (Prevalence %)

(<5y, < 10y, > 10y) < 5 Y < 10 Y > 10Y

Horses 65 (11,29,25) 2 (18.2) 2 (6.9) 4 (16)

Mules 33 (8,18,7) 1 (12.5) 2 (11.1) 2 (28.6)

Donkeys 332 (119,148,65) 6 (5.0) 17 (11.5) 8 (12.3)

Total 430 (138, 195, 97) 9 (6.5)a 21 (10.8)a 14 (14.4)a


Table-33 Sex related prevalence 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


Total Examined B. caballi positive T. equi positive Mix Infection

295 Male 64 (21.7%) 126 (42.7%) 29 (9.8%)a

135 Female 29 (21.5%) 51 (37.8%) 15 (11.11%)a

Total 93 (21.6%) 177 (41.2%) 44 (10.2%)


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4.8o. Hematological Analysis of Piroplasmosis Positive Equine Blood Samples

Results of hematological analysis of equines found sero-positve for T. equi and B. caballi are

described in Table-34, 35, 36 and 37.

Hematological analysis results regarding the horses (n=53) found sero-positive for

piroplasmposis (Theileria equi and Babesia equi) is presented in Table-34. Significantly lower

(P<0.05) than reference value mean white blood (WBC) cell counts were observed in horses

positive for T. equi (6.01+1.79), B. caballi (5.33+1.77) and mix infection (5.27+1.87). However,

these values were not statistically different (P>0.05) among horses positive for T. equi, B. caballi

and mix infections. A statistically not significant difference (P>0.05) was observed regarding

mean RBC counts among horses found positive for T. equi (5.61+1.26), B. caballi (5.31+1.15)

and mixed infection (5.25+1.38). Although, these values were numerically lower than reference

RBC count value (5.93+2.81) but this difference was not significant (P>0.05). Mean hemoglobin

concentration was significantly lower (P<0.05) than reference value (10.93+1.89) in T. equi

(9.55+2.00) and B. caballi (9.28+2.24) sero-reactive horses. However, mean Hb concentration

found in mix infection positive horses (9.81+2.7) was not significantly lower (P>0.05) than

reference value. There was not a significant difference (P>0.05) among horses found positive for

T. equi, B. caballi and mix infection. Packed cell volume (PCV) was significantly lower

(P<0.05) in horses sero-positive for T. equi (28.69+4.07), B. caballi (26.08+2.93) and mix

infection (27.63+2.91) than the reference value (37.75+4.57). Difference between mean packed


91

cell volume among horses sero-positive for T. equi, B. caballi and mix infection was not

significant (P>0.05). Erythrocytic indices indicated presence of microcytic hyperchromic type of

anemia in horses found seropositive for T. equi, B. caballi and mixed infection.

Hematological data regarding donkeys (n=191) found sero-positive for T. equi, B. caballi and

mix infection is presented in Table-35. Significantly lower (P < 0.05) than reference value (9.75 +

3.29) mean white blood count was observed in donkeys found sero-positive for T. equi (6.41 +

1.98), B. caballi (6.15 + 1.96) and mix infection (5.96 + 2.00). Mean red blood cell counts were

not significantly (P > 0.05) lower than the reference values with lowest mean RBC count was

observed in donkeys found positive for both T. equi and B. caballi (5.16 + 2.00) followed by B.

caballi (5.22 + 1.48) and T. equi (5.52 + 1.48) seropositive donkeys. Mean hemoglobin

concentration was not significantly (P > 0.05) different from reference value (9.01 + 2.65) in

donkeys sero-positive for B. caballi (9.01 + 1.86), T. equi (8.88 + 1.88) and mix infection (8.83 +

1.88). A significantly lower (P < 0.05) mean packed cell volume values were recorded in

donkeys cELISA positive for mix infection (29.57 + 5.42), T. equi (29.07 + 5.74) and B. caballi

(28.28 + 5.31). Mean corpuscular volume values were 60.88, 58.58 and 56.01 (fl) in mix

infection, B. caballi and T. equi positive donkeys respectively. Mean corpuscular hemaoglobin

(pg) was 18.55 in B. caballi positive donkeys followed by 18.18 and 16.99 in mix infection and

T. equi positive donkeys respectively. Mean corpuscular hemoglobin concentration (g/dL)


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numerically higher in donkeys seropositive for B. caballi (32.79) followed by 31.46 and 30.45 in

T. equi and mix infection positive donkeys respectively. On the basis of eryhtrocytic indices

macrocytic hyperchromic type of anemia was observed in all three types of sero-positive

animals.

Hematological data regarding piroplasmosis affected mules (n = 26) is presented in the Table-36.

Mean white blood cell counts were found to be significantly lower (P < 0.05) than reference

values (9.40 + 2.89) in mules seropositive for T. equi (6.94 + 2.28) , B. caballi (6.26 + 2.52) and

mix infection (5.92 + 2.68). However, difference between T. equi, B. caballi and mix infection

seropositive mules regarding WBC counts were statistically not significant (P > 0.05). Mean red

blood cell counts were not significantly lower (P > .05) than reference value in B. caballi (5.70 +

1.91), T. equi (5.27 + 1.3) and mix infection (5.00 + 0.55) sero positive mules. Mean

hemoglobin concentrations in mules seropositive for T. equi (9.69 + 2.01), B. caballi (9.00 +

2.31) and mix infection (9.32 + 2.15) were not significantly (P > 0.05) different than reference

value (10.10 + 0.73). Recorded values regarding mean packed cell volume for T. equi (29.45 +

4.04), B. caballi (29.22 + 4.42) and mix infection (31.16 + 4.01) sero reactive mules were

significantly lower (P < 0.05) than reference value (34.37 + 4.13). Mean corpuscular volume

values were 63.04, 55.10 and 59.67 (fl) in mix infection, B. caballi and T. equi positive mules

respectively. Mean corpuscular hemaoglobin (pg) was 16.57, 18.73 and 19.1 in B. caballi, mix


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infection and T. equi positive mules. Mean corpuscular hemoglobin concentration (g/dL) was

numerically higher in donkeys seropositive for T. equi (33.38) followed by 30.73 and 29.92 in B.

caballi and mix infection positive mules respectively.

Hematological data regarding the comparison of piroplasmosis affected horses (n =45), mules (n

= 21) and donkeys (n = 160) is presented in Table-37. Mean white blood cell (WBC) counts

were significantly (P < 0.05) lower in horses (5.90 + 1.79), donkeys (6.86 + 2.30) and mules

(6.41 + 1.96) than their respective reference values. Comparatively mean WBC count was

significantly (P > 0.05) lower in horses than piroplasmosis sero-positive mules and donkeys.

Mean red blood cell (RBC) counts were numerically but statistically not significantly lower (P >

0.05) than reference values in horses (5.57 + 1.21), donkeys (5.48 + 1.47) and mules (5.54 +

1.69). Mean hemoglobin concentration was significantly lower (P < 0.05) than reference value

(10.93 + 1.89) in piroplasmosis seropositive horses (9.41 + 1.95). However, Hb. concentrations

were not statistically significantly lower (P > 0.05) than reference values in seropositive donkeys

(8.92 + 1.81) and mules (9.45 + 2.15). Mean packed cell volume (PCV) values were significantly

lower (P < 0.05) than reference values in piroplasmosis seropositive horses (27.95 + 4.57),

donkeys (28.64 + 5.66) and mules (28.93 + 4.10).


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Table-34 Hematological values of horses found positive (n = 53) for piroplasmosis in 5 draught equine populated urban areas of Punjab

PIROPLASMOSIS

POSITIVE

PARAMETERS WBC/10³μL RBC/10μL Hb (g/dL) HcT (%) MCV (fl) MCH (pg) MCHC (g/dL)

REFERENCE 7.18 + 2.87 5.93 + 2.81 10.93 + 1.89 37.75 + 4.57 63.66 18.43 28.95

T. equi

(N = 37) Mean 6.01 + 1.79Sa

5.61 +

1.26a 9.55 + 2Sa 28.69 + 4.07Sa 53.65 17.82 33.73

B. caballi

(N = 16) Mean 5.33 + 1.77Sa

5.31 +

1.15a 9.28 + 2.24Sa 26.08 + 2.93Sb 52.34 18.69 35.59

Mix Infection

(N = 8) Mean 5.27+ 1.87Sa

5.25 +

1.38a 9.81+ 2.7NSa 27.63 + 2.91S* 57.27 20.41 35.79





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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

PIROPLASMOSIS

POSITIVE

PARAMETERS WBC/10³μL RBC/10μL Hb (g/dL) HcT (%)

MCV

(fl)

MCH

(pg)

MCHC

(g/dL)

REFERENCE 9.75 + 3.29 5.88 + 2.65 9.01 + 1.13 32.42 + 4.38 55.13 15.32 27.79

T. equi (N = 124) Mean 6.41 + 1.98 Sa 5.52 +1.48NSa 8.88 + 1.88NSa 29.07 + 5.74Sa 56.01 16.99 31.46

B. caballi (N = 67) Mean 6.15 + 1.96 Sa 5.22 + 1.48NSa 9.01 + 1.86NSa 28.28 + 5.31Sa 58.58 18.55 32.79

Mix (N = 31) Mean 5.96 + 2Sa 5.16 + 1.5NSa 8.83 + 2.09NSa 29.57 + 5.42Sa 60.88 18.18 30.45





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Table-36 Hematological values of mules found positive (n = 26) for piroplasmosis in 5 draught equine populated urban areas of Punjab

PIROPLASMOSIS

POSITIVE

Parameters WBC/10³μL RBC/10μL Hb (g/dL) HcT (%) MCV (fl) MCH (pg) MCHC (g/dL)

REFERENCE 9.40 + 2.89 5.74 + 2.62 10.10 + 0.73 34.37 + 4.13 59.88 17.59 29.38

T. equi

(N = 16) Mean 6.94 + 2.28Sa 5.27 + 1.3NSa 9.69 + 2.01NSa 29.45 + 4.04 Sa 59.67 19.1 33.38

B. caballi

(N = 10) Mean 6.26 + 2.52Sa 5.7 + 1.91NSa 9 + 2.31NSa 29.22 + 4.42Sa 55.1 16.57 30.73

Mix

(N = 5) Mean 5.92 + 2.68Sa 5 + 0.55NSa 9.32 + 2.15NSa 31.16 + 4.01Sa 63.04 18.73 29.92





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Table-37 Comparison of hematological values in equines (n = 226) found positive for piroplasmosis in 5 draught equine populated urban areas of Punjab

PIROPLASMOSIS

POSITIVE

Parameters WBC/10³μL RBC/10μL Hb

(g/dL)

HcT

(%)

MCV

(fl)

MCH

(pg)

MCHC

(g/dL)

Horses

(N = 45)

Reference 7.18 + 2.87 5.93 + 2.81 10.93 + 1.89 37.75 + 4.57 63.66 18.43 28.95

Mean 5.9 + 1.79 Sb 5.57 + 1.21 9.41 + 1.95S 27.96 + 4.1S 52.54 17.67 34.03

Donkeys

(N = 160)

Reference 9.75 + 3.29 5.88 + 2.65 9.01 + 1.13 32.42 + 4.38 55.13 15.32 27.79

Mean 6.41 + 1.96 Sa 5.48 + 1.47 8.92 + 1.81 28.64 + 5.66S 55.8 17.31 32.13

Mules

(N = 21)

Reference 9.40 + 2.89 5.74 + 2.62 10.10 + 0.73 34.37 + 4.13 59.88 17.59 29.38

Mean 6.86 + 2.3 Sa 5.54 + 1.69 9.45 + 2.15 28.93 + 4.1S 56.69 17.98 32.94





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4.8p. Seroprevalence of Equine Piroplasmosis in Association with Housing Pattern of Equines

Analysis of data regarding the housing pattern of piroplasmosis sero-positive (n=226) and

negative (n=204) equines (Table-38) showed that 105 (46.5%) sero-positive equines were kept

alone, 59 (26.1%) managed with other equine cohorts and 62 (27.4%) equines were kept with

animals other than equines (cattle, buffalo, sheep, goat & dog). Univeriate analysis indicated that

equines kept alone or with equine cohorts were more likely to be positive for piroplasmosis

(OR=1.30, 95%CI=0.84-2.01). However, this difference was statistically not significant, 2 (2df)

=2.061, P=0.357.

4.8q. Seroprevalence of Equine Piroplasmosis in Association with Presence of Ticks on Cohorts

Analysis of data related to presence and absence of ticks on sampled equines and their cohorts is

reflected in table-39. In total, presence of ticks in cohorts was observed in 71 animals (16.5%).

Out of these equines, 38 (16.8%) were found positive for piroplasmopsis and 33 (16.2%) were

sero-negative. No significant difference regarding the prevalence of piroplasmosis in equines

living with tick infested and tick free cohorts was observed, 2 (1df) =0.032, P=0.859. Out of

226 equines found seropositive for piroplasmosis ticks were present on equines cohorts of 15

(6.6%) animals. Whereas, non equine (cattle, buffalo, sheep, goat and dog) cohorts of 23 (10.2%)

sero-reactive equines were tick infested. Odds of contracting piroplasmosis were found to be

more in equines living with tick positive equine cohorts (OR: 1.19, CI 95% 0.65-2.18) and non

equine cohorts (OR: 2.04, CI 95% 0.73-5.69) as compared to the equines living alone or with

cohorts having no ticks. However, when presence and absence of ticks was compared along with


99

the housing pattern of studied equine population, no significant difference was observed, 2 (2df)

=1.898, P=0.387.

4.8r. Effect of Employing Tick Control Measures on Seroprevalence of Equine Piroplasmosis

Out of 111 equines where owners reported use of tick control measures 51 (45.9%) were found

seropositive for piroplasmosis. Whereas 54.8% (n=175) of those equines whose owners practiced

no tick control methods were positive for piroplasmosis (Table-40). Although equines belonging

to the owners who were not using tick control measures appeared to be more likely to contract

piroplasmosis (OR=1.43, 95% CI=0.93-2.21) but this difference was not significant, 2 (1df)

=2.624, P=0.105. A significant difference was observed regarding the seroprevalence among

equines of Lahore (2=4.326, df=1, P=0.038) when compared for the tick control used or not.

No significant difference (P>0.05) was observed regarding this among equines of other areas.

However, a significant difference (2=13.65, df=4, P=0.008) was observed regarding

seroprevalence of piroplasmosis between Lahore and other areas when compared for the tick

control.

4.8s. Treatment Chosen by the Veterinarians and Animal Health Workers against Piroplasmosis

Twenty veterinarians and animal health workers were randomly interviewed from each 5

sampled areas about their knowledge and understanding about recommended treatments for

Babesia caballi and Theileria equi (Table-41). Out of 100 respondents 88 veterinarians and

animal health workers told that they prefer to use recommended imidocarb treatment (2.2mg/kg,

two treatments at 24 hour intervals) in suspected cases. Only 12 veterinarians and animal health


100

workers showed awareness and desire to use recommended Theileria equi treatment (4 mg/kg, 4

times at 72 hour intervals). Only 30% of these respondents reported to look at the stained blood

smears before starting the treatment in suspected cases.

4.8t. Multivariable Analysis Regarding Prevalence of Equine Piroplasmosis

Regarding seroprevalence of equine piroplasmosis 7 variables were used for initial bivariable

screening. Analysis was conducted regarding sero-positivity of T. equi, B. caballi, mix infection

and piroplasmosis in general. Out of these variables, metropolis (P<0.001), equine species

(P=0.009) and use of tick control measures (P=0.017) were significantly associated with high

prevalence of T. equi (Wald P < 0.2) and sex (P=0.33), age (0.26), housing (0.58) and presence

of ticks on cohorts (0.75) were not significantly associated with sero-prevalence of T. equi (Wald

P>0.2) (Table-42). Variables yielding Wald P < 0.2 were entered into a multivariable regression

model which resulted in only one variable remaining significant i.e. metropolis (P<0.001) so no

further model building was possible (Table-43).

Only age (P=0.085), presence of ticks on cohorts (P=0.016) and housing pattern of equines

(P=0.034) were significantly associated (Wald P < 0.2) with seroprevalence of B. caballi and

were entered into a model (Table-44). Housing pattern and age were knocked out at first and

second step and only presence of ticks on cohorts was significantly associated with prevalence of

B. caballi (P=0.017) (Table-45). Therefore, there was not a multivariable model to present for B.

caballi prevalence.

During bivariable analysis regarding the sero-prevalence of mix infection (T.equi and B. caballi),

metropolis (P=0.167), age (P=0.136), presence of ticks on cohorts (P=0.014), housing (P=0.186)


101

and the use of tick control measures (P=0.119) were selected (P<0.2) for multivariate model

building (Table-46). Variables housing pattern, use of tick control, metropolis and age were

removed from the model at each step and only presence of ticks on cohorts was found

significantly associated with prevalence of mixed infection (P=0.016) (Table-47). This showed

that no multivariable model building was possible in this case.

Bivariable analysis regarding seroprevalence of piroplasmosis indicated metropolis (P<0.001),

equine species (P=0.003), age (P=0.182) and use of tick control (P=0.106) were the variables

significant enough (Wald P < 0.2) to be included in multivariable analysis (Table-48). However,

use of tick control measures and age were deleted from the model at subsequent steps. Resulting

final model suggested (Table-49) the prevalence of piroplasmosis in various equine species

might vary based on the different metropolis. For further analysis, categorical variables

(metropolis and equine species) were collapsed and tested for interaction term. For this purpose,

two new variables were created one comparing the prevalence of piroplasmosis in Lahore with

all other metropolis and other comparing the prevalence in horses with mules and donkeys. The

model was re-run with these new variables and interaction term to assess the inference that effect

of metropolis varies by equine species. However, inclusion of the interaction term did not

improve the fit of the model.


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Table-38 Comparison of housing pattern of equines (n=430) found positive (n=226) for equine piroplasmosis in 5 draught equine populated urban areas of Punjab

Values with same superscript alphabet are not significantly different, 2 (2df) =2.061, P=0.357

Area Sampled

Housing Pattern observed in piroplasmosis positive equines

Total equines Positive for piroplasmosis

Alone With Equines

With other animals

Multan 18 7 6 31



Lahore 20 16 18 54


Total 105a (46.5%) 59a (26.1%) 62a (27.4%) 226


103

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

Area Sampled

Piroplasmosis positive and tick

infested cohorts

Piroplasmosis negative and ticks

infested cohorts

Ticks on

Equine

Cohorts

Ticks on

other

Cohorts

Total Ticks on Equine

Cohorts

Ticks on

other

Cohorts

Total

Faisalabad 6 8 14 3 5 8

Lahore 4 7 11 1 2 3


Multan 2 1 3 0 8 8


Total (%) 15a 23a 38 (16.8) 8 25 33 (16.2)

Values with same superscript alphabet are not significantly different, 2 (2df) =1.898, P=0.387.


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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

Area Sampled

Piroplasmosis Negative Piroplasmosis Positive

Tick Control Used

Tick Control not Used

Tick Control Used

Tick Control not Used

Multan 13 24 14 17



Lahore 5 10 6 48


Total (%) 60 144 51 175


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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

Location

Veterinarians / animal health workers interviewed

regarding preferred treatment against suspected

equine piroplasmosis cases

Prefer B. caballi treatment

(%)

Prefer T. equi treatment

(%)

Faisalabad 18 (90.00) 2 (10.00)

Lahore 15 (75.00) 5 (25.00)

Gujranwala 19 (95.00) 1 (5.00)

Multan 17 (85.00) 3 (15.00)

Bahawalpur 19 (95.00) 1 (5.00)

Total 88 (88.00) 12 (12.00)


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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

S. No. Exposure Variable Comparison Chi Square df Wald P Odds Ratio 95% CI Lower Upper

1 Metropolis 26.41 4 <0.001 Multan Gujranwala 1 0.349 1.403 0.691 2.848 Bahawalpur Gujranwala 1 0.452 1.314 0.645 2.678 Faisalabad Gujranwala 1 0.042 1.862 1.023 3.391 Lahore Gujranwala 1 0.00 5.143 2.528 10.464 2 Species of equine 9.39 2 0.009 Horse Donkey 1 .004 2.217 1.293 3.8 Mule Donkey 1 .213 1.579 0.77 3.237 All others Donkey Sex 3 Female Male 0.931 1 0.335 0.814 0.536 1.236 4 Age groups 1.568 2 0.457 5 or less More than 10y 1 0.257 0.736 0.434 1.25 6-10 Years More than 10y 1 0.774 0.931 0.57 1.521 5 Tick on Cohorts No ticks on cohorts 0.105 1 0.746 0.918 0.546 1.544 6 Housing 1.077 2 0.583 Alone Non equine cohorts 1 0.793 1.065 0.663 1.712 With Equine Non equine cohorts 1 0.508 0.836 0.493 1.419 7 Tick Control Not Used Used 5.731 1 0.017 1.742 1.103 2.752


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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

Variables


95% C.I.for EXP(B)

Lower Upper

Step 2a Metropolis 22.685 4 .000

Multan(1) .418 .365 1.307 1 .253 1.519 .742 3.107

Bahawalpur(2) .339 .366 .854 1 .355 1.403 .684 2.876

Faisalabad(3) .617 .307 4.049 1 .044 1.854 1.016 3.383

Lahore(4) 1.619 .364 19.824 1 .000 5.049 2.476 10.299

Tick Controlled -.462 .243 3.624 1 .057 .630 .392 1.014

Constant -.848 .262 10.475 1 .001 .428

a. Variable(s) entered on step 1: Metropolis, Tick Control Used.


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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

S. No. Exposure Variable Comparison Chi Square df Wald P Odds Ratio 95% CI Lower Upper

1 Metropolis 4.092 4 0.394 Multan Gujranwala 1 0.457 0.731 0.32 1.669 Bahawalpur Gujranwala 1 0.331 0.658 0.284 1.528 Faisalabad Gujranwala 1 0.91 0.962 0.495 1.87 Lahore Gujranwala 1 0.387 1.393 0.658 2.948 2 Species of equine 2.218 2 0.33 Horse Donkey 1 0.422 1.292 0.691 2.412 Mule Donkey 1 0.178 1.72 0.781 3.786 Sex 3 Female Male 0.002 1 0.96 0.987 0.602 1.62 4 Age groups 4.938 2 0.085 5 or less More than 10y 1 0.029 0.414 0.171 0.999 6-10 Years More than 10y 1 0.323 0.716 0.347 1.477 5 Tick on Cohorts No ticks on cohorts 5.816 1 0.016 1.978 1.128 3.468 6 Housing 6.782 2 0.034 Alone Non equine cohorts 1 0.018 0.519 0.301 0.894 With Equine Non equine cohorts 1 0.037 0.523 0.284 0.963 7 Tick Control Not Used Used 0.073 1 0.788 1.076 0.633 1.828


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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



95% C.I.for EXP(B)

Variables Lower Upper

Step 3a Ticks on Cohorts .682 .286 5.672 1 .017 1.978 1.128 3.468

Constant -1.418 .133 113.295 1 .000 .242

a. Variable(s) entered on step 3: Ticks on Cohorts.


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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

S. No. Exposure Variable Comparison Chi Square df Wald P Odds Ratio

95% CI Lower Upper



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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



95% C.I.for EXP(B)


Step 5a Ticks on Cohorts .864 .360 5.759 1 .016 2.372 1.172 4.801

Constant -2.359 .188 157.618 1 .000 .095

a. Variable(s) entered on step 1: Ticks on Cohorts.


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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

S. No. Exposure Variable Comparison Chi Square

df Wald P

Odds Ratio

95% CI Lower Upper



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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


95% C.I.for EXP(B)


Step 1a Metropolis 22.572 4 .000

Multan(1) .154 .343 .201 1 .654 1.167 .595 2.286

Bahawalpur(2) .093 .344 .073 1 .787 1.097 .559 2.152

Faisalabad(3) .489 .291 2.825 1 .093 1.631 .922 2.886

Lahore(4) 1.616 .379 18.151 1 .000 5.031 2.392 10.578

Animal 11.139 2 .004

Horse(1) .912 .300 9.265 1 .002 2.489 1.384 4.476

Mule(2) .667 .390 2.931 1 .087 1.949 .908 4.182

Constant -.525 .247 4.494 1 .034 .592

a. Variable(s) entered on step 1: Metropolis, Animal.


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Plate-VII Photograph showing the communal housing pattern used by owners of draught donkeys in the study areas


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Plate-VIII Photograph showing the roof type being used in the construction of the communal draught equine housing systems in the study area


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Plate-IX Photograph of tick infestation on the medial aspect of the thigh in a horse sampled during the study

CHAPTER-V (DISCUSSION)

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CHAPTER-V

DISCUSSION

Current project was conducted to access the seroprevalence of three OIE (International Animal

Health Organization) listed equine diseases in 5 draught equine populated urban areas of Punjab,

Pakistan. Checking of current disease status through prevalence studies is important to

judge/estimate the disease load in a population and make some practical control strategies.

Prevalence studies are always costly and require exclusive resources for the travelling and

sample collection, therefore, multiple disease epidemiological studies are advocated to address

these problems. This study was focused to estimate the seroprevalence of equine infectious

anemia (EIA), glanders and piroplasmosis in draught equines of selected areas through testing

their sera by utilizing respectively enzyme linked immunosorbant assay (EIA), Rose Bengal

plate agglutination test (Glanders) and competitive ELISA (Piroplasmosis). Draught equine

population (horses, mules and donkeys) of these areas is usually kept in suboptimal management

and housing conditions that favors the spread of communicable diseases. Moreover, poor

nutrition and draught stress compromise their immune system and favor the diseases spread.

In the areas under study, draught equine population was dominated by donkeys followed by

horses and mules. This high donkey population was due to the economical limitations of owners,

less feeding and space requirements by these animals along with more docile temperament. It is

foreseen that this donkey population will rise further in future as waning financial conditions of

the owners and decline in use of horse drawn carriages (Tangas) in the cities make horse keeping

unaffordable for them. Equine owners of the studied areas preferred to keep female horses over


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males because of less behavioral problems like dominance and fighting were associated with

them. However, male donkeys were kept in higher numbers as compared to horses because of

less frequent behavioral problems associated with them.

5.1. Equine Infectious Anemia: Sero-epidemiological Findings

Equine infectious anemia posses a great diagnostic challenge to the scientific community. Over

the years many tests have been devised and validated for the diagnosis and control of the disease

but no test came near the qualities of a perfect diagnostic solution regarding this disease. The

ability of EIA virus to undergo antigenic drift causes harm to sensitivity and specificity of many

tests (Issel and Cook, 1993). The diagnostic dilemma continued till the development of some

more specific and sensitive ELISAs against some static regions of the virus. These tests are more

suited for the screening purposes as compared to AGID with certain advantages of being quick

and more reliable in diagnosing the early infections (Parė and Simard, 2004; Piza et al., 2007).

The commercial ELISA (VMRD, Inc., USA) used in the current study detects the antibodies

against EIA virus p26 (static region) gene in affected equine sera. The sensitivity (100%) and

specificity (100%) was well established by different laboratory (Susan et al., 2008) and field

studies (Cullinane et al., 2007). The test is approved by the United States Department of

Agriculture (USDA) for the field and laboratory diagnosis of EIA.

Although, heamatological values indicated towards presence of anemia in most of the equine

during this study, sampled draught equine population of the studied metropolises found to be free

from antibodies against equine infectious anemia virus on the basis of enzyme linked

immunosorbant assay (ELISA). Cause of these anemic findings can be attributed to the


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nutritional deficiencies and other blood parasitic disease like piroplasmosis and trypanosomiasis.

This finding is in accord with results reported by Gill et al. (2008) as they found no sero-reactive

animal for EIA in randomly selected equines of Faisalabad region may be ascribed to the failure

of the organism to mount a response or the absence of the disease in the sampled equine

population, which was also suggested by the preceding studies of some Turkish workers (Turan

et al., 2002; Ataseven and Arslan, 2005 and Kirmizigȕl et al., 2009). Further investigations in

this regard with new locale, wider sample size with molecular techniques are required to

strengthen the claims of disease free status regarding EIA of the selected areas and Pakistan.

5.2. Equine Glanders: Sero-epidemiological Findings

Over the years glanders has been controlled in many countries though strict implementation of

test and slaughter policy but the disease still persists in equines of many developing countries

and threatens the public health through its zoonotic potentials and as a potential agent of

bioterrorism and warfare (Gregory and Waag, 2007; Pawaiya and Chauhan, 2008). Many sero-

diagnostic assays like complement fixation test (CFT), indirect hemagglutination test (IHAT),

counter immunoelectrophoresis test (CIET) indirect fluorescent antibody test (IFAT) and enzyme

–linked immunosorbent assays (ELISAs) have been developed and evaluated for their diagnostic

efficiency in different forms of the disease to address the clinical and bacteriological difficulties

in diagnosis (Neubauer et al., 2005; Naureen et al., 2007; Saqib et al., 2008). Over the years,

need has been felt for the development of some simple, quick and field diagnostic assay to

complement mallien testing (Naureen et al., 2007) in order to address the limitations of the

mallein in terms of its sensitivity, cross-reactivity with streptococcus aureus (causative organism

of strangles) to produce false positive results and time consumption (48 hours) to reach the


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diagnosis. In this study Rose Bengal plate agglutination test (RBT) was used to estimate

seroprevalence of glanders. The test was developed by the Russian workers (OIE, 2004) which

further recently evaluated by Naureen and co-workers (2007) and was found to carry good

diagnostic efficiency.

On the basis of Rose Bengal Plate Agglutination (RBT) test, the overall seroprevalence of

glanders was found to be 7.9% in the studied equine population. This finding indicates towards

the possible endemic nature of glanders in the draught equines in the selected metropolises. This

finding concord with past research studies and reports of a few recent clinical outbreaks in the

Punjab province, where the authors indicated towards possible high prevalence of glanders

(Nasreen, 1977; Vaid, 1981; Bashir, 1984; Muhammad et al., 1998; Saqib et al., 2003, Naureen

et al., 2007; Saqib et al., 2008).

5.2a. Area Based Seroprevalence of Equine Glanders

Study indicates that seroprevalence of equine was not significantly different in 5 selected

metropolis (2=2.84, 4df, P=0.58) that indicates towards endemic nature of this disease in

selected areas. One possible reason for this could be that most of the equines belonged to the

poor owners who share almost identical socioeconomic status and husbandry practices.

However, higher seroprevalence of glanders in Lahore may be attributed to the outbreak of

glanders in recent years as described by Naureen et al. (2007) and Saqib et al. (2008). Moreover,

being a large industrial city and major hub of economic activities the metropolis attracts owners

along with their draught equines from neighboring towns and cities to seek the work

opportunities. Within city, equines are usually put to work in close groupings and kept under


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inadequate management which probably precipitates the spread of glanders in this area.

Moreover, relatively consistent seroprevalence found in studied metropolises could be attributed

to the climatic and draught stress, poor nutrition and care provided to draught equines by the

owners owing to their poor economic situation. Similar has been reported by various authors

elsewhere (Muhammad et al., 1998; Manso, 2003; Neubauer et al., 2005; Al-Ani and Roberson,

2007; Gregory and Waag, 2007)

Study indicates towards possible rise in the current prevalence of glanders in future as the

diseased animals are well recognized by the owners and they prefer to sale their sick animals to

unaware owners of other areas/cities over getting them destroyed by the authorities for a meager

compensation. Furthermore, chances of spread also increase through the buying and selling of

apparently healthy carriers sold in the large markets present in each area, where animals and

buyers come across from distant areas. (Muhammad et al., 1998; Saqib, 2000)

5.2b. Clinical Signs Observed in RBT Positive Equines

Glanders sero-positive animals showed clinical signs of the disease in variable intensity. Because

majority of these animals were not in the clinical stage of the disease, most of the signs observed

were not specific: like congested mucous membranes, loss of stamina, fever, anorexia. More

specific signs indicating a respiratory tract involvement were cough, nasal discharge and

dyspnea. Orchitis was found in only one male donkey. These signs are comparable with previous

reports by different workers (Saqib et al., 2003; Al-Ani and Roberson, 2007; Gregory and Wagg,

2007; Naureen et al.2007 and Saqib et al.2008).


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5.2c. Species, Age and Sex based Seroprevalence of Glanders

Significant difference (P<0.01) was observed regarding the seroprevalence of glanders between

horses and donkeys but the differences between horse vs. mule and mule vs. donkey were not

significant (P>0.05). This could be attributed to the differences in presentation of disease in these

equine species as reported by the other workers (Al-Ani et al., 1998; Muhammad et al., 1998;

Al-Ani and Roberson 2007, Gregory and Wagg, 2007) who reported that the course of disease is

usually chronic in horses and donkeys mostly suffer from acute disease, whereas, the disease can

manifest itself in both acute and sub acute forms in mules. Another possible reason for this

difference could be that equines owners are well familiar with clinical picture of glanders and

avoid bringing equines in clinical state (usually donkeys and mules) for work and veterinary

advice.

No significant (P>0.05) difference in seroprevalence of glanders was observed among 3 different

age groups of equines. Relatively higher prevalence rates were observed in age group-II (6-10

years) and I (less than equal to 5 years) than older equines of group-III (more than 10 years). In

general the seroprevalence was found to be higher in young and middle aged animals as

compared to old. Incidence of glanders as documented elsewhere (Dungworth, 1993; Radostits et

al., 2007) found to be higher in old age and equines older than 2 years of age were found more

susceptible (Al-Ani and Roberson, 2007). This high prevalence in relatively young equines could

also be attributed to the fact that equines in these areas were put to work in early age and poorly

managed, which might be favouring spread of the disease. In addition, clinical form of the

disease is frequently seen in aged equines so they are usually culled or not put to work

intentionally in order to avoid them being noticed and destroyed by the authorities as owners are


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well familiar with the Glanders and Farcy Act. Same has been reported by Al-Ani et al. (1987),

Al-Ani and Roberson (2007), Naureen et al. (2007) and Pawaiya and Chauhan (2008).

Seroprevalence of glanders was significantly higher (2 =5.94 (1df), P=0.014) in female equines

as compared to males. Univariate analysis has indicated the same that female equines were more

likely to be positive for glanders (OR 2.3, 95%CI 1.16-4.77). This finding differs from the

reports of Al-Ani et al. (1987), Neuber et al. (2005) and Al-Ani and Roberson (2007) who

reported no significant association between sex and glanders. However, the higher

seroprevalence found in female equines in this study could be attributed to the relatively less

numbers of females enrolled in the sampled equine populations. Moreover, the female equines

usually face an added stress of breeding along with malnutrition and heavy draught work that

might weaken their immune response to endemic diseases.

5.2d. Prevalence of Equine Glanders with Reference to Housing Pattern of Equines

Seroprevalence of glanders was not found to be associated (P>0.05) with housing pattern of the

equines sampled. Numerical differences regarding seroprevalence were observed among 3 types

of housing practices. However, comparatively higher percentage of equines found sero-positive

for glanders were kept alone or with equine cohorts. This difference could be due to the

congested and unhygienic conditions of the communal housing systems in case of equines kept

with equines. Moreover, draught equines although kept alone but working in local tanga stands,

grain and green markets have higher chances of contracting the disease because of greater

interaction with other infected equines. Similar findings has been reported by other researcher as

well (Lovell, 1935; Al-Ani et al., 1987; Muhammad et al., 1998; Saqib et al., 2003; Al-Ani and


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Roberson, 2007) who reported that overcrowding, unhygienic conditions and poor nutrition

make the equine more susceptible to the disease. The higher seroprevalence in equines kept with

non equine cohorts could be attributed to the fact that they are usually kept for the fodder

carriage and tend to get less attention regarding feeding and management by the owners who

consider them animals of less economical importance. However, had this been a clinical study

the effect of housing may have been different in various housing patterns adopted by the owners.

5.2e. Prevalence of glanders with reference to watering habits of the equine owners

Seroprevalence of glanders was found more in equines having access to the communal water

troughs either at work or home however this difference was not significant (P>0.05). However,

univariate analysis indicated that equines drinking water through communal water troughs were

more likely to be sero-positive (OR 2.7, 95% CI 0.931-7.86). Water can become contaminated

with B. mallei through a diseased equine and organism can survive there for the weeks to come

(Miller et al., 1948; Gangulee et al., 1966). Usually these water troughs are without running

stream and stagnant water sources is shared by many equines working on the roads. Moreover,

sharing of buckets for watering is fairly common in equine owners. These watering methods

used by the owners might be contributing towards the spread of disease. Similar has been quoted

by the different workers (Hutyra et l., 1949, Gangulee et al., 1966; Al-Ani and Roberson, 2007;

Radostits et al., 2007) who stated that contaminated utensils, food, water and congested housing

are causes for spread of glanders.

5.3. Equine Piroplasmosis: Sero-epidemiological Findings


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Piroplasmosis caused by Theileria equi and Babesia caballi is an important disease of horses

with serious health and economic impact. For the years, diagnosis of the disease remained a

challenge for the researchers and veterinarians as the conventional Giemsa stained smears are

rarely conclusive and most of the serological tests have the many problems including reporting

false positive / negative results (Donnelley et al., 1980; Ribeiro et al., 1999; Abdelkebir et al.,

2001; Akkan et al., 2003; Zinora et al., 2007). Similar had been observed during the study where

examination of Giemsa stained blood smears were found inconclusive for either T. equi or B.

caballi.

Competitive ELISAs used in the current study were recognized by OIE on the basis of

validations provided by the works of Knowles et al. (1991), Shkap et al. (1998), Kappmeyer, et

al. (1999) and Katz et al. (2000).

Seroprevalence of piroplasmosis (T. equi, B. caballi or mixeded infection) was found to be

52.5% in the studied metropolises which shows the widespread nature of the problem. Moreover,

seroprevalence of Theileria equi (41.2%) was significantly higher (2=38.07 (1df), P<0.01) as

compared to B. caballi (21.6%). These findings are in line with those found by various authors in

Brazil, (Barbosa et al., 1995; Ribeiro et al., 1999), Japan (Ikadai et al., 2001), Mongolia

(Boldbaatar et al., 2005), Spain (Camacho et al., 2005), Trinidad (Asgarali et al., 2007) and

Turkey (Akkan et al., 2003; Karatepe et al., 2009). These authors invariably reported the

comparatively higher prevalence of T. equi than B. caballi in different endemic regions of the

world. However, some workers (Xuan et al., 2001; Chahan et al., 2006; Torina et al., 2007)

reported an equitable distribution in some regions of China owing to similar distribution of

vector populations. A serologically higher seroprevalence of T. equi noted in this study could be


126

due to the fact that infected equines usually become life-long carriers of the infections and

infections with B. caballi usually subsides in 4-5 years (Shkap et al., 1998; Vial and Gorenflot,

2006; Rüegg et al., 2007; Rüegg et al., 2008).

The prevalence of piroplasmosis was significantly higher (P<0.01) in equines of Lahore as

compared to the Faisalabad, Multan, Gujranwala and Bahawalpur. This difference could be due

to the more favorable climatic conditions that favor the spread of tick vectors. Furthermore, the

trend of using tick control measures was significantly lower (P<0.01) in equine owners of Lahore

than other areas. Similar findings have been reported elsewhere, with authors reporting

differences in prevalence of disease in the areas based upon presence of tick vectors and faulty

management (Shkap et al., 1998; Asgarali, et al., 2007; Karatepe et al., 2009). Another possible

reason for this higher prevalence could be attributed to the presence of more carrier animals in

the area as equines born and raised in endemic areas usually enter into the carrier state of

piroplasmosis (Abdelkebir et al., 2001; Camacho et al., 2005).

5.3a. Clinical Signs Found in Equines Tested Positive for Piroplasmosis

Though the aim of the study was not to pick the clinical cases of piroplasmosis but data

regarding seropositive equines was analyzed to seek the most frequent clinical signs observed in

these animals to suggest possible clinical indicators of piroplasmosis. Anorexia and debility are

two non specific signs found frequently in piroplasmosis positive equines. The specific clinical

signs found were pale mucous membranes, fever, congested mucous membranes, eyelid edema

with muco-purrulent discharge, edema of the distal limb, enlarged lymph nodes and patechial

hemorrhages on the mucous membrabes. These observations have also been reported by Roberts


127

et al. (1962), Hailat et al. (1997), Seifi et al. (2000) in clinical and sub clinical forms of the

piroplasmosis. However, many authors reported that equines in the endemic areas may adapt to

the piroplasmosis where the disease would prevail in its latent form (Abdelkebir et al., 2001;

Camacho et al., 2005; Chahan et al., 2006; Asgarali et al., 2007) and the stress may lead to

development of clinical disease in these equines.

5.3b. Seroprevalence of Theileria equi: Sero-epidemiological findings

Current study suggested that under the prevailing conditions in the sampled metropolises,

Theileria equi is the major contributor (41.2%) of piroplasmosis. This finding is in agreement

with those reports depicting that in general prevalence of T. equi infection is usually higher in the

endemic regions of the world (Barbosa et al., 1995; Ribeiro et al., 1999; Boldbaatar et al., 2005;

Camacho et al., 2005; Asgarali, et al., 2007; Karatepe et al., 2009). However, some studies

document the relatively less difference in prevalence of T. equi and B. caballi (Shkap et al.,

1998; Heuchert et al., 1999) and the possible reason for this difference could be selection of the

equines population for study. In these studies target equine population usually belonged to

privately owned well managed horses receiving good nutrition and owners/managers could be

well aware of utilizing vector control methods. However, the current study differs from above

because all the equines selected were kept for draught purpose and usually were managed under

poor management, improper housing and high draught stress, which could be a possible cause of

this higher prevalence found.

Seroprevalence of T. equi was found to be significantly higher (P<0.01) in equines of Lahore

than of other four cities. This could be due to the difference observed in tick control measures


128

adopted, as the owners reported significantly lower use of tick control measures than other areas.

Similar findings have been reported by various authors (Heuchert et al., 1999; Skhap et al.,

1998; Acici et al., 2008; Karatepe et al., 2009: Salim et al., 2008) in their respective studies

based upon the differences found in the climatic / geographic conditions, vector population,

draught stress and equine keeping practices.

Seroprevalence of T. equi infection found in horses was significantly higher (P<0.01) as

compared to donkeys and mules. Possible reason for this could be that the horses have less vigor

and strength to bear the heavy draught burden which can be more easily managed by the mules

and donkeys. These findings regarding T. equi prevalence are in line with those reported by

Torina et al, (2007) and Acici et al, (2008) who reported higher seroprevalence of B. caballi in

donkeys as compared with horses in Sicily and Turkey. Moreover, prevalence of T. equi

infection in equine species was higher in Lahore as compared to other 4 areas. So the higher

draught stress, significantly lesser tick control employed and malnutrition may compromise the

immune system of animals and make them more vulnerable to already prevailing T. equi

infections.

Seroprevalence of T. equi infection observed in this study was not found significantly (P>0.05)

associated with age of equines. However, seroprevalence increased with the age of equines and

more prevalence was observed in older equines as compared to young ones. Similar has been

presented by various authors (Bruning, 1996; Oliver and Garcia, 2001; Asgarali, et al. 2007;

Rüegg et al., 2007; Rüegg et al., 2008; Karatepe et al., 2009) in their respective studies and

possible reason could be that once infected with T. equi equines remain carriers throughout their


129

life. However, a few authors have not mentioned any significant relationship between prevalence

of T. equi and B. caballi infections and age of equines (Tenter et al., 1988; Shkap et al., 1998).

No significant (P>0.05) difference was found between either sexes of equines on the basis of T.

equi seroprevalence and these findings are in accord with those reported by Oliver and Garcia

(2001), Asgarali, et al. (2007) and Karatepe et al,(2009). However, Shkap et al. (1998) and

Rüegg et al. (2007) found a higher sero-positivity in mares and geldings as compared to stallions

which could be due to the different level of care, grooming and attention these animals were

getting according to their importance for owners. The numerically higher sero-positivity found in

males as compared to females could be due to the large proportion of male equines enrolled for

sampling as compared to females.

5.3c. Prevalence of Babesia caballi: Sero-epidemiological Findings

Significantly (P<0.01) lower seroprevalence of B. caballi (21.6%) was found in the present study

as compared with T. equi and this finding is in line with those reported by Heuchert et al., (1999)

and Karatepe et al. (2009) in different regions of Brazil and Turkey. This is also in accord with

those researchers who reported that less prevalence of B. caballi could be attributed to the fact

that it clears away from the body in few years and generally responds well to the recommended

dosage of imidocarb treatment (Bruning, 1996; Ali et al., 1996; Rüegg et al., 2007; Rüegg et al.,

2008).

Seroprevalence of B. caballi infection found in the study was not significantly different (P=0.39)

in 5 selected metropolises and similar has been reported by researchers from other countries

(Heuchert et al., 1999; Karatepe et al., 2009). However, a numerically higher number of B.


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caballi sero-positive equines were found in Lahore possibly owing to more suitable conditions

prevailing there for the persistence and spread of this parasite.

There was not association found between seroprevalence of B. caballi infection and equine

species (P=0.33). Only numerical differences were found possibly due to the differences in the

sampled proportions of these equines. However, horses, mules and donkeys of metropolis Lahore

suffer more from the disease as compared to the equines of other locations.

Age was not found to be significantly associated with the seroprevalence of B. caballi infection

(P=0.08), however, the rate of sero-positivity was generally higher in old equines as compared to

young animals. This finding was in accord with those found by Abdelkebir et al. (2001), Balkaya

and Erdogmus (2006) and Acici et al. (2008) who reported the prevalence of B. caballi is

independent of age of equines in their studied areas.

The prevalence of B. caballi in the studied areas was not dependent (P>0.05) upon the sex of

equines and similar findings are also quoted elsewhere by different workers (Oliver and Garcia,

2001; Asgarali, et al., 2007; Karatepe et al., 2009).

5.3d. Mixed Infection of Theileria equi and Babesia caballi: Sero-epidemiological Findings

Sero-positivity for mixed infection with T. equi and B. caballi was found in 10.2% samples.

Mixed infection of piroplasmosis is a common feature and has frequently been reported in

varying intensities by different workers according to the prevailing conditions, vector

populations and circulating prioplasms from Brazil (Heuchert et al., 1999), Iran (Seifi et al.,


131

2000), Japan (Ikadai et al., 2002), Spain (Camacho et al., 2005), Trinidad (Asgarali et al., 2007)

and Turkey (Acici et al., 2008).

No significant difference (P=0.167) was observed in the seroprevalence of mixed infection

regarding 5 selected areas. Moreover, mixed infection of piroplasmosis was found to be

independent of equine species. This has been reported also by Acici et al. (2008) who found no

difference regarding mixed infection in three species in belonging to different areas of Black Sea

region of Turkey. Similarly, no significant differences (P > 0.05) were observed regarding mixed

infections in different age groups and sex of equines and this finding is in accord with those

reported by Balkaya and Erdogmus, (2006), Acici et al. (2008), Rüegg et al. (2007) and Rüegg et

al. (2008).

5.3e. Seroprevalence of Piroplasmosis and Housing Pattern of Equines

Different housing pattern were not found associated with seroprevalence of piroplasmosis

(P>0.05), however, computed odds ratio indicated that equines living alone/with equine cohorts

in these metropolises were more likely to be positive for piroplasmosis (OR 1.30, 95% CI 0.84-

2.01) as compared to those managed with non equine cohorts and usually kept for the domestic

draught purpose. Similar has been reported by many studies as well that chances of contracting

the prioplasmosis were higher in those areas where equines have greater interaction with each

other or other domestic animals which favors the spread of tick infestation (Heuchert at al.,

1999; Camacho et al., 2005; Karatepe et al., 2009).

5.3f. Seroprevalence of Piroplasmosis with Reference to Presence of Ticks and Use of Tick Control Measures


132

Adjusted analysis for possible confounding factors revealed that equines living with tick infested

equine cohorts (OR = 1.19, 95% CI 0.65–2.18) and non equine cohorts (OR = 2.04, 95% CI

0.73-5.69) were more likely to be sero-positive for piroplasmosis. However, in statistical terms

this difference was not significant (P>0.05). Seroprevalence of piroplasmosis was not

significantly associated with the used of tick control measures by the equine owners. However,

seroprevalence was found to be higher in equines and areas where owners were not giving much

emphasis on using tick vector control (OR=1.43, 95%CI=0.93-2.21). These findings are in

agreement with those reported by Heuchert et al. (1999), Barbosa et al. (1995), Abdelkebir et al.

(2001) and Chahan et al. (2006) who found out a positive correlation between piroplasmosis and

tick infestation in equines and their equine or non equine cohorts. However, there is still a need

to establish the possible tick vector responsible for the spread of equine piroplasmosis in

Pakistan.

5.3g. Treatment Chosen by the Veterinarians and Animal Health Workers against Suspected Piroplasmosis in Equines

A structured interview of practicing veterinarians and animal health workers responsible for

equine disease management in their respective areas indicated that a lacunae regarding the

correct diagnosis and treatment of equine prioplasmosis exists, which could be a leading cause

for higher seroprevalence found in this study. As indicated in the results, that majority of

veterinarians and animal health workers were not familiar with the separate treatment protocol

recommended for B. caballi and T. equi, were using only the therapeutic dose of imidocarb

diapropionate recommended for the treatment of B. caballi infections. This is due to the fact that

they were relying just only upon the clinical signs for making the presumptive diagnosis and

almost always were not using any Giemsa stained smear examination or other serological tests


133

for diagnosing the condition. Conducting workshops, lectures and seminars regarding

piroplasmosis its diagnosis and correct treatment as reported by several authors (Ali et al., 1996;

Hailat et al., 1996; Seifi et al., 2000; Vial and Gorenflot, 2006) can address this issue in future.

5.3h. Comparison of Hematological Findings in Equine Species

A comparison of the hematological values of equines originating from 5 different locations

revealed significantly higher mean white blood cell (WBC) counts in the animals of Bahawalpur

and Multan. This may relate to the relatively higher seroprevalence of glanders in the equines of

these areas. Similar findings regarding the alterations in WBC counts associated with glanders

have also been reported previously by Saqib (2002). Mean red blood counts (RBC) were found

to be significantly lower in equines of Lahore as compared to equines of other areas and this

could be associated with the higher seroprevalence of piroplasmosis in the equines of this

metropolis. Mean heamoglobin concentration, hematocrit, MCV and MCH were sifnigicantly

different in equines of Bahawalpur and Multan as compared to the other areas and this could be

attributed to the nutritional status of these animals. Because of the extreme heat and dryness and

deprivation from proper nutrition, the equines of these areas tend to suffer more from heat related

disorders like anhydrosis or non perspiration also described by Jenkinson et al. (2007).

Moreover, the study was conducted during the hot and dry months of the year (July – August) in

the said locations when the draught equines were suffering more from heat stress and

dehydration. In short, these variations can be due to the difference in the climatic, nutritional,

management and disease burden of the equines in the study areas.


134

Differences were observed among horses, mules and donkeys regarding WBC and RBC counts,

hemoglobin concentration and packed cell volume. Comparison of hematological values found in

this study revealed significantly (P < 0.05) lower mean white blood cell counts than reference

values in donkeys and mules. Although, some workers (Nayeri, 1978; Reece, 1997) found total

WBC numbers of donkeys highest among domestic animals but lower values were also observed

elsewhere by Zinkl et al. (1990). Moreover, these difference could be due to the physiological

and pathological variations like age, nutritional and health status, pregnancy and lactation stage

as described by Reece (1997). However, this difference was not significant (P > 0.05) among

horses. Mean RBC counts were significantly lower in horses as compared to mules and donkeys

which could be due to the fact that these values are species specific (Gul et al., 2007). Generally

the horses of the studies areas had significantly lower mean Hb concentration than reference

values but mules and donkeys had mean Hb concentrations comparable with reference values.

These differences could be due to the fact that horses generally are more frequently underfed as

compared to mules and donkeys. Mean hematocrit was found to be significantly lower (P < 0.05)

than the reference values in all the equine types which could be due to the effect of mild

dehydration (Thrall, 2004). A not significant difference (P > 0.05) was observed among horses,

mules and donkeys regarding MCV, MCH and MCHC.

Hematological alterations regarding mean WBC and RBC counts were not significantly different

(P > 0.05) between male and female equines. Similar findings were observed by Nayeri (1978)

Reece (1997), Al-Busadah and Homeida (2005) and Gul et al. (2007) where they found a not

significant difference regarding hematological values between healthy male and female equines.

Mean hemoglobin concentration, packed cell volume and erythrocytic indices were found to be


135

towards higher end in females as compared to males which could be due to the lower numerical

proportion of sampled females as compared to male animals as well as females tend to get more

care as compared to males due to their breeding potentials.

Analysis of hematological values regarding the age of equines suggested that mean WBC and

RBC counts and hemoglobin concentration were significantly higher (P < 0.05) in young animals

and tend to decrease with age. Similar findings were reported by Zinkl (1990) and Gul et al.

(2007).

5.3i. Hematological analysis of equine blood samples found sero-positive for glanders and piroplasmosis

Glanders

Mean white blood cell (WBC) counts were not significantly higher than reference values in

horses but in mules these values were below normal and significantly low in donkeys. Mean red

blood cell counts (RBC) were also found below than reference values in horses, donkeys and

mules showing anemia. Mean hemoglobin concentrations were found higher than reference

values in all 3 equine types. Mean packed cell volume (PCV) was found lower than reference

values in horses, mules and donkeys. On the basis of erythrocytic indices macrocytic

hyperchromic type of anemia was established in horses, mules and donkeys. These

hematological findings are in line as described by other researchers (Saqib 2000; Saqib et al.,

2003; Al Ani and and Roberson, 2007; Saqib et al., 2008)


136

Piroplasmosis

Hematological analysis of the prioplasmosis sero-positive equines revealed marked decrease in

white blood cell counts (WBC), packed cell volume (PCV) and hemoglobin (Hb.) concentrations

regarding horses and mules. However, there was no decrease of Hb. Concentration below

reference values in donkeys. Erythrocytic indices indicated presence of microcytic hyperchromic

type of anemia in horses found seropositive for T. equi, B. caballi and mixed infection, while in

donkeys macrocytic hyperchromic was noted. Erythrocytic indices in mules revealed normocytic

hyperchromic, microcytic hyperchromic, and macrocytic hyperchromic type of anemia in T.

equi, B. caballi and mixed infections. These findings are in accord with those reported by various

researchers (de Waal 1992; Hailat et al., 1997; Seifi et al., 2000; Camacho et al., 2005; Asgarali

et al., 2007; Zobba et al., 2008) regarding RBC counts, Hb concentration and PCV, they found

values of varying degree of lower levels according to clinical and subclinical infections.

Similarly more severe anemia was recorded in equines suffering from T. equi infections when

compared with those suffering from B. caballi infections.


137

CONCLUSIONS

Equine Infectious Anemia

1. All equines were found sero-negative for equine infectious anemia (EIA) tested by

enzyme linked immunosorbent assay (ELISA).

Equine Glanders

1. The RBT based prevalence (7.9%) of equine glanders indicates towards endemic nature

of this disease in Pakistan.

2. Seroprevalence of equine glanders was not significantly associated with sampling locales,

equine species and age.

3. Female equines were more likely to be sero-positive for equine glanders (OR=2.3,

95%CI=1.13-4.69)

4. Equines sharing communal watering sources (toughs & buckets) are more likely to be

sero-positive for glanders (OR=2.71, 95%CI=0.94-7.83)

Equine Piroplasmosis

1. Seroprevalence of piroplasmosis was found alarmingly high (52.5%) in the sampled

equine population

2. Seroprevalence of piroplasmosis was found to be significantly higher in equines of

Lahore than those of other metropolises

3. Seroprevalence of piroplasmosis was significantly higher in horses than other equine

species

4. Mixed infection with Theileria equi and B. caballi was found to be 10.2% in the equines

of sampled areas

5. Seroprevalence of Theileria equi (41.2%) was significantly higher than that of B. caballi

(21.62%)


138

6. Seroprevalence of Theileria equi was significantly different with reference to areas

sampled (higher in Lahore than other areas) and equine species sampled (higher in horses

than mules and donkeys).

7. Equines belonging to owners who don’t practice tick control were found more likely to

be sero-positive for T. equi (OR=1.74, 95% CI=1.10-2.75)

8. Seroprevalence of B. caballi was independent of location, species, sex and age.

9. Equines living with tick infested cohorts were more likely to be sero-positive for B.

caballi (OR=1.97, 95% CI=1.13-3.47) and mixed infection (OR=2.37, 95% CI=1.17-

4.80)

10. Equines living alone or with equine cohorts were more likely to be sero-positive for B.

caballi infection (OR=1.92, 95%CI=1.17-3.15)

11. Lack of tick control measures adopted by equine owners and lack of knowledge among

animal health professionals about the correct diagnosis and treatment of T. equi and B.

caballi might be responsible for the high seroprevalence of piroplasmosis in studied

areas.


139

UNIQUE ATTRIBUTES OF THE PRESENT STUDY

1. Prevalence of equine infectious anemia (EIA), glanders and piroplasmosis was

investigated over a wide geographic region for the first ever time in Pakistan

2. Risk factors for responsible for spread equine glanders and piroplasmosis were

scientifically investigated

3. Some of the lacunae and drawbacks of previous studies were addressed in this study


140

FUTURE OUTLOOK AND RECOMMENDATIONS

1. Sero-negative findings regarding equine infectious anemia indicate towards the possible

EIA free status of the studied draught equine populated urban areas of Punjab, which

should be further certified by conducting more epidemiologic studies with different

settings and locales to attain a possible disease free status regarding EIA.

2. Potential role of communal water troughs in the spread of equine glanders requires

further investigations through carefully designed experimental studies. Equine owners,

vets and animal health workers should be given awareness about possible spread of

equine glanders through communal water troughs and faulty practices.

3. Glanders & Farcy Act should be revised regarding compensations & implemented strictly

to control and eradicate glanders in Pakistan

4. Future studies are required to identify the possible tick vectors responsible for spread and

maintenance of piroplasmosis in the region through conventional and molecular

techniques to understand the epidemiology of disease and devise control measures

5. Equine owners should be educated to adopt proper tick control measures to check the

spread of piroplasmosis

6. Veterinarians and animal health workers should administer the standard treatment

regimen recommended for each type of piroplasm (T. equi or B. caballi) after proper

diagnosis to control the disease

CHAPTER-VI (SUMMARY)

141

CHAPTER-VI

SUMMARY

Equine infectious anemia (EIA), glanders and piroplasmosis (Theileria equi and Babesia caballi

infections) are the three World Animal Health Organization (OIE) listed equine diseases with

serious economic and health effects. Keeping in view the scarce epidemiological information

available regarding these diseases in Pakistan, a cross-sectional sero-epidemiological study was

designed to investigate prevalence of these diseases in 5 draught equine populated urban areas of

Punjab province, Pakistan. Subordinate objectives included determination of hematological

alterations associated with these diseases and formulating a conceptual framework of control

measures on the basis of epidemiological data generated.

Four hundred and thirty (430) blood and serum samples were randomly collected from 5 selected

urban areas of Punjab (Lahore, Gujranwala, Faisalabad, Multan and Bahawalpur). Sample size

was calculated for the expected prevalence of 50 percent (unknown disease status) with

confidence limits of 95% and a desired absolute precision of 5%. Relevant information on each

disease was collected on a predesigned proforma. Hematological parameters studied were red

blood cell (RBC) count, hemoglobin concentration (Hb), white blood cell (WBC) count packed

cell volume (PCV) and erythrocytic indices. Giemsa stained thin and thick blood smears were

also examined microscopically for the presence of piroplasms.

Serum samples were subjected to commercial enzyme linked immunosorbant assay (ELISA,

VMRD, Inc., USA) for EIA, Rose Bengal plate agglutination test (RBT) for glanders and

commercial competitive ELISA (cELISA, VMRD, Inc., USA) for piroplasmosis status. Positive

and negative ELISA results were interpreted as per the criteria given by the manufacturers of the


142

commercial kits. For RBT, positive samples were identified on the basis of degree of agglutination

(+++ and ++++ considered positive). Data were analyzed using softwares (WINPEPI, EPiCalc,

Survey Tool Box and SPSS) for contributing risk factors by calculating Chisquare values,

multivariable analysis and calculations of odds ratio (OR).

None of the serum sample was found positive for EIA which is reflective of a possible disease free

status of the selected equine communities or the failure of the disease to mount an immune

response.

Sero-prevalence 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 higher (P<0.01) prevalence was recorded in horses (16.9%) and mules

(12.1%) than in donkeys (5.7%). Sero-prevalence recorded in young (<5 years; 7.9%), adult

(<10years; 9.2%) and old (>10years; 5.2%) equines were statistically not significantly different

(P>0.05). Significantly higher (P<0.05) seroprevalence was recorded in females (12.6%) as

compared to males (5.76%). Analysis for the possible confounding factors indicated that females

equines (OR = 2.3, 95% CI= 1.16–4.77) 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.

Over all 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 (mix infection). Significantly higher (P<0.05) prevalence was recorded in Lahore

(95.6%, n=66) followed by Faisalabad (64%, n=96), Multan (52.9%, n=36), Gujranwala (50.7%,

n=38) and Bahawalpur (50.0%, n=34). Seroprevalence of T. equi infection was significantly

different among equine species (P<0.01) and in equines of Lahore (P<0.01). Equine age and

gender based differences in seroprevalence of T. equi infection were non-significant (P>0.05). B.


143

caballi and mixed infection seroprevalence was not found significantly different (P>0.05) in

selected areas, equine species, 3 age groups and both sexes of equines. 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 tick infested equine cohorts (OR=1.19, 95%CI=0.65-2.18) or 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.

In general, hematologic alterations observed in the equines were indicative of anemia and chronic

infections with significant differences observed (P<0.05) in mean WBC counts, mean RBC

counts, mean Hb concentration, PCV, MCV and MCH in selected metropolises. Hematological

alterations observed in RBT positive (glanders positive) equines were indicative of mild

leukocytosis (P>0.05) in horses and decreased WBC counts in mules (P>0.05) and donkeys

(P<0.05). Mean RBC counts were significantly (P<0.05) lower in sero-positive horses and

donkeys but this difference was not significant (P > 0.05) in mules. Mean Hb concentrations were

higher (P>0.05) in horses, mules and donkeys than reference values. Mean PCV was lower

(P>0.05) in RBT positive individuals of 3 equine species. Differences among species regarding

mean WBC counts, RBC counts, Hb. concentrations and PCV (hematocrit) were not significant

(P>0.05). Macrocytic hyperchromic anemia was observed in all RBT positive horses, mules and

donkeys.

Hematologic analysis of equines sero-positive for prioplasmosis indicated a marked decrease

(P<0.05) in WBC counts, lower (P>0.05) mean RBC counts, significantly (P < 0.05) lower mean

PCV and lower (P>0.05) mean Hb concentration. Erythrocytic indices pointed towards microcytic

hyperchromic type of anemia in sero-positive horses, donkeys and mules sero-positive for

piroplasmosis.


144

In conclusion, the study results indicated towards possibility of acquiring disease free status

regarding EIA in studied areas particularly. Therefore, further studies with different

epidemiological settings, larger sample size and use of molecular techniques is obviously

warranted. The alarmingly high prevalence of glanders underscores the need to step up efforts for

control / eradication of disease from the region through revisiting of Glanders and Farcy Act and

strict implementation of ‘Test and Slaughter’ policy. Future epidemiological studies based upon

molecular techniques should be undertaken to determine prevalence of the disease in other regions

of Pakistan and scientific evaluation of possible risk factors involved in spread of glanders in

Pakistan. The present study highlights the lack of knowledge among veterinarians and animal

health workers regarding epidemiology, treatment and control of piroplasmosis. Furthermore,

education of equine owners regarding control of ticks and improvements in management practices

is required to control the alarmingly high seroprevalence of piroplasmosis. Future studies to

identify the potential vectors (e.g., ticks) responsible for spread and maintenance of equine

piroplasmosis as well as control program suited to the local context is also obviously warranted.

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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

sero-survey of equine infectious anemia, glanders and

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