STUDIES ON THE PREVALENCE AND MANAGEMENT OF PARASITIC INFECTIONS IN ZOO ANIMALS

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1 STUDIES ON THE PREVALENCE AND MANAGEMENT OF PARASITIC INFECTIONS IN ZOO ANIMALS Dissertation Submitted to Guru Angad Dev Veterinary and Animal Sciences University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in VETERINARY PARASITOLOGY (Minor Subject: Veterinary Pathology) By Aman Dev Moudgil (L-2011-V-20-D) Department of Veterinary Parasitology College of Veterinary Science Guru Angad Dev Veterinary and Animal Sciences University Ludhiana

2 CERTIFICATE I This is to certify that the thesis entitled, STUDIES ON THE PREVALENCE AND MANAGEMENT OF PARASITIC INFECTIONS IN ZOO ANIMALS submitted for the degree of Ph.D, in the subject of Veterinary Parasitology (Minor subject Veterinary Pathology) of the Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, is a bonafide research work carried out by Aman Dev Moudgil (L-2011-V-20-D) under my supervision and that no part of this thesis has been submitted for any other degree. The assistance and help received during the course of investigation have been fully acknowledged. (Dr. L. D. Singla) Major Advisor Professor-cum-Head Department of Veterinary Parasitology Guru Angad Dev Veterinary and Animal Sciences University Ludhiana (Punjab) 2

3 CERTIFICATE - II This is to certify that the thesis entitled, STUDIES ON THE PREVALENCE AND MANAGEMENT OF PARASITIC INFECTIONS IN ZOO ANIMALS submitted by Aman Dev Moudgil (L-2011-V-20-D), to the Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, in partial fulfillment of the requirements for the degree of Ph. D in the subject of Veterinary Parasitology (Minor Subject: Veterinary Pathology) has been approved by the Student s Advisory Committee after an oral examination on the same, in collaboration with an external examiner. (Dr. L. D. Singla) Major Advisor (Dr. S. K. Gupta) External Examiner Professor-cum-COE Department of Veterinary Parasitology College of Veterinary Science, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar (Haryana) (Dr. L. D. Singla) Head of the Department (Dr. Simrat Sagar Singh) Dean, Postgraduate Studies 3

4 ACKNOWLEDGEMENTS God understand our prayers even when we can t find the words to spell them With limitless humility, I bow my head with reverence and dedicatedly award my recondite gratitude to the ALMIGHTY, who bestowed me with such affectionate parents, whose sacrifice and limitless blessings have made me stand against every up and down phase of my life. I want to dedicate this work to my father whose dream has been formulated in the form of thesis today. My feelings of deep gratitude to my major advisor, Dr. L. D. Singla, Professor-cum- Head, Department of Veterinary Parasitology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana for his apt, sagacious, inspiring guidance, persistent encouragement, continuous moral support, creative suggestions, parental attitude, constructive criticism and dedication towards the work had prompted me for the successful culmination of my research work. I consider myself fortunate enough and privileged having worked under such a gracious and unique personality. I feel privileged to express my sincere thanks to Dr. Manish Kumar, Director, MCZP, Chhatbir, Punjab and Dr. M. P. Singh, Senior Veterinary Officer, MCZP Chhatbir, Punjab for their ever-willing guidance, suggestions and support throughout my research work by providing all the necessary facilities to carry out the research. I am also thankful to Chief Wildlife Warden of Punjab for providing the permission to carry out the research. I also express my sincere gratitude to the worthy members of my advisory committee; Dr. Kuldip Gupta, Associate Professor, Department of Veterinary Pathology, Dr. C. S. Randhawa, Professor, Department of Veterinary Medicine, Dr. M. S. Bal, Assistant Scientist, Department of Veterinary Parasitology and Dr. S. K. Uppal, Professor, Department of Veterinary Medicine, (Dean PGS nominee) for their expert advice, invaluable suggestions and never ending cooperation during the course of this investigation. Special thanks are due to Dr. Kuldip Gupta and Dr. M. S. Bal for their practical suggestions, prompt support and immense help at all stages of my research work. I express my deep regards to other faculty members of the Department: Dr. S. S. Rath, Late Dr. H. S. Rai, Dr Paramjit Kaur, Dr N. K. Singh, Dr. Jyoti and Dr. Harkirat Singh for inspirational guidance, encouragement and invaluable support throughout the course of my study. I cherish the indispensible help, cooperation, motivational zeal and all-time presence of my dear friends namely Dr(s) Deepak, Abhishek, Prashant and Amandeep for their moral support and constant encouragement and to create a comfortable environment during my research. I am also thankful to my loving juniors Dr(s) Siddharth, Hitesh, KD, Ajay, Adarsh, Gaurav, Manoj, Bhaskar, Akhilesh, Anubhav, Debasis, Nishant, Ankaj, Gursharan, Harsimran, Abhijit, Ramandeep, Ekta, Shalini, Kaushalendra and Rahul for their enthusiastic moral support. I wish to thank my seniors Dr(s) Suhail, Dinesh Krofa and Atul Gupta for their invaluable suggestions and company. Special thanks are also due to Ms. Amrita Sharma, Senior Research Fellow for her great technical help. I render my thanks to Mr. John, Raghu, Tony and Kaka, who helped me a lot during my stay and work at MCZP, Chhatbir. I also extend my gratitude to technical/ supporting staff (Mr. Subodh, Ashish, Ankur, Sarabjeet, Chajju Ram, Paras Ram and Mrs. Santosh) of 4

5 Department of Veterinary Parasitology, CVS, GADVASU, for their kind support during my study. I want to express my deep gratitude to Department of Science and Technology, New Delhi for providing INSPIRE fellowship during the program. All words in the lexicon will be futile and meaningless if I fail to acknowledge the constant prayers, ever encouraging moral support and selfless sacrifices extended by my respected Grandmother, late father and mother. Mere words of acknowledgement will never express the sense of regards and indebtedness toward my father-in-law, mother-in-law and Pranav. I shall always remember the deep concern, good wishes and true affection rendered by my brother, Naman and his wife Bhawna. Last but not the least; I would love to exhibit my deep sense of gratitude to Pallavi, my better-half for her firm emotional support, love, care and encouragement throughout the research work. All may not have been mentioned but none is forgotten. Place: Ludhiana Date: (Aman Dev Moudgil) 5

6 Title of the Thesis : Studies on the prevalence and management of parasitic infections in zoo animals Name of the student : Aman Dev Moudgil Admission No. : L-2011-V-20-D Major Subject : Veterinary Parasitology Minor Subject : Veterinary Pathology Name and Designation of Major Advisor : Dr. L. D. Singla Professor-cum-Head Degree to be Awarded : Ph.D Year of award of Degree : 2015 Total Pages of Thesis : VITA Name of University : Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana (Punjab), India ABSTRACT An investigation was carried out for the detection, identification, prevalence and management of parasitic infections of zoo animals and birds of various zoological/ deer parks of Punjab. Out of 909 animals and 549 birds faecal samples screened in three different seasons (winter, summer and monsoon) of two years in Mahendra Mohan Choudhury Zoological Park, Chhatbir, Punjab, 232 animals and 206 birds samples were found positive, showing an overall prevalence of and 37.52%, respectively. The animals of Bir Motibagh Deer Park Patiala, Bir Talab Deer Park Bathinda, Tiger safari Ludhiana and Deer Park Neelo exhibited an overall gastrointestinal parasitic burden of 87.43, 66.67, and 94.84%, respectively. Whereas, the birds of Bir Motibagh Deer Park Patiala, Patiala aviary, Bir Talab Deer Park Bathinda, Tiger safari Ludhiana and Deer Park Neelo exhibited an overall gastrointestinal parasitic burden of 25.54, 37.50, 45.39, 67.64, 16.88%, respectively. Faecal culture from the positive cases with strongyles revealed two prominent species of gastrointestinal nematodes, Haemonchus contortus and Cooperia oncophora. Based on PCR analysis, the eggs retrieved from sloth bear and Asiatic lions were confirmed as eggs of Baylisascaris transfuga and Toxascaris leonina, respectively. The embryonation studies carried out in controlled environmental conditions on eggs of Baylisascaris transfuga and Toxascaris leonina exhibited an interval of 12 and 9 days post incubation in 0.4% formalin solution and 14 and 9 days post incubation in faecal samples, respectively to acquire final infective stages. Serological studies revealed exposure of Toxoplasma gondii to lions and tigers and Trypanosoma evansi to elephants. Ctenocephalides felis and Afrimenopon waar was the ectoparasites recovered from wild cat and pigeon, respectively. The histopathological studies revealed pathogenic effects of adult Capillaria obsignata, larvae of Ascaridia columbae and adult of Ornithostrongylus quadriradiatus in the intestines and Paratanaisia bragai in the kidneys of pigeons. The drug efficacy studies carried out against strongyle infections in small and large captive wild ruminants, against Toxascaris leonina in Asiatic lions and Trichuris sp. in non human primates revealed lower faecal egg count reduction with fenbendazole (with standard drug schedule), whereas, an extended drug schedule proved effective. The alternative drug (ivermectin) used also proved effective for the control of helminthic burden. In case of birds also the regularly used drug (albendazole) proved ineffective as per the regular schedule, but proved effective at an extended dose period schedule. Toxocara canis, Baylisascaris transfuga, Strongyloides fuelleborni and Spirometra species were the major parasites of zoonotic importance recovered during the study. The data generated by the present study will prove beneficial for the management of parasitic infections in captive wild animals and birds and for future specific explorations. Keywords: Deer parks, drug efficacy, embryonation, ectoparasites, faecal culture, histopathology, PCR, prevalence, Punjab, serological studies, zoological park Signature of Major Advisor Signature of the Student 6

7 CONTENTS CHAPTER TOPIC PAGE NO. I INTRODUCTION 1 5 II REVIEW OF LITERATURE 6 47 III MATERIAL AND METHODS IV RESULTS AND DISCUSSION V SUMMARY REFERENCES VITA 7

8 LIST OF TABLES Table no. Title Page no. 1 Distribution of species of animals and birds of different locations under study 2 Season wise distribution of faecal samples collected from different locations 3 Criteria to identify viability and stage of development of ascarid eggs by microscopy 4 Description of the drugs used as per the animal and birds groups and seasons 5 Season wise prevalence of parasitic infection in animals and birds of MC Zoological Park, Chhatbir, Punjab 6 Captivity based prevalence in animals and birds of MC Zoological Park, Chhatbir, Punjab 7 Prevalence of parasitic infection in animals and birds based on feeding behaviour 8 Prevalence of endoparasitic infections in herbivores of MC Zoological Park, Chhatbir 9 Prevalence of endoparasitic infections in omnivores of MC Zoological Park, Chhatbir 10 Prevalence of endoparasitic infections in carnivores of MC Zoological Park, Chhatbir 11 Prevalence of endoparasitic infections in birds of MC Zoological Park, Chhatbir 12 Morphometric description of parasitic eggs/ oocysts recovered from herbivores 13 Morphometric description of parasitic eggs/ oocysts recovered from omnivores 14 Morphometric description of parasitic eggs/ oocysts recovered from carnivores 15 Morphometric description of parasitic eggs/ oocysts recovered from birds 16 Season wise prevalence of parasitic infection in animals and birds of Bir Motibagh Deer Park, Patiala 17 Season wise prevalence of parasitic infection in birds of Patiala aviary 18 Season wise prevalence of parasitic infection in animals and birds of Bir Talab Deer Park, Bathinda 19 Season wise prevalence of parasitic infection in animals and birds of Tiger Safari, Ludhiana 20 Season wise prevalence of parasitic infection in animals and birds of Deer Park Neelo

9 Table no. Title Page no. 21 Serum biochemical alterations in Infected, Suspected and Noninfected Royal Bengal tigers and Asiatic lions 22 Haematological values of Infected, Suspected and Non-infected Royal Bengal tigers and Asiatic lions Serum biochemical observations of elephants Haematological assessment of the elephants Drug efficacy assessment against strongyles in large ruminants (n=6) Drug efficacy assessment against strongyles in small ruminants (n=20) 27 Drug efficacy assessment against toxascariosis in lions (n=3) Drug efficacy assessment against Trichuris species in non human primates (n=6) Drug efficacy assessment against ascarids in Galliformes (n=6) Drug efficacy assessment against ascarids in Columbiformes (n=6) 119 9

10 LIST OF FIGURES Fig. No. Title 1 Map of Punjab depicting the Zoological and Deer Parks targeted under the study 2 Season wise prevalence of parasitic infection in animals and birds of MC Zoological Park, Chhatbir, Punjab 3 Captivity based prevalence in animals and birds of MC Zoological Park, Chhatbir, Punjab 4 Season wise prevalence of parasitic infection in animals and birds based on feeding behaviour 5 Photomicrograph of Haemonchus contortus egg from Indian gazelle 6 Photomicrograph of Cooperia sp. egg from black buck 7 Photomicrograph of Trichostrongylus sp. egg from blue bull 8 Photomicrograph of Trichuris sp. egg from swamp deer 9 Photomicrograph of Moniezia benedeni egg from gaur 10 Photomicrograph of Strongyloides papillosus egg from black buck 11 Photomicrograph of ascarid egg from porcupine 12 Photomicrograph of Trichuris sp. egg from porcupine 13 Photomicrograph of strongyle egg from elephant 14 Photomicrograph of Strongyloides sp. egg from elephant 15 Photomicrograph of Eimeria pellita oocyst from goral 16 Photomicrograph of Eimeria kamoshika oocyst from goral 17 Photomicrograph of Eimeria cylindrica oocyst from blue bull 18 Photomicrograph of ascarid egg from sloth bear 19 Photomicrograph of Trichuris sp. egg from non human primate 20 Photomicrograph of Strongyloides fuelleborni egg from non human primate 21 Photomicrograph of Isospora felis oocyst from jungle cat 22 Photomicrograph of Toxascaris leonina egg from Asiatic lion 23 Photomicrograph of Isospora revolta oocyst from leopard 24 Photomicrograph of Spirometra sp. egg from leopard cat 25 Photomicrograph of Toxocara canis egg from hyaena 10

11 Fig. No. Title 26 Photomicrograph of Ascaridia sp. egg from black pigeon 27 Photomicrograph of Capillaria sp. egg from white pigeon 28 Photomicrograph of Syngamus trachea egg from dove 29 Photomicrograph of Ornithostrongylus quadriradiatus egg from white pigeon 30 Photomicrograph of Amidostomum sp. egg from Mongolian pheasant 31 Photomicrograph of Hymenolepis sp. egg from Indian peafowl 32 Photomicrograph of Eimeria mayurai oocyst from Indian peafowl 33 Photomicrograph of Echinostoma revolutum egg from painted stork 34 Photomicrograph of Echinuria uncinata egg from sarus crane 35 Photomicrograph of Eimeria labbeana oocyst from sarus crane 36 Photomicrograph of Eimeria sp. oocyst from red jungle fowl 37 Photomicrograph of Eimeria sp. oocyst from kalij pheasant 38 Season wise prevalence of parasitic infection in animals and birds of Bir Motibagh Deer Park, Patiala 39 Season wise prevalence of parasitic infection in birds of Patiala Aviary 40 Season wise prevalence of parasitic infection in animals and birds of Bir Talab Deer Park, Bathinda 41 Season wise prevalence of parasitic infection in animals and birds of Tiger Safari, Ludhiana 42 Season wise prevalence of parasitic infection in animals and birds of Deer Park, Neelo 43 Microphotograph of larva of Haemonchus contortus (10X) 44 Microphotograph of larva of Cooperia oncophora (40X) 45 Embryonic developmental stages of Baylisascaris transfuga and Toxascaris leonina in 0.4% formalin solution and faecal sample at 27±1 C temperature 46 Photomicrograhs of various developmental stages of ascarids 11

12 Fig. No. Title 47 The adult male and female B. transfuga worms retrieved from sloth bear 48 Photomicrograph of anterior end B. transfuga showing triradiate lips (A), cervical alae (B) and oesophagus (C) 49 Photomicrograph of the curved caudal end of male B. transfuga with precloacal papillae (P) 50 Microphotograph of posterior end of male B. transfuga: cloacal opening (C) and tail knob (T) 51 Photomicrograph depicting anterior end of adult Ascaridia columbae with triradiate lips (black arrow), filariform oesophagus (yellow arrow) and wide cephalic alae (white arrow) 52 Photomicrograph depicting posterior end of adult male Ascaridia columbae with precloacal sucker (white arrow) and spicules (black arrow) 53 Photomicrograph of female Capillaria obsignata 54 Female C. obsignata characterized by slightly prominent vulva (white arrow), just posterior to the union of the oesophagus and the intestine (black arrow) 55 Anterior end of Ornithostrongylus quadriradiatus with vesicular enlargement (white arrow) 56 Posterior end of female O. quadriradiatus with vulva (white arrow) and spine (black arrow) 57 PCR amplification for identification of Baylisascaris transfuga targeting ITSs 58 Phylogenetic relationship of Baylisascaris transfuga recovered from Melursus ursinus with related species of Baylisascaris genus (The values/ numbers at the nodes of the branches depict confidence level) 59 PCR amplification for identification of Toxascaris leonina 60 Seasonal distribution of the antibody titres of the wild felids and their segregation into infected, suspected and non-infected groups 12

13 Fig. No. Title 61 Biochemical parameters of the tigers depicting alterations in uric acid (UA), potassium (K), calcium (CA) and phosphorus (P) values 62 Biochemical parameters of tigers depicting alterations in creatinine kinase (CK), sodium (Na), chloride (Cl) and iron (Fe) values 63 Card agglutination test for trypanosomosis 64 Photomicrographs depicting cut section of Capillaria obsignata (white arrows) and Ornithostrongylus quadriradiatus (black arrow) in the lumen of the intestine (10X) 65 Photomicrographs depicting cut section of C. obsignata (black arrows), Ascaridia columbae (brown arrow) and O. quadriradiatus (red arrow) in the lumen of the intestine (10X) 66 Photomicrograph of pigeon s kidney showing presence of brownish parasitic eggs (white arrows) with tubular damage (10X) 67 Photomicrograph depicting cut sections of the parasites trapped in renal glomeruli (red arrow) of pigeon, damaging renal tubules (black arrows) and causing marked haemorrhages and infiltration of mononuclear inflammatory cells (yellow arrow) (4X) 68 Photomicrograph of the anterior end of Ctenocephalides felis felis 69 Photomicrograph of C. felis felis depicting first spine of the genal comb approximately equal to the second one 70 Photomicrograph of C. felis felis depicting only one spine on lateral metanotal area 71 Photomicrograph of the louse recovered from pigeon 72 Photomicrograph depicting morphometry of the head of the louse recovered from pigeon 73 Photomicrograph of the posterior end of the louse 13

14 ABBREVIATIONS % : per cent %P : Per cent positivity & : and C : degree centigrade F : degree fahrenheit µ : micron µg : microgram µg/ dl : microgram per deciliter µl : : at the rate of < : less than = : equal to > : more than µm : micro meter ALB : Albumin ALT : Alanine aminotransferase ANOVA : Analysis of variance AST : Aspartate aminotransferase BLAST : Basic Local Alignment Tool bp : base pairs BUN : Blood urea nitrogen CRTN : Creatinine DNA : Deoxy ribonucleic acid DLC : Differential leucocyte count 14

15 dntps : deoxynucleoside triphosphate ELISA : Enzyme linked immunosorbent assay et al : and others Fe : Iron Fig. : Figure g : gram g% : gram per cent g/ dl : gram per deciliter GLO : Globulins Glu : Glucose Hb : haemoglobin i.e. : that is ITS : Internal transcribed spacer kda : kilo Dalton kg : kilo gram KOH : Potassium hydroxide L 3 : third stage larvae mg : milligrams mg/ dl : milligram per deciliters MgCl 2 : Magnesium chloride min. : minutes ml : milliliter mm : millimeter n : number PBS : Phosphate buffer saline 15

16 PCR : Polymerase chain reaction PCV : Packed cell volume PLT : Platelets rpm : revolutions per minute rdna : ribosomal deoxy ribonucleic acid sec. : seconds sp. : species sq. cm : square centimeter TBE : Tris-Borate-EDTA TBIL : Total bilirubin TEC : Total erythrocyte count TLC : Total leucocyte count TP : Total protein UA : Uric acid U/ L : Units per liters V/ cm : Volt per centimeters wt. : weight χ : Chi 16

17 CHAPTER I INTRODUCTION India is blessed with great climatic diversity, naturally favourable for existence of vast spectrum of animals and birds species (Chhabra and Pathak 2013). But, during these days, wildlife (both flora and fauna) is under the shadow of threats due to increased climatic changes, overexploitation of natural resources/ habitats by human beings and overall environmental degradation, which has posed a serious threat to the life of these precious organisms and hence, numerous species are enlisted as endangered. Zoological parks are evolving institutions with respect to the conservation of biological diversity and are centres for public recreation and education (Cuaron 2005). The zoological gardens/ parks thus can be considered as the centres of ex situ conservation of wild animals outside their natural habitat/ environment for aesthetic, educational, research and recreational purposes (Varadharajan and Pythal 1999). To give proper direction and thrust to the management of zoos in the country, the national zoo policy was framed and adopted by the government of India in the year The main objective of the zoos under the national zoo policy is to complement and strengthen the national efforts for the conservation of biodiversity of the country, particularly wild fauna. Animal health in zoological parks plays a vital role in achieving the primary objective of conserving wild fauna. Exact diagnosis of the cause of illness of the zoo animals and their effective treatment is a challenging task. Paucity of adequate information on the diseases and parasites of wild animals is a major challenge to maintain the sound health status of the captive wild animals (Varadharajan and Pythal 1999). 1

18 Like domestic animals, zoo animals are also affected by various infectious and non-infectious diseases. Moreover, presence of diseases in a zoo always poses a risk for domestic animals as well as humans in its periphery and vice-versa. In their natural habitat, the wild animals might possess natural resistance or live in balanced system with their parasites (Thawait et al 2014). The initiation, development and spreading of the parasitic diseases is governed by the interrelationships among the parasites, their host and micro environment (Panayotova-Pencheva 2013). But, the change in environment and change in living conditions from freedom to captivity influences the animal s ecology and might increase the sensitivity to diseases, especially parasitic infections (Goossens et al 2005). In captivity, the health status of the animals depends on many factors, like feeding, animal management and environmental conditions. Ecto and endoparasites affect directly or indirectly the health status of wild animals. These parasites could be held responsible for multitudinous problems for wildlife. Although it often appears that wildlife have adapted to the presence of parasites, but they fail to adapt to the adverse effects of parasitism (Bliss 2009). Parasites can affect host survival and reproduction directly through pathological effects and indirectly by reducing the host's immunity and affecting the physical condition (Thiangtum et al 2006). Information on parasitic infections of wild animals is meagre due to paucity of systematic investigations (Varadharajan and Kandasamy 2000). In the absence of an adequate surveillance and monitoring system, there had been only sporadic reports and reviews (Chakraborty et al 1994) on the problem in question and majority of these reports had informations based on animals maintained in captivity in regional zoological gardens or parks. Geo-helminths could be 2

19 considered as more often cause of massive parasitoses in zoo animals rather than biohelminths because in the confined living space, geo-helminths have the optimum conditions for development and can quickly lead to re-infection (Panayotova- Pencheva 2013). Thus, these infections constitute one of the major managemental problems and may even cause mortality in wild animals in captivity (Rao and Acharjyo 1984). Parasitic diseases reported in captive wild animals mainly include infections due to gastrointestinal parasites (Singh et al 2006a) as well as haemoprotozoans. Many of these parasitic diseases are zoonotic also such as trichinellosis, hydatidosis, giardiosis, ancylostomosis, toxocarosis, fasciolosis, toxoplasmosis etc. In the past, sporadic studies on the parasitic infections in wild animals (Kumar and Rao 2003, Patel et al 2003 and Singh et al 2006a) have been carried out, but these studies were not comprehensive enough to furnish complete picture of parasitism in captive animals and birds in various parts of the country, especially at Mahendra Mohan Choudhury Zoological Park (MCZP), Chhatbir, Punjab. This zoo possesses various wild herbivores (family Cervidae, Bovidae, Elephantidae, Equidae and Hippopotamidae), wild omnivores (family Ursidae and primates), wild carnivores (family Felidae, Hyaenidae and Canidae) as well as birds of indigenous and exotic origin. Mishra et al (2008) reported the presence of Babesia species in a white tigress died at MC Zoological Park, Chhatbir, Punjab. Various chemotherapeutic studies (mainly case reports) against parasitic infections have also been carried out by various workers from time to time in different zoos of the country (Suresh et al 2000, Islam and Nashiruddulah 2000, Upadhye et al 2001, Singh et al 2006b) and these studies have proved beneficial for controlling the parasitic infections in captive wild animals in different parts of the country. 3

20 Historically, most studies describing parasitic effects on individual hosts or host populations focused on single parasite species, despite the fact that a majority of hosts simultaneously get infected with multiple parasites (Petney and Andrews 1998). More recently, however, both the number and specific identity of co-infecting parasites have been implicated as potentially critical determinants of the relative impact of parasites on hosts (Behnke et al 2005, Craig et al 2008, Telfer et al 2010). Hematological and biochemical profiles may indicate a direct result of parasite-induced blood and energy losses (Colditz 2008), up-regulation of host immunity in response to infection, and even the repair of collateral damage caused by host immune mediators (Lochmiller and Deerenberg 2000, Colditz 2008). Even nonbloodsucking helminths (e.g. Cooperia species and Trichostrongylus species in ruminants) can also alter host haematology by limiting essential nutrients (e.g. amino acids, copper and proteins) or as a byproduct of the immune response to chronic infection (Feldman et al 2000). Ultimately, parasite-induced haematological changes can increase host morbidity and/or mortality (Qiu et al 2010, Rodrigues et al 2010), and decrease reproductive output (Ramakrishnan 2001). Thus, examining hematological and biochemical parameters, in addition to standard body condition indices, classical parasitological identification and molecular detection of parasites may provide an integrated, short-term measure of the effect of parasites on hosts. Nowadays, emerging infectious diseases have become an important challenge for wildlife ecologists and managers. Management actions to control these diseases are usually directed at the parasite, the host population, or a key component of the environment with a goal of reducing disease exposure and transmission (Wobeser 2006). Control methods directed at the host population, however, remain limited in 4

21 approach (e.g. vaccination, population reduction and test-and-remove) due to financial, logistical and political constraints. Keeping in mind the importance of parasitic infections in wild animals and their potential threat to domestic animals and human beings, the present study was envisaged with the following objectives: 1) To conduct the qualitative and quantitative copro-parasitological analysis of gastrointestinal parasitism in zoo animals and birds. 2) To study the prevalence of haemoprotozoa in captive wild felids and family elephantidae. 3) To conduct chemotherapeutic studies against parasitic infections. 5

22 CHAPTER II REVIEW OF LITERATURE The present study was conducted on diagnosis, prevalence and therapeutic management of gastrointestinal and blood parasites of zoo animals and birds kept in zoological and deer parks of Punjab state. Except for few detailed studies carried out in 70 s and 80 s of past century, only case reports of gastrointestinal parasites, haemoprotozoans, ectoparasites and their therapeutic management in zoo animals and birds from different parts of India had been reported. 2.1 National scenario Gastrointestinal helminthosis in zoo animals Post mortem investigation of 153 vertebrates of 36 species at the Baranga zoo (Patnaik and Acharjyo 1970) revealed 127 animals infected with 16 trematode, 17 cestode, 34 nematode and 3 acanthocephalan species. Tripathy et al (1971) surveyed intestinal parasitic infection of different animals and birds of the State Biological Park, Odisha through coprology from Dec 1968 to June 1969 and recorded higher intensity of Toxocara infection in wild felids. Rathore and Khera (1983) conducted a countrywide survey for recording the causes of mortality in various species of wild animals and birds living freely or in capitivity from 1975 to 1977 in all zoological parks, national parks, sanctuaries and reserve forests. Deaths were reported due to a variety of diseases including trypanosomosis, babesiosis, coccidiosis, tuberculosis, rinderpest, anthrax, salmonellosis, Johne s disease, ascariosis and fasciolosis. Rao and Acharjyo (1984) studied the common diseases in captive animals at Nandankanan zoo and observed that parasitic diseases proved fatal (21.55%) in different species of zoo animals. 6

23 Choudary et al (1987) reported a case of hydatidosis in an American bison, died at Nehru Zoological Park, Hyderabad. Post mortem examination revealed presence of tennis ball sized cysts in lungs and almost whole of the liver was occupied by small and large hydated cysts which were filled with hydatid sand. Padhi et al (1987) reported the occurrence of amphistomosis in spotted deer in Nandankanan zoo, Odisha. The post mortem examination revealed that a large number of amphistomes were attached in the rumen, which was later on identified as Paramphistomum cervi, Gastrothylax crumenifer and Fischoederius elongatus. Borthakur et al (1988) reported a case of distomiasis in elephant. The scatology revealed the presence of Fasciola eggs. Agarwal and Ahluwalia (1989) reported occurrence of Cysticercus tenuicollis in spotted deer/ cheetal (Cervus axis) at Prince of Wales Zoological Garden, Lucknow. Post mortem revealed the presence of cyst in the peritoneal cavity, having an invaginated scolex and was attached to the large intestine, which was later identified as Cysticercus tenuicollis. Dutta and Bordoloi (1989) reported prevalence of intestinal helminthic infection in elephants of Tiger Project of Manas (Assam). Faecal examination revealed that 80 per cent of the elephants (Elephas maximus) were infected by intestinal helminths. Highest infection (29.17%) was due to Fasciola sp., followed by mixed infection (20.83%) of Fasciola sp. and Strongyloides sp., Paramphistomum sp. (8.33%) and other parasites (4.17 %) such as Ascaris sp. and Oesophagostomum sp. Dutta et al (1990) reported occurrence of various helminthic parasitic infections in Rhinoceros unicornis at Assam State Zoo, Guwahati. The various parasitic eggs recovered were of Fasciola sp., Paramphistomum sp., Ascaris sp. and Strongyloides sp. 7

24 Arora and Ramaswamy (1990) did a survey to assess the parasitic load among spotted deer (Axis axis) in different National Parks in India. They found that Muellerius capillaris infection was widely prevalent in 2 out of 3 national parks surveyed. The overall prevalence rate was 30.96%. Maske et al (1990) conducted a coprological survey on 28 animals of Maharaj Bagh zoo, Nagpur. Out of these, 17 animals exhibited the presence of parasitic infections. Toxascaris sp. and Ancylostoma sp. were the predominant species identified in lions and tigers, especially in rainy and winter seasons. Isospora felis, Paragonimus westermanii and Taenia pisiformis were the other species recorded in wild felids in winters. Strongyle eggs were found in the faecal samples of elephants and cestode egg was identified in scat of python. Rao and Acharjyo (1991) recovered P. westermanii from tigers, mongoose and golden cat during routine necropsy conducted on carnivores at Nandankanan zoo, Odisha during At necropsy, cysts containing one or more reddish brown oval shaped flukes were seen in one or both lungs. Sarode et al (1991) reported the occurrence of Oesophagostomum eggs and worm in faecal samples from 6 elephants. Blood examination of these elephants revealed decrease in total leukocyte count, total erythrocyte count and haemoglobin levels, whereas increase in eosinophil count was recorded. Bordoloi et al (1991) reported occurrence of various intestinal helminthic infections in captive deer at Assam State Zoo, Guwahati. A total of 33.33% of the deer were infected with intestinal helminths. The highest infection was recorded due to Trichostrongylus sp. (50%), followed by Bunostomum sp. (25%) and Dicrocoelium sp. (25%). 8

25 Rao et al (1991) reported the occurrence of lung fluke (Paragonimus sp.) in tiger of Nandankanan zoo. Multiple cysts were found in the lungs during post mortem examination. Chakraborty (1992) examined 214 large intestines of wild herbivores collected at postmortem from Assam State Zoo and found that 10 (4.67%) animals of 6 different species were having Trichuris sp. infection. The parasites were mainly seen in caecum and colon. Rao and Acharjyo (1993) recorded occurrence of Dirofilaria immitis in different wild carnivores on routine necropsy and histopathological examination at Nandankanan zoo during It was reported that 17 cases involving 8 species harboured the parasite in the right ventricle of heart and all animals were above 5 years of age. Gogol (1994) reported the presence of hookworm infection in a tiger at Zoological Park, Itanagar. Islam (1994a) did a survey on gastrointestinal parasites of free living one horned Indian rhinoceros (R. unicornis) at Rajiv Gandhi Wildlife Sanctuary, Assam. Various parasitic eggs/ oocysts encountered were of strongyles, Paramphistomum sp. and coccidian oocysts. Islam (1994b) reported the occurrence of Bivetellobilharzia nairi in captive Asian elephants (Elephus maximus) from Kaziranga National Park and Assam State zoo. Out of 44 samples tested, 7 were found positive for B. nairi infection. Chakraborty et al (1994) recorded the occurrence of parasitic infection in captive wild herbivores on post-mortem of 214 animals in Assam State zoo. The various nematodes found were Haemonchus sp., Ascaris sp., Gongylonema sp., Chabertia sp., Trichostrongylus sp., Oesophagostomum sp., Setaria sp., Dioctophyma sp., Cooperia sp., Onchocerca sp., Trichuris sp., Kululima sp., Necator sp., Bunostomum sp., Dictyocaulus sp., Habronema sp., Chonigium sp. and 9

26 Grammocephalus sp. The various trematodes recorded were Fasciola sp., Paramphistomum sp., Gastrothylax sp., Fischoederius sp., Cotylophoron sp., Gigantocotyle sp., Homologaster sp., Pseudodiscus sp., Pfenderius sp. and Brumphtica sp. Various cestodes found were Moniezia sp., Anoplocephala sp., hydatid cyst and Cysticercus sp. Various protozoa found were Sarcocystis sp., Eimeria sp., and Balantidium coli. Modi et al (1995) identified Ascaris sp., Ancylostoma sp., Fasciola sp., Trichuris sp., Oesophagostomum sp., Strongyloides sp., Paramphistomum sp., Entamoeba sp. and coccidia in various species of zoo animals in India including elephant, rhinoceros, hippopotamus, mithun, nilgai, sambar, black buck, spotted deer, capped langur, golden langur, common langur and gibbon. Varma et al (1995) reported the occurrence of hydatid cyst in giant squirrel (Ratufa indica) from National Zoological Park, New Delhi. Hydatid cysts collected from liver and spleen were used for experimental infection of 6 pups (6-8 weeks old) with 1500 viable protoscolices. After 10 weeks, pups were sacrificed and mature Echinococcus granulosus worms were recovered from intestine. Chakraborty and Gogoi (1995) recovered different parasites on postmortem examination of 12 rhinoceros, including Kululima goodeyi, Chabertia sp., Necator americanus, Bunostomum sp., Paramphistomum sp., Anoplocephala sp., Balantidium coli and hydatid cyst. Arunachalam et al (1996) reviewed the parasitic diseases in Indian elephants (Elephas maximus indicus). A variety of parasites have been reported in elephants including trematodes like amphistome: Fasciola jacksoni, Bivetellobilharzia nairi, Ornithobilharzia nairi, cestodes like Anoplocephala manubriata and nematodes like Amira pileata, Decruzia aditicta, Equinurbia siphunculiformis, Quilonia travencra, 10

27 Q. rennie, Murshidia murshida, M. falcifera, Haemonchus contortus, Grammocephalus elathratus, Syngamus indicus and Strongyloides sp. Islam and Lahkar (1996) reported a case of eye worm of Thelazia sp. in an elephant. Chakraborty and Islam (1996) conducted a coprological examination for endoparasitic infections in hog deer (Axis porcinus), swamp deer (Cervus duvauceli), water buffalo (Bubalus bubalis) and elephant (Elephas maximus) at Kaziranga National Park, Assam. A total of 171 samples were screened, of which 40.35% were found positive for parasitic infections. The various parasitic eggs recovered were of Paramphistomum sp., Fasciola sp., strongyles, Trichuris sp., Oesophagostomum sp., Strongyloides sp. and Ascaris sp. Islam et al (1996) reported a case of grammocephalosis due to Grammocephalus veredatus in an African elephant (Loxodonta africana) at Assam State Zoo, Guwahati. This hookworm unlike other hookworms was found in liver and a number of these parasites were also recovered from bile duct. Ganorkar et al (1997) reported the presence of 36 cysts of Echinococcus granulosus in the liver of a lion in a zoo in Nagpur, Maharashtra, India, at post mortem. Modi et al (1997a) studied the seasonal influence on the parasitic infection in both herbivore and carnivorous animals of Bihar zoo and found that maximum percentage of infection was observed in monsoon season i.e and per cent, respectively and minimum in summer and per cent, respectively. Modi et al (1997b) investigated the effect of age on the prevalence of gastrointestinal parasitism in zoo animals, which were grouped as young (<1 year old) and adult (>1 year old) animals. The faecal sample examination revealed that per cent of the herbivores and 50 per cent of the carnivores had protozoal and 11

28 helminthic infections. The young carnivores harboured significantly fewer parasites than the older ones, but no significant effect of age was observed in the herbivores. Panda and Ganguly (1997) reported the occurrence of Ascaris lumbricoides infection in captive monkeys and zoo personnel of Zoological Garden, Calcutta. The infection of A. lumbricoides was detected in and per cent samples from captive monkeys and zoo personnel and their families, respectively. On post mortem examination of a tiger, several tapeworms were recovered from duodenum and jejunum (Rao and Singh 1998). These worms were later identified as Diphyllobothrium latum and cestode of Cyclophyllidium sp. Kolte et al (1998) reported a big white coloured lemon sized cyst of Cysticercus tenuicollis on the peritoneum in a spotted deer (Axis axis). Gupta et al (1999), at necropsy of a lion, a lioness and a tigress found a bunch of heartworms in the right ventricle and pulmonary artery. Islam et al (1999) recovered both immature and mature Gastrodiscus secundus and Pseudodiscus collinsi from the caecum of 2 captive Asian elephant at Kaziranga National Park, Assam, India. Oedema, pin head size haemorrhages and ulcerative patches in the caecal mucosa were prominent. Ravindran and Pillai (1999) reported a case of pinworm infection in an Indian palm squirrel (Funambulus palmarum). The necropsy of the animal revealed that a large number of small nematodes were occluding the posterior part of intestine. The worms were later identified as Syphacia sciuri. Sabapara (1999) described a case of nematode infection in a young rustyspotted cat (Prionailurus rubiginosus), found in the wild and brought to a zoo at Gujarat for treatment. It died after 23 days. At post mortem nematodes were found in 12

29 the intestines and the faeces contained ova of Toxascaris leonina, Trichuris sp. and Ancylostoma sp. None of these species had been previously reported in this host. Varadharajan and Pythal (1999a) revealed that examination of 32 faecal samples from free living feral bonnet macaque (Macaca radiata), 30 (90%) were found positive for one or another parasite. The various parasitic eggs recovered were of strongyles, Strongyloides sp. and Ascaris sp. Coccidian oocysts were also recovered and some animals also had B. coli infection. The coprological study by Varadharajan and Pythal (1999b) in Zoological Garden, Thiruvananthapuram revealed that 74 and 23 per cent of the wild animals were harbouring helminthic and protozoan infections, respectively. Strongyles, amphistomes, Strongyloides sp. and Fasciola sp. in herbivores; Ancylostoma sp., Toxascaris sp., Diphyllobothrium sp. and Paragonimus sp. in carnivores and strongyles, Strongyloides sp. and Hymenolepis sp. in omnivores were the prominent infections recorded. Jayathangaraj et al (2000) recorded the presence of Bunostomum trigonocephalum in black bucks in Chennai, Tamilnadu, India. The black bucks were having history of inappetance, weakness, persistent diarrhoea and anemia and having a rough coat. On faecal examination, eggs and larvae of B. trigonocephalum were observed. Varadharajan and Kandasamy (2000) screened 60 faecal samples of wild animals at V.O.C Park and Mini Zoo, Coimbatore and found a high percentage (58%) of captive animals positive for helminth parasitic infection and only 6% of the animals were positive for protozoan infection. Strongyles, Trichuris sp., Strongyloides sp. as well as coccidia were recorded in herbivores and Toxocara sp., Ancylostoma sp. and Artyfechnostomum sp. in case of carnivores. 13

30 Xavier et al (2000) on analysis of faecal samples of 7 mammal groups at Silent Valley National Park, Kerala found that lion tailed macaque, Nilgiri langur, leopard, porcupine, Indian civet and toddy cat were hosts to nematodes. Cestode infestation was recorded in porcupine and Indian civet, while trematodes were found in leopard and toddy cat. The various parasitic eggs reported were of Trichuris sp., Oesophagostomum sp., Ancylostoma sp., Ascaris sp., Hymenolepis sp., Diphyllobothrium sp. and Paragonimus sp. During a post mortem examination, Agrawal and Chauhan (2001) found Ancylostoma duodenale and A. paraduodenale in a 7-year-old lion (Panthera leo) at a zoological park located at Gwalior. Chakraborty (2001) reported F. gigantica infection in 28 animals of different species, including Axis axis (spotted deer), Bos frontalis (gayals) and Bubalus bubalis (wild water buffalo), died at Assam State Zoo, Guwahati, India. Chakraborty and Goswami (2001) conducted necropsy of 85 captive nonhuman primates at Assam State Zoo and recovered Trichuris trichiura, filarial parasite, Enterobius vermicularis, Oesophagostomum aculeatum, Cysticercus sp. and F. gigantica. Coprological examination revealed the presence of other parasites viz. Ancylostoma sp., Ascaris sp., Strongyloides sp., Entamoeba sp., Balantidium sp. and Giardia sp. Similarly, Nashiruddulah and Chakraborty (2001) conducted postmortem of 60 captive wild carnivores at the Assam State Zoo. The various parasites recovered were Toxascaris leonina, Toxocara cati, T. canis, Ancylostoma caninum, Uncinaria sp., Rictularia sp., Gnathostoma spinigerum and Galonchus perniciosus in the gastrointestinal tract of various animals. Bronchostrongylus subcrenatus, Capillaria aerophila and Toxocara canis larva were recovered from lungs of Royal Bengal tiger and Dirofilaria immitis in heart of a lion. 14

31 Suresh et al (2001) examined clinical records for a period of 10 years ( ) to determine the prevalence of strongylosis in Asian elephants of Nehru Zoological Park, Hyderabad in relation to season, age and sex. The old records revealed that strongylosis was predominant in summer (52.63%) and prevalence was lower in animals below 15 years of age. Varadharajan et al (2001) investigated the prevalence of helminth parasites in wild mammals in the Thrissur zoo, Kerala. The overall prevalence of helminthic infections was found to be 68.05%. Carnivores exhibited the highest prevalence (75.34%) as compared to herbivores (64.47%) and omnivores (65.35%). They also observed a higher prevalence in adult animals (70.50%) than younger animals (54%). Dhoot and Upadhye (2002) found a large hydatid cyst in the liver of a lion on post mortem examination. It was identified as metacestode stage of Echinococcus granulosus. Hussain et al (2002) conducted a study to observe the prevalence of helminthic infection in axis deer at Nagpur. Out of 176 faecal samples examined, only 17 (9.71%) were found to be non-infected. Maximum percentage of infection was of Strongyloides sp. (30.86%) followed by mixed infection of strongyles and Trichuris sp. (27.44%), Trichuris sp. (12.57%), Nematodirus sp. (8.00%), Haemonchus sp. (6.29%) and Oesophagostomum sp. (5.14%). Jayathangaraj et al (2002) observed the occurrence of hookworm infection in civet cat (Viverricula malaccensis) at Arignar Anna Zoological Park, Vandalur, Tamil Nadu. Murata et al (2002) reported a fatal infection in a captive chimpanzee with human pinworm Enterobius vermicularis. At postmortem the animal had multifocal caecal nodules and histopathologically large number of pinworms was seen in the 15

32 nodular lesions in caecum and intestinal wall. Pinworms were also observed in the portal venule and parenchyma of the liver. Shrikhande et al (2002) reported the occurrence of schistosomosis in lions (Panthera leo persica) maintained by Gemini Circus, Nagpur. The animals were having a history of anorexia and diarrhoea for 2 days. The faecal samples were tested and were found to be positive for the eggs of Schistosoma spindale. Kashid et al (2003) analyzed faecal samples of various species of wild and zoo animals for presence of gastrointestinal helminths from 6 different locations of India. The worms recovered were: amphistomes, strongyles, Trichuris sp., Moniezia sp., Ascaridia galli, T. leonina, Raillietina tetragona, Paragonimus westermanii, Filaroides osleri, F. hirthi, Taenia taeniaeformis and Subulura sp. The animal species affected were lions, tigers, leopards, elephants, spotted deer, monkeys, peacocks, geese and ducks. Varadharajan and Subramanian (2003) examined faecal samples for the prevalence of parasitic infections in wild mammals in captivity with respect to their age at Thrissur zoo Kerala. Out of 961 faecal samples examined during the study period (May1998-April 1999), 654 samples (68.36%) were found positive for helminthic infection. Prevalence of helminthic infection in herbivores (71.62%) and omnivores (65.90%) was higher in animals aged 1 year and above as compared to their younger counter parts of age lesser than 1 year. Patel et al (2003) screened 156 pooled faecal samples collected at random from foxes, wolves and golden jackals at Ahmedabad, Baroda and Junagarh zoos in Gujarat. They found 132 (84.61%) samples positive for helminthic infections, of which 107 (80.06%) had nematodes and 25 samples (18.94%) had cestode infection. 16

33 Irrespective of the season, the predominant infection in wild canids was of hookworms (34.61%), followed by ascarids (24.35%). Kumar and Rao (2003) found that out of the 621 faecal samples examined from felids in Animal Rescue Centre at Vizag Zoo, Visakhapatnam, Andhra Pradesh, 96 samples (15.4%) were observed positive for helminthic infection. They observed that animals aged one year and less had higher prevalence (41%) as compared to 13.3 and 15.0% prevalence in the animals of the age of 2-5 years and more than 5 years, respectively. Nighot et al (2004) found that overall 26.12% population of Pench National Park, Maharashtra was showing helminthic infection. Out of which 19.59% were wild herbiovores and 34.17% were cattle. In wild herbivores highest prevalence was observed in bison (29.17%) followed by chital deer (28.57%). Strongyles (27.59%) were more commonly found in wild herbivores. Banerjee et al (2005) reported 17.7, 41.6, 73.6 and 63.6% samples of spotted deer, nilgai, wild pigs and elephants, respectively, as positive for either single or mixed parasitic infections through microscopic examination in a study carried out on samples collected from forests of Tarai region in South- Uttaranchal. Ravindran et al (2006) assessed the parasitic infection in captive lions at Wayanad, Kerala. Out of the 18 samples screened, 15 samples revealed presence of mixed infections. Singh et al (2006a) carried out a study to assess the gastrointestinal parasitic prevalence in wild herbivores of MCZP, Chhatbir, Punjab. The overall occurrence of gastrointestinal parasites based on screening of 389 faecal samples was found to be per cent. The most commonly detected parasitic infection (89%) was of strongyles followed by Trichuris sp. and amphisotomes. Singh et al (2006b) in the 17

34 same year also conducted a study to assess the parasitic prevalence in wild carnivores and observed an overall prevalence of 58.68%. Thilakan et al (2006) recovered a spirurid round worm, Parabronema smithi on necropsy of wild elephant calf in Mudumalai wild life sanctuary. Subramaniam et al (2006) carried out a scatological examination to assess gastrointestinal parasitic infection in lion tailed macaque at Parambiculam wild life sanctuary, Kerala. Out of 139 faecal samples screened, 64.75% were found positive for one or another infection. Mohan and Coumarane (2007) while screening the faecal samples of 18 spotted deer kept at Department of Forest and Wildlife, Puducherry, found that eight samples exhibited either individual or mixed parasitic infection. The most common eggs recovered were of the parasites Trichostrongylus axei, Cooperia punctata and Capillaria bovis. Concurrent infection of Gnathostoma spinigerum and Ancylostoma braziliense in a tigress at necropsy was reported by Thilakan et al (2007) at Vandaloor zoo, Chennai. Chandranaik et al (2008) found that out of 360 faecal samples screened over a period of one year from more than hundred lions at Bannerghatta Biological Park, 56.3% samples were found positive for T. leonina eggs. Sahoo et al (2009) carried out scatological study to assess gastrointestinal helminthic infection in animals of Nandankanan Zoological Park, Odisha. With 51.57% overall prevalence, maximum prevalence was recorded during monsoon (57.49%) followed by post-monsoon (49.10%) and pre-monsoon (47.62%) seasons. Highest degree of parasitic burden was recorded in carnivores (89.77%). Nematode parasites comprising Toxocara sp., Ancylostoma sp., strongyles, Trichuris sp. and Strongyloides sp., either singly or in concurrence were recorded in 97.03% animals. 18

35 Singh et al (2009a) assessed that out of 317 samples screened, 92 were found to be positive for helminthic eggs exhibiting a prevalence of 29.02% in 13 different wild omnivore species of MC Zoological Park, Chhatbir, Punjab. Singh et al (2009b) found an overall gastrointestinal parasitic prevalence of 38.17% in free ranging herbivores of Van Vihar National Park, Bhopal. Mahali et al (2010) studied the incidence and seasonal variation of gastrointestinal parasitic infections in captive wild carnivores in Nandankanan zoological park Odisha. Out of the 228 faecal samples (74 in rainy, 73 in winter and 81 in summer) examined, 138 (60.52%) were found positive for ova and oocysts of different gastrointestinal (GI) parasites. These included two trematodes, Schistosoma sp. (lion) and Euparyphium sp. (jungle cat); one cestode, Hymenolepis sp. (sloth bear); five nematodes, Ancyclostoma sp. (tiger, lion, leopard, hyaena and jungle cat), Toxocara sp. (tiger, lion, leopard and jungle cat), Toxascaris sp. (tiger, lion and jungle cat), Trichuris sp. (hyaena) and Physaloptera sp. (tiger); and oocysts of Isospora (tiger, lion and leopard). Incidence of Ancylostoma sp. was more common, followed by Toxocara sp. and Toxascaris sp. The overall incidence was highest during rainy season (63.51%), followed by summer (62.96%) and winter (54.29%). In addition to faecal sample examination, post-mortem examination of one common palm civet revealed the presence of Rictularia cahirensis. The gastrointestinal parasitic prevalence of big cats (tiger, lion and leopard) was studied coprologically by Singh et al (2010) in Van Vihar National Park, Bhopal. Out of 25 samples of captive felids (11 tiger, 8 lion and 6 leopard), 56% were positive for single or mixed parasitic infection. The prevalence of ascarids, strongyles and coccidia was 44, 32 and 16%, respectively. 19

36 Gupta et al (2011) conducted a study to determine the prevalence of gastrointestinal parasites in wild ruminants around Jabalpur, India. Coprological examination of different wild herbivores depicted the highest rate of parasitic infection in Sambar (90.0%) followed by Neelgai (86.67%) and Chital (80.0%). Sarmah et al (2011) studied the occurrence and morphology of Strongylus species in a free ranging one horned rhino (R. unicornis) from Kaziranga National Park, Assam. The morphological features of the recovered worms were compared and found to have taxonomical resemblance to the type species Strongylus edentates. Nath et al (2012) reported an incidence of 13.63% due to helminth parasites in non human primates of Assam State zoo. Singh et al (2012) assessed the endoparasitism in rescued antelopes at Van Vihar National Park, Bhopal and found an overall prevalence of 42.10% of gastrointestinal parasites including single or mixed parasitic infections. Molecular detection and characterization of T. leonina and T. cati was performed by Pawar et al (2012). Vatsya et al (2012) revealed the presence of Taenia taeniaeformis adult parasites during necropsy of a leopard of Rajaji National Park, Dehradun. Microscopic examination of its intestinal contents revealed ova and oocysts of gastrointestinal helminths and parasitic protozoa, respectively. Eggs of Paragonimus and Opisthorchis species were found in the sediments. Thawait et al (2014) studied the prevalence of gastrointestinal parasites in captive wild animals of Nandan Van zoo, Raipur (Chhattisgarh). Out of 210 faecal samples screened, 97 (46.2%) were found positive for the gastrointestinal parasitic eggs. The prevalence of gastrointestinal parasites was higher in primates (60%) followed by herbivores (45.6%) and carnivores (45.2%). 20

37 2.1.2 Gastrointestinal helminthosis of zoo birds The birds in zoo are often subjected to an additional stress of caged captivity, overcrowding and the environmental conditions which are conducive for the development of parasites. As a result, the birds in captivity generally harbour more parasitic infections as compared to their counterparts living freely. The incidence of helminthic parasites in all birds was observed higher than the zoo animals (carnivores, omnivores and herbivores) by Dhoot et al (2002). As such, despite a regular deworming regime, parasitic infections varying between 25 to 99% had been reported in zoo birds at various locations of India (Parsani et al 2007). Most of the studies carried out in past in India were mainly conducted on the pooled faecal screening of the captive birds. Patnaik and Acharjyo (1970) found that out of 80 birds of 18 species, 71 (88.75%) were infected with 28 different helminths (5 trematodes, 12 cestodes, 10 nematodes and one acanthocephalan) in Baranga zoo, Odisha. Patnaik et al (1970) reported a trematode Chaunocephalus ferox in open bill stork, found associated with intestinal nodular disease. The helminthic eggs in faecal samples of several avian species at Lucknow and Delhi zoos were detected by Chauhan et al (1973), who identified one or more of spiruroid, ascarid and capillarid types. The Indian peafowl (Pavo cristatus), Burmese peafowl (P. munticus) and guineafowl (Numida meleagris) were found infected with all three types of infections. Other than these, Strongyloides avium was found in golden pheasant (Chrysolophus pictus) and in parakeet (Psittacula eupatsia) and hymenolepid in sarus crane (Grus antigone) and common crane (G. grus). All of these parasitoses were without apparent clinical symptoms. 21

38 Prevalence of gastrointestinal parasites in captive birds was reported coprologically by Reddy et al (1992) at Bannerghatta National Park, Bangalore. The examination revealed the presence of nematodes (31%), comprising of Ascaridia sp., strongyle and Capillaria sp. in almost equal proportion, followed by the tapeworm Hymenolepis (6.9%). In birds maintained at the zoos in Gujarat, per cent faecal samples were found positive (Patel et al 2000). A study in Maharajbagh zoo, Nagpur (Dhoot et al. 2002) exhibited the presence of Ascaridia and Capillaria species predominantly in birds. Raillietina tetragona and Subulura brumpti were also recorded from cockatoo (Cacatua galerita) and R. tetragona and Davainea sp. in peafowl. Kashid et al (2003) reported an incidence of 88.33% parasitic infections in peacocks, predominantly helminthic in nature in six different locations of Maharashtra. Faecal screening for endoparasites among free-ranging peafowl at Tirunelveli and Kanyakumari in Tamil Nadu (Subramanian et al. 2003) revealed the infection with a range of nematodes (Heterakis, Ascaridia, Capillaria, Syngamus, and Strongyloides species), an acanthocephalan and an unidentified cestode egg. Selvaraj et al (2003) recovered acuarid worms from the gizzard and proventriculus, which were identified as Skrjabinocara squamata at necropsy of three cormorants (Phalacrocorax niger) in Chennai (Tamil Nadu). The detailed scatological assessment of parasitic infections in captive zoo birds in Ahmedabad, Baroda, Junagadh and Rajkot (Parsani et al 2003) revealed 57.32% (133/ 232) pooled faecal samples positive, where eggs of Ascaridia and Capillaria sp. were predominant. Trichostrongylus sp., Strongyloides sp., spiruroid, Dispharynx and Heterakis sp. were also identified. 22

39 Examination of 92 viscera of pigeons in and around Thrissur revealed an overall prevalence of 75.0% of helminthic infection (Senthilvel and Pillai 2005). Mixed infection of helminths (55.43%) was observed predominantly than single infection (19.57%). Parsani et al (2007) assessesd parasitic prevalence of captive zoo birds in Gujarat (Kamla Nehru Zoological Garden, Kankaria Zoo, Ahmedabad; Shri Sayaji Bagh, Baroda; Sakkarbagh Zoo, Junagadh and Rajkot Municipal Corporation Zoo, Rajkot). Out of 232 pooled faecal samples (cage wise) examined from different wild birds, 132 (57.32%) were found positive for parasitic infections. Eggs of Ascaridia sp. and Capillaria sp. were observed in 56 (51.85%) and 35 (32.40%) pooled faecal samples, respectively. At Maharajbagh zoo, Nagpur, heavy parasitic infection of pigeons with gastrointestinal helminths, viz. Capillaria sp., Ascaridia sp. and Heterakis sp. was recorded (Borghare et al 2009). Islam et al (2009a) described the prevalence and pathology of Chaunocephalus ferox in a free living open billed stork (Anastomus oscitans) from Kaziranga National Park, Assam. In the same year, Islam and Talukdar reported the presence of Contracaecum spiculigerum in a wild spotted billed pelican from Assam. Also, Islam et al (2009b) reported extensive nodular lesions on the wall of small intestine of Greater Adjutant stork (Leptoptilos dubius) at necropsy, which were induced by the echinostomatid parasite Balfouria monogama. Parasitic infections in captive birds at Sayaji Bagh, Baroda through faecal examination, exhibited 73.01% positivity (Parsani and Momin 2009), where trematode infection was recorded high (80.69%) in carnivorous birds. Fatal Ascaridia galli infection in guinea fowl (Numida meleagris) was reported from Jammu region (Sudhan et al 2009). Postmortem examination of 23

40 intestinal tract of the common myna (Acridotheres tristis) at Namakkal, Tamil Nadu (Ponnudurai et al 2009) exhibited Diplotriaena and Raillietina species infection. Sahoo et al (2009) assessed the prevalence of gastrointestinal parasites of captive birds of Nandankanan zoo, Odisha. Forty-four species of birds were screened for presence of gastrointestinal parasitic infection during three seasons. The overall prevalence rate was 29.54% (39/ 132). The nematode parasites were predominant, where Ascaridia, Capillaria, Strongyle and Strongyloides species were diagnosed in 43.10, 18.97, 6.90 and 3.45% birds, respectively. A report from Maharajbagh zoo, Nagpur also reported the presence of Prosthogonimus sp. in captive wild birds (Gawande et al 2010). Roy et al (2011) studied the prevalence of endoparasites in pigeons of Kamakhya Temple premises. Single or mixed infection of cestode, nematode and protozoan parasites were recorded during post-mortem examination of 40 pigeons. Infection with Raillietina sp. (45%) was most predominant followed by Capillaria obsignata (40%), Ornithostrongylus quadriradiatus (22.5%), Strongyloides avium (15.0%), Hymenolepis sp. (10%), Cotugnia sp. (5.0%) and Ascaridia columbae (2.5%). Parsani et al (2014) conducted a study to assess the prevalence of helminth parasites of domestic, wild and zoo pigeons in Gujarat, India, by coprology and postmortem examination. Qualitative examination of 78 faecal samples revealed 71 (91%) with parasitic infections of nematodes (85%), cestodes (31%) and Eimeria sp (77%). On post-mortems, 85% of the birds showed parasitic infections. Two species of nematodes (Ascaridia columbae and Capillaria obsignata) and five species of three genera of cestodes (Raillietina echinobothridia, R. tetragona, R. cesticillus, Cotugnia digonopora and Hymenolepis sp.) were identified. 24

41 Kamdi et al (2015) reported the presence of Ascaridia galli infection in cattle egret (Bubulcus ibis) at necropsy from Nagpur Gastrointestinal protozoans in captive wild animals Patnaik and Acharjyo (1970) reported two coccidia belonging to genus Eimeria from intestinal scrapings of 3 dead slow loris (Nycticebus bengalensis) and two Isospora species (Isospora leonina and Isospora felis) in the faecal samples of an African lion from Nandankanan Zoo. Patnaik and Acharjyo (1971) performed screening of the mammalian stock of the Nandankanan zoo (Odisha) for coccidian infestation and described 4 new species i.e. Isospora pardusi from leopard (Panthera pardus), Isospora felina and I. bengalensis from leopard cat (Felis bengalensis) and Eimeria gauresi from Indian bison (Bos gaurus). In addition, they detected Eimeria felis, E. felina and Isospora leonina from Indian lion, Eimeria harmani and E. novowenyoni from tiger, Isospora levinei from striped hyaena and a number of different Eimeria oocysts from herbivores. Khan et al (1981) reported occurrence of infection of Balantidium coli in various species of monkeys kept at the Nehru Zoological Park, Hyderabad. The findings revealed that out of the 23 species of monkeys, 10 species were found to be positive for B. coli during faecal examination. Chaudhuri and Choudhary (1982) described the relationship between sporulation and effect of temperature and chemical exposure on coccidian oocysts recovered from a white tiger in Zoological Garden of Calcutta. The time required for sporulation (80%) was found to be ranging between hrs at room temperature (25-35 C) with proper aeration. The average size of oocyst was measured to be 46.0 m 31.0 m. 25

42 Khan et al (1983) reported amoebiasis in non human primates of Nehru Zoological Park, Hyderabad. Out of the 25 species of monkey, 7 were found to be positive for Entamoeba histolytica. Five of these 7 species showed no symptoms, whereas the chimpanzee and spider monkeys showed signs of fever, anorexia and dysentery. Reddy et al (1984) recorded the occurrence of balantidiosis among white rhinos at Nehru Zoological Park, Hyderabad. Two rhinos (1 male and 1 female) developed signs of foetid diarrhoea with mucus, loss of appetite, weakness and emaciation. Acharjyo and Rao (1988) on histological examination of different tissues of 128 wild ruminants belonging to 10 species, died at Nandankanan zoo between , found sarcocysts in sambar, nilgai, four horned antelope and mithun. It was suggested that all 4 species might have got infection from feed contaminated with faeces of carnivores. Rao et al (1992) reported balantidiosis in an orangutan, presented with history of watery diarrhoea. Gangadharan et al (1996) recorded occurrence of Sarcocystis cysts in the heart muscles of the Indian gaur, which was brought for necropsy from Parambikkulam Wildlife Sanctuary, Kerala. The presence of Isospora viverrina was reported by Agrawal and Chauhan (1993) during coprology of fishing cat. Pande and Pal (1996) studied the association of B. coli with other infections in captive monkeys. The results revealed that B. coli infection was maximally associated with E.coli and Trichuris trichiura followed by Ancylostoma sp., Trichomonas tenax and Entamoeba histolytica infections. Sinha and Prasad (1999) reported a case of intestinal amoebiasis in a lioness. 26

43 Entamoeba coli had also been found in faeces of chimpanzees and bonnet macaques (Acharjyo 2000). Three protozoan parasites, viz. Entamoeba sp., Balantidium sp. and Giardia sp. were recorded from non human primates of Assam State zoo (Chakraborty and Goswami 2001). The species recorded from chital (Axis axis) with dhole (Cuon alpinus) as definitive host, was identified as Sarcocytis axicuonis (Jog et al 2003) from Mudumalai National Park and wildlife sanctuary. Eimeria as a part of gastrointestinal parasites had been observed in nilgai and spotted deer in Uttaranchal (Banerjee et al 2005) and in sambar at MC Zoological Park Chhatbir, Punjab (Singh et al 2006) and more recently in sambar of Periyar Wildlife Sanctuary in Kerala (Ravindran et al 2011). Isospora felis had been reported during scatological studies carried out on the faecal samples of tigers and lions by Singh et al (2006) in MC Zoological Park, Chhatbir and Mahali et al (2010) at Nnandankanan zoo, Odisha. Isospora species was reported coprologically in leopards at Nandankanan zoo (Mahali et al 2010) and Kerala zoo (Ravindran et al 2011) Gastrointestinal protozoans in captive wild birds The most prevalent protozoan infections in zoo birds have been incriminated to coccidia in the past. Chauhan et al (1973) carried out a study on birds of Delhi and Lucknow zoos and identified Eimeria dubeyi and E.vanmurghavi from red jungle fowl (Gallus gallus); Isospora choudarii from grey jungle fowl (G. sonneratii); E. gorakhpuri from guinea fowl; E. mayurai, E. pavonis, E. mandali and I. mayuri from peafowl; E. teetari from black partridge (Francolinus francolinus) and chakor partridge (Caecabis chakor); E. pictus from golden pheasant (Chrysolophus pictus) and silver pheasant (Lophura nyathemera nippone); E.meleagridis or E.adenoeides in 27

44 Lady Amherst s pheasant (Chrysolophus amherstiae); E. tropicalis from white pigeon (Columba livia); E. grusi from sarus crane (Grus antigona); I. koreani from Korean pheasant (Phasianus colchicus); E. choudari from ring dove (Streptopelia decasto) and I. mandari from Mandarin duck (Aix glericulata). Reddy et al (1992) also reported the presence of coccidian oocysts in captive peacocks, rock pigeon, parrot (Psittacula krarneri), silver pheasant and grey jungle fowl at a zoological park in Bangalore. A study on the prevalence of Toxoplasma antibodies in sera of chickens, pigeons, turkeys, geese and quails revealed the highest positivity in pigeons (Geetha et al 2002). Eimeria and Isospora oocysts had been identified in free-ranging Indian peafowl in Tamil Nadu (Subramanian et al 2003). A study performed by Parsani et al (2003) on gastrointestinal parasites of captive birds at a zoo in Ahmedabad revealed 85.48% of 138 pooled faecal samples positive for coccidian oocysts. Bandopadhyay et al (2006) reported coccidia species in the blue rock pigeon (Columa livia). Eimeria columbae, E. kapotei, E. labbeana, E. tropicalis, Wenyonella columbae and E. janovyi were the species observed. Parsani et al (2007) reported per cent of 232 pooled faecal samples (cage-wise) of different wild birds of four zoos of Gujarat positive for Eimeria oocysts. However, the species were not identified. Incidence of coccidiosis in captive wild birds was studied by Jayathangaraj et al (2008) at Arignar Anna Zoological Park, Vandalur. Among the samples examined, eight samples from Pariah kites (Milvus migrans) and three samples from love birds (Agapornis sp.) revealed the presence of coccidiosis. 28

45 Recently, coccidian oocysts had been reported in the faecal samples of the captive zoo birds of Gujarat (Parsani et al 2009), Nandankanan zoo, Odisha (Sahoo et al 2009) and in pigeons of Kamakhya Devi Temple, Assam (Roy et al 2011). Karabasanavar and Deshpande (2012) found that out of 70 scat samples examined, about 40 (57.14%) were found to be positive for Isospora sp. infections in sparrows (Passer domesticus) of Maharashtra Haemoparasitic infection in captive wild animals and birds in India Khurana (1969) reported a case of babesiosis in white tiger at Delhi Zoological Park. The tiger was having high temperature (104.6 F), was dull, off feed and passed dark coloured urine. Blood smears examination revealed haemoprotozoa resembling piroplasms. Reddy et al (1975) recorded the outbreak of surra in tigers of circus stationed at Vijaywada. A total of 6 tigers were reported ill. On clinical examination, temperature was observed between 106 F F and blood smears revealed Trypanosoma evansi in all the tigers. Khan et al (1980) reported the cases of trypanosomosis in various species of wild cats at the Nehru Zoological Park, Hyderabad. Out of total 15 cats, 8 were positive for parasites in the blood smear and rest showed clinical signs. All animals responded well to the treatment with Antrycide and Berenil. Gopalakrishanan (1982) reported a case of trypanosomosis in a tigress at Madras Zoo. The animal was presented with a history of high fever (104.2 F F), dullness, lethargy. The blood smear examination revealed that animal was positive for T. evansi. Khan et al (1982) reported trypanosomosis in a white tiger at Nehru Zoological Park, Hyderabad, India and treated it successfully with Berenil (2.4 g as 29

46 total dose) intramuscularly. Khan et al (1985) reported a case of surra in jackal at Nehru Zoological Park, Hyderabad and treated it successfully with Antrycide prosalt. The other jackals were given prophylactic dose of Antrycide 2 mg/ kg body wt. subcutaneously. Choudary et al (1986) reported death of a male tiger cub due to trypanosomosis in Nehru Zoological Park, Hyderabad. T. evansi was demonstrated in the heart blood smear at post mortem. Sarode and Pawshe (1990) recorded a case of anaplasmosis in a captive lioness. Blood examination revealed leukocytosis, decreased heamoglobin (8 g%) and presence of Anaplasma marginale in blood smears. The animal was treated with 10 mg/ kg body wt. in dextrose saline by intravenous route. Ahmed et al (1990) had placed on record a very rare case of feline anaplasmosis in a white tiger. The infection seemed to be chronic in nature, which resulted ultimately into severe anemia, icterus and death due to coli septicemia. Rao et al (1992) reported an outbreak of pigeon malaria (Haemoproteus columbae) in which 25 pigeons died at a zoo in Hyderabad. The haemosporids identified as intraerythrocytic gametocytes and were observed concurrently with Ornithostrongylus quadriradiatus infection. Ziauddin et al (1992a) reported death of a tigress in zoo due to trypanosomosis. The tigress showed high temperature (104 F) and blood smears revealed parasites similar in morphology to Trypanosoma evansi. The animal died the next day despite of the treatment, which was attributed to the fact that time between onset of symptoms and death was very short due to acute stage of disease. Similarly, Ziauddin et al (1992b) also reported a case of death due to trypanosomosis in a wolf 30

47 at Mysore zoo. On post-mortem, the impression smears from heart, lung, liver and spleen revealed presence of T. evansi. Rao et al (1995) reported surra outbreak in circus tigers belonging to National circus at Kakinada (Andhra Pradesh). Five adults and 4 tiger cubs were infected by T. evansi as revealed after blood smear examination. The temperature was recorded to be between 104 F- 106 F. The animals were treated with g/ 100 kg body wt. intramuscularly. Temperature started decreasing from day 2-post treatment onwards and reached normal by day 3-post treatment. Singh et al (1997) reported a case of trypanosomosis in a tigress at Lucknow zoo. The animal was taking feed partially and her body temperature was 104 F. Blood smear revealed T. evansi and she was treated with 0.8 g/ 100 pounds body wt and a week later Berenil was again 0.4 g/ 100 pounds body wt and on subsequent day the blood smears were observed negative for T. evansi. Singh et al (2000) conducted haematological and biochemical profile of a white tiger suffering from trypanosomosis. The blood examination revealed lower heamoglobin, total erythrocyte count (TEC), packed cell volume (PCV) and mean corpuscular volume (MCV) than the values of respective parameters recorded in healthy tigers. Erythrocyte sedimentation rate values were higher than the healthy tigers and the anemia recorded was microcytic normochromic type. Sinha et al (2000) reported a case of babesiosis in a tigress. The tigress was brought with the history of being off feed and passing red coloured urine. The body temperature was recorded to be 105 F. Hyper pyrexia confirmed clinically a case of babesiosis, so animal was treated with 1.5 g dissolved in 15 ml distilled water and given intramuscularly. The colour of urine and appetite became normal at very next day. 31

48 Upadhye and Dhoot (2000a) reported a case of trypanosomosis in a tiger at Maharajbag zoo, Nagpur. The animal showed sign of rapid respiration, panting with inclination to press the head on the bars of the cage and body temperature was F. The blood smears revealed the presence of T. evansi. Tiger showed complete recovery on the 6 th day post treatment with 3 g intramuscularly. Upadhye and Dhoot (2000b) reported a case of sudden death of a leopard due to babesiosis. On post mortem, the peripheral and heart blood smears revealed Babesia sp. infection. The animal didn t show the typical signs of babesiosis like hyperpyrexia, jaundice, haemoglobinurea and haemolytic anemia. Parija and Bhattacharya (2001) reported an outbreak of trypanosomosis, in which 12 tigers (8 white and 4 normal) died despite of treatment at Nandankanan Zoological Park, Odisha. Devasena and Shobhamani (2006) reported trypanosomosis in a tigress and treated it successfully with diminazine aceturate. A few cases of babesiois had also been reported in the past decade, especially in captive wild felids, such as low grade parasitaemia was reported in a lion in Lucknow zoo by Haque (2007). Mishra et al (2008) reported the presence of Babesia sp. in a white tigress died at MC Zoological Park, Chhatbir, Punjab. A report on clinical trypanosomosis concurrent with tuberculosis accompanied by stress of cold weather in black bucks and the successful treatment has been presented from a deer park near Bathinda (Gupta et al 2009). Recently, and 6.76 per cent prevalence of Haemoproteus and Plasmodium species respectively, was reported in 266 rock pigeons (Gupta et al 2011) Ectoparasites in captive wild animals and birds in India Herbivores: Various reports of the infestation of the herbivores with fly larvae, fleas and ticks have been recorded in the past. The deer warble Hypoderma diana (Islam et 32

49 al 2003) had been recorded from hog deer in two national parks in Assam. Fleas recorded include Ancistropsylla sp. from sambar and barking deer in the Nilgiris (Joseph and Mani 1980) and Vermipsylla sp. fleas from swamp deer at Nandankanan zoo, Odisha. Geevarghese et al (1997) enlisted Amblyomma testudinarium parasitizing sambar deer and Haemaphysalis birmaniae in barking deer and serow. Rhipicephalus haemaphysaloides and Otobius megnini were also found prevalent in Artiodactyla. Geevarghese et al (1997) also enlisted several tick species infesting wild boar, viz. Dermacentor auratus, Haemaphysalis (5 species recorded from various parts of the country), Hyalomma dromedarii and Nosomma monstrosum. Easwaran et al (2003) identified Amblyomma sp. and pig louse Haematopinus suis from wild boar at Thekkady, Kerala. Omnivores: Most commonly, monkeys were found infested with ectoparasites including mites and ticks. Sarcoptic mange had been recorded in a large number of Hanuman langurs (Semnopithecus entellus) around Jodhpur, Rajasthan (Chhangani et al 2001). Demodicosis associated with alopecia in a rhesus monkey (Macaca mulatta) had also been reported from Pune (Nighot et al 2011). Whereas, Geevarghese et al (1997) had listed some tick species of genus Haemaphysalis, which were found parasitizing monkeys, viz. H. bispinosa, H. obesa, H. cuspidata, H. kinneari, H. wellingtoni and H. turturis. Carnivores: Sarcoptes scabiei mange infestation in lions at Bikaner zoo as reported by Gaurav and Singh (1999). Nashiruddullah and Chakraborty (2001) reported myiasis in footpad wound due to Sarcophaga sp. in lion at Assam State Zoo. Severe infestation with flea Ctenocephalides felis felis in leopard cat (F. bengalensis) was reported from Assam (Islam 2010). 33

50 Geevarghese et al (1997) enlisted the ticks of genus Haemaphysalis from wild felids such as H. silvafelis on jungle cat, H. kinneari on tiger, H. cuspidata on leopard and also Amblyomma hebraeum was observed on tiger. An unidentified Amblyomma sp. tick was also reported from tiger at Assam State Zoo (Nashiruddullah and Chakraborty 2001). Birds: Phthirapteran ectoparasites had been observed most commonly on Indian birds (Saxena et al 2007, Khan et al 2009). The common myna (Acridotheres tristis) was reported heavily infested with Menacanthus eurysternus (Chandra et al 1990). Menacanthus stramineus had been found on free-ranging peafowl in Tamil Nadu (Subramanian and Raman 2002) and on bush-dwelling grey partridge (Perdix perdix) in Tamil Nadu (Ponnudurai et al 2003). Singh et al (2009) observed a brown francolin (Francolinus pondicerianus) infested with two species i.e. M. kalatitar and Goniocotes jirufi in Uttarakhand. Four species of lice were found prevalent in wild pigeons in Rampur, Utter Pradesh (Khan et al 2009) viz. Columbicola columbae, Companulotes bidentatus compar, Calpocephalum turbinatum and Hohoretiella lata in descending order of prevalence. More recently, prevalence of Myrsidea salimalii an amblyceran louse was found in striated babblers (Turdoides earlei) in U.P. (Ahmad et al 2011). Ectoparasites reported on free-ranging peafowls included the lice Menacanthus sp., flea Ctenocephalides felis orientalis and tick Haemaphysalis sp. (Subramanian and Raman 2002). Mite infestation with Dermanyssus gallinae in 6.52% psittacid and phasiamid birds at Baroda, Gujarat was observed by Parsani and Momin (2009). 34

51 2.2 International scenario Silva et al (1973) on necropsies and coprological investigations of wild animals from the Lisbon Zoo, Portugal, found that 52.8% of the animals had been infected with parasites, out of which 83.5% of the parasites were helminths and the remaining 16.5% were ectoparasitic ticks swallowed with the food. During the necropsy of an elephant, succumbed to an intestinal infection, four nematode species had been found in the digestive tract, including Choniangium magnostomum, Equinurbia sipunculiformis, Murshidia murshida and M. falcifera from Lisbon zoo (Silva 1974). Periodical investigations of the predatory animals from the East Berlin Zoo had shown that the most widely distributed parasites among the Felidae, Ursidae, Canidae and Procyonidae were also nematodes, mainly ascarids (Tscherner 1974). In the captive wild canids, ancylostomids were found predominantly and in Asian and South-American cats, cestodes of the Spirometra genus were predominant. Other data for the parasitological status of the animals from the East Berlin zoo had been reported by Priemer (1980) and Priemer and Tscherner (1992). Priemer (1980) had detected larval cestodosis caused by the Mesocestoides genus in snakes of the species Elaphe schrencki, Boiga cyanea, Agkistrodon rhodostoma and monkeys of the species Macaca sylvana and Cercopithecus aethiops. Priemer and Tscherner (1992) had investigated cestode fauna of 97 avian species of different orders. They reported 57 cestode species belonging to following genera: Ligula, Diphyllobothrium, Schistocephalus, Nematoparataenia, Cladotaenia, Diploposthe, Davainea, Cotugnia, Fimbriaria, Aploparaksis (Monorchis), Armadoskrjabinia, Cloacotaenia, Dicranotaenia (Wardium), Diorchis, Drepanidotaenia, Flamingolepis, Hispaniolepis, Microsomacanthus, Oshmarinolepis, Passerilepis, Retinometra, Skrjabinoparaxis, 35

52 Sobolevicanthus, Variolepis, Anomotaenia, Biuterina, Dilepis, Gryporhynchus, Lateriporus, Malika, Monopylidium, Neogryporhynhus, Paradilepis, Paruterina (Notopentorchis) and Polycercus. Data about occurrence of trematodes of the suborder Paramphistomata in animals from some European zoos was presented in the study of Pacenovsky and Krupicer (1975). The following paramphistomids had been found: Tugumea heterocaeca in elephant (Vienna zoo), Gigantocotyle explanatum in antelope Damaliscus albifrons (Rotterdam zoo), Liorchis scotiae in European bison (Beloveja Reserve), Stichorchis subtriguertus in Eurasian beaver (a reserve in Bern), Calicophoron calicophorum in antelope Connochaetes gnu (Basel zoo). Scatological examination of 48 mammals and four birds revealed 95 percent of the samples to be positive at the Dublin Zoological Garden (Geraghty et al 1982). In the Wroclaw zoo, helminthological studies on monkeys by larvoscopical methods and necropsies had been performed by Krynicka et al (1979). The following parasitic species were recovered: Trichocephalus trichiuris, Strongyloides papillosus, S. stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, Capillaria sp., Enterobius sp., Hymenolepis diminuta, Oesophagostomum apiostomum and Dicrocoelium dendriticum. The eggs of H. diminuta had been found in monkey s faeces, the adults in the rats, and the cycticercoids in the cockroachs of the same zoo. These findings confirmed the role of invertebrates and rodents in the transmission of parasites among zoo animals. Another study on the intestinal parasites of monkeys in the Wroclaw zoo (Oculewich and Kruczkowska 1988) had depicted an 80% prevalence rate. Six species of nematodes had been recovered during the study. The intensity of infections had been low except for Enterobius vermicularis in chimpanzees. 36

53 During the examination of dead animals from the Antwerp Zoological Garden, some capillariids had been found, including Capillaria contorta at the tongue-root of a raven and C. hepatica in the liver of a monkey (Kumar et al 1983). Hartmanova et al (1987) found Trichiuris trichiura and Enterobius vermicularis in monkeys and Toxascaris leonina in large felines at Brno Zoological Garden. In Sofia zoo, from almost all monkey species (chimpanzees, marmosets, macaques, baboons, mandrills, capuchin monkeys and others) Trichocephalus sp. had been recovered by Matevsky (1988). In some chimpanzees strongyloid larvae were also found. Tigin et al (1989) during investigation of the helminthological status of some mammals and birds in the Ankara zoo, found that infection rate was higher in omnivores (50%). The birds had been found infected with Heterakis sp., Strongyloides sp., Ascaridia sp., Capillaria sp. and Trichostrongylus sp. In carnivores, T. canis, T. cati, Toxascaris leonina, Uncinaria sp., Strongyloides sp., Trichuris sp., Taenia sp. and Dipylidium caninum were major species and in herbivores: Trichostrongylidae, Muellerius capillaris, Cystocaulus ocreatus, Protostrongylus sp., Trichuris sp., Skrjabinema ovis, Strongylus sp. and Passalurus ambiguus were encountered. In omnivores, Trichuris sp., Strongyloides sp. and Enterobius sp. were reported. Series of examination on the helminth fauna of different animals from the Belgrade zoo had been carried out (Pavlovic et al 1990, Pavlovic et al 1991, Nesic et al 1991, Nesic et al 1992). In pheasants, subclinical infections with Raillietina tetragona, Ascaridia columbae, Syngamus tracheae, Heterakis gallinae, Acuaria hamulosa, Ornithostrongylus quadriradiatus, Capillaria columbae and C. gallinarum had been registered. The presence of infections was observed despite of good hygiene 37

54 and modern technologies of breeding and incriminated to the contact between the pheasants and the local wild birds (Pavlovic et al 1990). Subclinical infections with species of Trichonema and Strongylus had been found in an elephant and of Oesophagostomum columbianum in a giraffe (Pavlovic et al 1991). Similarly subclinical infections of Ascaris lumbricoides in chimpanzees and mangabey monkeys, Enterobius vermicularis in chimpanzees, rhesus monkeys and lemurs, Trichostrongylus sp. in Diana monkeys (Nesic et al 1991), Trichostrongylus capricola, Nematodirus filicollis, Chabertia ovina in chamois and mouflons, Haemonchus contortus in chamois, red deer and roe deer, O. venulosum in mouflons and roe deer, Trichostrongylus sp., N. filicollis, Ostertagia circumcincta in red deer, and Ostertagia sp. and Trichostrongylus colubriformis in roe deer (Nesic et al 1992) had been reported. Kharchenko and Marunchin (1992) on coprological investigations of animals kept at Kiev zoo found helminth infestation in cats to be 52%, including T. leonina (40%), strongyles (8.7%), Toxocara mystax (8.7%), Trichuris sp. (4.3%) and Diphylobothrium sp. (4.3%). Low infection of polar fox with Diphylobothrium sp. had been observed. Only one brown bear had been found infected with Toxascaris transfuga. Helminths had not been recovered from primates. Kharchenko and Marunchin had also performed necropsies of dead animals and observed four nematode species- Trichuris trichiura and Subulura distans (in a southern pigtail macaque), Murshidia murshida (in an Indian elephant), T. globulosa (in a giraffe), one fluke- Hawkesius hawkesi (in an Indian elephant) and one species of Acanthocephala- Prostenorchis elegans (in a common squirrel monkey). A 12-month survey of the gastrointestinal helminths of antelopes, gazelles and giraffids kept at two zoos in Belgium (Goossens et al 2005) revealed 36.5% of the 38

55 faecal samples infected with nematode eggs. Faecal egg counts had been recorded higher during the mid-grazing season (July) and had peaked at the end of the grazing season (October). The nematode species recovered from some dead animals included Camelostrongylus mentulatus, Trichostrongylus retortaeformis, Nematodirus fillicollis, Capillaria sp. and Trichuris sp. Seroprevalences to detect antibodies of Neospora caninum and Toxoplasma gondii was conducted in 13 Czech and Slovak zoos (Sedlák and Bártová 2006). Antibodies to N. caninum had been found in 5.6% of zoo animals including Eurasian wolf (Canis lupus lupus), maned wolf (Chrysocyon brachyurus), fennec (Vulpes zerda), cheetah (Acinonyx jubatus), jaguarundi (Herpailurus yaguarondi), Eurasian lynx (Lynx lynx), Indian lion (Panthera leo), fisher (Martes pennanti), black buck (Antilope cervicapra), European bison (Bison bonasus), lechwe (Kobus leche), African buffalo (Syncerus caffer caffer), eland (Taurotragus oryx), sitatunga (Tragelaphus spekei gratus), Thorold s deer (Cervus albirostris), Eastern elk (Cervus elaphus canadensis), Vietnam sika deer (C. nippon pseudaxis) and Pere David s deer (Elaphurus davidianus). Antibodies against T. gondii had been found in fairly more number (34.7%) and species of animals. The highest prevalence of parasites had been found in carnivores i.e. 50% for N. caninum in the family Mustelidae and 100% for T. gondii in Hyaenidae, Mustelidae, Ursidae and Viveridae. Melfi and Poyser (2007) while studying the factors governing the prevalence of Trichuris sp. in Abyssinian colobus (Colobus guereza kikuyensis) at Paignton Environmental Park, observed that shedding of the eggs had been significantly higher in the afternoon than in the morning and in dominant adult male colobus. The season wise survey of gastrointestinal parasites had been carried out for a year in Almunecar Zoological Garden, Spain (Cordón et al 2008). Primates, 39

56 Carnivora, Perissodactyla, Artiodactyla, Rodentia, Diprotodontia, Galliformes, Anseriformes and Struthioniformes had been examined. One or more intestinal parasites had been identified in 72.5% of the animals. The most frequent pathogenic endoparasites recorded were Eimeria sp. (17.3%), Trichuris sp. (5.1%), Strongyloides sp. (4.5%), Cyclospora sp. (4.5%), Cryptosporidium sp. (3.2%) and Isospora sp. (2.6%). Multiple parasitic infections had been common, where 70% of the animals had been presented with at least two parasites. Lim et al (2008) examined 197 faecal samples, collected randomly from various primates (99), hoofed mammals (70) and feline (28) from zoological garden in Malaysia. It was observed that 89.3% of feline, 54.5% of primates and 45.7% of hoofed mammals were infected with intestinal parasites. Birds from Psittacidae, Cacatuidae, Phadianidae, and Anatidae from the Almunecar Ornithological Garden (Grenada, Spain) had been surveyed for intestinal and blood parasites (Cordón et al 2009). Intestinal parasites had been found in 51.6% and blood parasites in 26.8% of the samples. Nejsum et al (2010) had searched for explanation of a persistent ascarid infection among the chimpanzees in the Copenhagen zoo. It had been unknown whether the recurrence of the infection had been provoked by the reintroduction of eggs from an external source or by a sustained transmission cycle within the zoo. Molecular- genetic analyses had revealed that the chimpanzees are infected with pig ascarid, Ascaris suum, which in spite of control efforts had established itself into a permanent transmission cycle in the zoo s chimpanzee troop. The prevalence of gastrointestinal parasites had been investigated in mammals (primates, carnivores, perissodactyls, artiodactyls and proboscideans) housed in two of the main Italian Zoological Gardens: the Zoo Safari of Fasano- province of Bindisi, 40

57 Apulia and the Giardino Zoologico of Pistoia- Tuscany (Fagiolini et al 2010). Overall, 10% (Cryptosporidium sp.) and 43.3% (Toxocara cati, Strongyloides stercoralis, Toxascaris leonina and hookworms) of carnivores, 66.7% (Cryptosporidium sp.) and 100% (Trichuris sp. and Strongyloides flleborni) of primates, 25% (Eimeria sp., Cryptosporidium sp.), and 57.1% (Trichuris sp., Neoascaris vitulorum, gastrointestinal strongyles, and Paramphistomidae) of artiodactyls had been infected with protozoa and helminth parasites, respectively. The potential of captive wild ruminants to serve as reservoirs for zoonotic parasites, Cryptosporidium and Giardia had been studied by a commercial immunofluoresence assay in different animal species from the Antwerp Zoo and in American bison (Bison bison) from a commercial breeding farm in Belgium (Geurden et al 2009). The Cryptosporidium prevalence had been found to be 7.5% in the Antwerp Zoo animals and 3.7% in the bison from the breeding farm, whereas, the prevalence of Giardia had been 8.9% and 23.2%, respectively. A survey about spreading of metastrongyloid lungworms in the red panda population had been conducted within 20 European zoos (Bertelsen et al 2010). It had been found that 35% of the animals from 37% of the zoos shed metastrongyloid first stage larvae. Morphologically they had been divided into three species including Crenosoma sp. (4.3%), Angiostrongylus vasorum (2.6%), and an unidentified metastrongyloid species (27.8%). Beck et al (2011) reported Giardia sp. in Artiodactyla, primates, Rodentia, Hyracoidea and Carnivora at Zagreb zoo, Croatia. A phylogenetic analysis of obtained sequences had shown that isolates from captive mammals housed at the zoo were genetically different from isolates of human and domestic animal origin. 41

58 The presence of Giardia in captive mammals had been also studied in two Spanish zoological gardens located in Valencia and Madrid (Martínez-Díaz et al 2011). Around 16 different species of nonhuman primates were targeted, in which 70% of the samples had been recorded positive for Giardia. Berrilli et al (2011) had carried out molecular identification of G. duodenalis and Entamoeba sp. from nine species of primates housed in the Zoological Garden of Rome, aiming to ascertain their occurrence and zoonotic potential. It was found that all G. duodenalis isolates belonged to the zoonotic assemblage B. Three Entamoeba species i.e. E. hartmanni, E. coli and E. dispar had also been reported. Solarczyk and Majewska (2011) found the cysts of Giardia sp. in a giant toad (Bufo marinus), in a tamandua (Tamandua tetradactyla) and a cactus mouse (Peromyscus eremicus) from Poznan Zoological Garden. Molecular characterization of G. duodenalis from the tamandua had also been performed. Mukarati et al (2013) conducted a coprological survey in a population of captive African lions (Panthera leo) at a recreational game park in Zimbabwe and found an overall prevalence of helminth and protozoan parasites infections to be 100 (30/ 30) and 80% (24/ 30), respectively. Barmon et al (2014) found that out of 127 faecal samples of deer, 88 (69.29%) were infected with gastrointestinal parasites in Bhola district of Bangladesh. Maesano et al (2014) estimated that out of 71 pooled samples, 48% of the samples were positive and 47% of samples showed multiple infections in groups of mammals kept in the Warsaw zoological garden (Poland). Mamun et al (2014) observed an overall incidence of 92.9% of intestinal helminths in Asiatic lions (Panthera leo persica) at Dhaka Zoo, Bangladesh. Among 42

59 these, 78.6% (11/14) samples were positive for Spirometra sp., 57.1% (8/14) for T. leonina, 28.6% (4/14) for hookworms and 21.4% (3/14) for Strongyloides sp. Raja et al (2014) recorded an overall coprological prevalence of gastrointestinal parasites in carnivores and small mammals at Dhaka zoo, Bangladesh to be 78.72%, with a prevalence rate of 51.06% for helminths and 27.66% for protozoa. 2.3 Therapeutic management of parasitic diseases in India The safest drug has been used most of the times against the parasitic infections in the zoo animals. Fenbendazole had sufficed the purpose as drug of choice in most of the occasions in case of helminthic gastrointestinal infections. Khurana (1969) treated a case of babesiosis in white tiger at Delhi Zoological Park with Berenil (2g) and the blood smear was observed negative for protozoa on 6 th day after treatment. Reddy et al (1975) treated surra in 6 tigers of a circus stationed at Vijaywada. Four animals were administered with 0.8 g/ 100 pounds body wt. intramuscularly and the other 2 were given Antrycide 500 mg subcutaneously. By 7 th day post treatment no evidence of infection was seen. A second dose of Berenil and Antrycide prosalt were given to respective group of animals on 7 th day at half the dose rate. All the tigers recovered well. Gopalakrishanan (1982) treated successfully a tigress infected with trypanosomosis with Antrycide (Quinapyramine 1 g dissolved in 10 ml distilled water subcutaneously. Khan et al (1982) reported trypanosomosis in a white tiger at Nehru Zoological Park, Hyderabad, India and treated it successfully with 2.4 g 43

60 intramuscularly. Khan et al (1985) reported a case of surra in jackal at Nehru Zoological Park, Hyderabad and treated it successfully with Antrycide prosalt. Borthakur et al (1988) treated a case of Distomiasis in elephants using 100 g/ kg body wt. orally in 2 divided doses. The animal started taking feed 3 days post treatment and scat was found negative even after1 month post treatment. Roy and Mazumdar (1988) tested the efficacy of fenbendazole (Panacur) against natural infection of Murshidia murshida in zoo elephants. Oral administration of the 5 mg/ kg body wt. as a single dose treatment was found to be efficacious without any side effects. Gogoi and Dutta (1989) treated an elephant with fenbendazole 30 g as a single dose treatment orally, which was declared positive for ascariasis on the basis of recovery of eggs and worms in the faeces. The elephant s faecal sample was checked for ova on 7 th and 30 th day post treatment, which revealed absence of any ova or worms. Rao et al (1990) used fenbendazole (Panacur) against strongyle infection in elephants. Panacur was administered g/ animals mixed in wheat flour and jaggery. Faecal samples examined at day 7 post treatment revealed absence of any egg. Toxocara cati infection in snow leopards was treated successfully in Darjeeling zoo by Maity et al (1994). Qureshi et al (1994) administered triclabendazole medicated corn bait to white tailed 11 mg/ kg body wt./ day for 7 days to control Fascioloides magna infection. Tiwari and Rao (1996) used fenbendazole (Panacur) 5 mg/ kg body wt. against amphistome infection in elephants as a single dose treatment at National 44

61 Park Bhandhavgarh. Islam (1996) administered 9 mg/ kg body wt (not exceeding a total dose of 7.2 g/ animal) and 7.5 mg/ kg body wt (total dose not exceeding 6.8 g/ animal) against clinical fasciolosis in elephants and found them to be 100 and per cent effective, respectively till 21 days post treatment based on faecal egg count reduction. Chandrasekaran (1996a) used Cestonil tablets (Praziquantel mg/ kg body wt. as a single dose treatment for tapeworm infection in elephants incriminated to Anoplocephala manubriata and found the drug to be 100 per cent efficacious as egg per gram value was reduced to zero at day 3 post treatment. Chandrasekaran (1996b) used Distodin tablets (Oxyclozanide 1 g) in 22 elephants, found positive for Pseudodiscus collinsi, P. hawkesi and Gastrodiscus secundus. Drug was 3.5 mg/ kg body wt. orally as a single dose and was found to be 100 per cent effective based on faecal egg count reduction by day 3 post treatment. Tripathy et al (1997) treated chronic case of murshidiasis caused by Murshidia murshida in a female elephant by using fenbendazole (Panacur) 50 g total dose, which was repeated after a month. Absence of strongyle eggs in faeces and improvement in body condition was considered as the criteria for evaluation. Thiruthalinathan et al (1998) treated Spirometra infection in lion, tiger, wolves and panther by using 75 mg/ kg body wt. orally. Sinha and Prasad (1999) treated a case of intestinal amoebiasis in a lioness with Metrogyl (metronidazole). Suresh et al (2000) used 200 μg/ kg body wt. for the treatment of ancylostomosis in a leopard by intramuscular route using blowpipe. The scatological examination at day 7 post treatment revealed absence of eggs. 45

62 Islam and Nashiruddulah (2000) used 6 doses of piperazine against ascarid infection in Himalayan black bear and inferred that piperazine is clinically safe at 100 mg/ kg body wt. No ascarid egg or worms were observed in the faeces of the animals till 30 th day post treatment. Upadhye et al (2001) concluded that pyrantel pamoate (Rantac and Cisapride) was effective against Spirocerca lupi infection in tiger at a dose rate of 30 mg/ kg body wt. Ivermectin was used to treat mixed infection of Toxocara, Ancylostoma and tapeworm infection in tigers of Sunderbans brought to Alipur zoo (Sur et al 2001). Ivermectin was also used to evade toxocarosis in lions which were proved resistant for piperazine at MC Zoological Park, Chhatbir, Punjab (Singla et al 2003). Singh et al (2006a) used different groups of drugs to control Toxocara cati, Toxascaris leonina and hookworm infection in tiger cubs, adult tigers, lion cubs and adult tigers including piperazine adipate and pyrantel pamoate. Whereas, panthers and leopards infected with hookworms were treated with ivermectin. Piperazine adipate proved completely effective to control ascarids even after 30 days post treatment. On the other hand reoccurrence of T. leonina was recorded in lions at day 30 post treatment with pyrental pamoate depicting its inefficacy to control the parasitic infection. Singh et al (2006b) treated captive wild herbivores having single nematode infection (strongyles) with albendazole (Tab. Suprazole 3 g) at the rate of 15 mg/ kg body wt. for 3 days. The reduction in faecal egg count was recorded 100% by day 3 post treatment and no reoccurrence was reported till day 55 post treatment. Fenbendazole (Vetfen 600) was used successfully to treat multiple nematode infections (Trichuris sp. and strongyles) and combination of fenbendazole and 46

63 sulphadimidine (100 mg/ kg body wt.) was used for the treatment of mixed infection of nematodes and coccidia. Singh et al (2009a) treated Trichuris sp. and Hymenolepis diminuta infected omnivores with Prazital (containing praziquantel 50 mg, pyrental pamoate 144 mg and fenbendazole 150 mg) 1.5 tablets/ animal for 3 days mixed in feed. The drug proved 99.25% and 100% effective on day 5 post treatment based on faecal egg count reduction values. The animals infected with Trichuris sp. only, were treated successfully with pyrantel pamoate (tablet Nemocid ) only. Triquin (quinapyramine sulphate and quinapyramine chloride) was successfully used to treat trypanosomosis in wild felids (Gupta et al 2009, Sahoo et al 2009). Dehuri et al (2013) found ivermectin effective for the control of ascarids and hookworms in lions, but reoccurrence of the parasites observed few days later. 47

64 CHAPTER III MATERIALS AND METHODS The present study was conducted on the detection, identification, prevalence and management of parasitic infections of zoo animals and birds kept at different zoological/ deer parks in Punjab. The study involved the detection of gastrointestinal parasitism, haemo and ectoparasites of the captive wild animals and birds. The classical, molecular and serological techniques were employed for the detection of blood and gastrointestinal parasites. The infected animals were then treated with most appropriate drugs as per the recommendations of Central zoo authority, India. 3.1 Zoological parks under study Punjab state is located between N to N latitude and E to E longitudes at an altitude ranging between 180 to 300 metres above sea level. The total area covered by the state is 50, 362 km 2. Being a part of tropical country, this state supports three well defined seasons (summer, winter and monsoon) and two transitional seasons i.e. pre and post monsoon season. There is wide climatic, geographical variation in different parts of the state, which could be attributed to its subtropical latitudinal and continental location. The parks (zoological and deer parks) from which samples of wild animals and birds had been used in the present study are situated in three different climatic zones of Punjab based on homogenicity and rainfall pattern. The complete study targeting the assessment of prevalence of gastrointestinal parasites and their therapeutic management was carried out in Mahendra Mohan Choudhury Zoological Park (MCZP), Chhatbir, Punjab. Whereas, only gastrointestinal parasitic prevalence studies were carried out in Deer parks located at Patiala (Bir Motibagh), Bathinda (Bir Talab), Neelo and Tiger safari, Ludhiana (Fig. 1). The M C Zoological Park comprised of animals and birds belonging to around 61 48

65 different species, some of which were rare or endangered. The deer parks mainly housed the deer species and a few bird and other animal species. Thus, in the present study all the animals (omnivores, carnivores and herbivores) and birds (exotic and indigenous) were included for the assessment of parasitic infection. Various species of animals and birds were targeted from different locations (Zoological Park, Deer Park and Tiger Safari) for the assessment of gastrointestinal parasitism. The most common species of animals and birds included in the present study were: Animals: (a) Herbivores: Blue bull (Boselaphus tragocamelus), Goral (Nemorhaedus goral), Gaur (Bos gaurus), Hog deer (Axis porcinus), Swamp deer (Cervus duvaucelii), Black buck (Antelope cervipara), Spotted deer (Axis axis), Fallow deer (Dama dama), Hippopotamus (Hippopotamus amphibius), Wild boar (Sus scrofa), Zebra (Equus quagga boehmi), Four horned antelope (Tetracercus quadricornis), Indian gazelle (Gazelle bennetti), Barking deer (Muntiacus muntjak), Manipuri deer (Cervus eldi), Porcupine (Hystrix indica), Elephant (Elephas maximus indicus); (b) Carnivores: White tiger (Panthera tigris tigris), Royal Bengal tiger (Panthera tigris tigris), Leopard (Panthera pardus), Jaguar (Panthera onca), Hyaena (Hyaena hyaena), Jackal (Canis aureus), Asiatic lion (Panthera leo persica), Leopard cat (Prionailurus bengalensis), Civet cat (Viverra zibetha), Jungle cat (Felis chaus); (c) Omnivores: Himalayan black bear (Ursus thibetanus), Sloth bear (Melursus ursinus), Hamadryas baboon (Papio hamadryas), Rhesus macaque (Macaca mulatta), Bonnet macaque (Macaca radiata), Common langur (Semnopthicus entellus), Lion tailed macaque (Macaca silenus) and Pig tailed macaque (Macaca nemestrina). Birds: (a) Galliformes: Ring necked pheasant (Phasianus colchicus), Red jungle fowl (Gallus gallus), Silver pheasant (Lophura nycthemera), Kalij pheasant (Lophura 49

66 leucomelanos), Grey partridge (Francolinus pondicerianus), Black partridge (Francolinus francolinus), Indian peafowl (Pavo cristatus), Lady Amherst s pheasant (Chrysolophus amherstiae), Golden pheasant (Chrysolophus pictus); (b) Gruiformes: Sarus crane (Garus antigone); (c) Anseriformes: Common goose (Anser cygnoides), Black swan (Cygnus atratus), Spot billed duck (Anas poecilorhyncha); (d) Psittaciformes: Budgerigar (Melopsittacus undulatus), Sulphur crested cockatoo (Cacatua galerita), Alexandrine parakeet (Psittacula eupatria), Rose ringed parakeet (Psittacula krameri), Shikra (Accipiter badius), Cockatiel (Nymphicus hollandicus), Love bird (Agapornis genus); (e) Ciconiiformes: Painted stork (Mycteria leucocephala); (f) Columbiformes: White pigeon (Columba livia domestica), Black pigeon (Columba livia domestica), Mourning dove (Zenaida macroura); (g) Pelecaniformes: Black headed ibis (Threskiornis melanocephalus); and (h) Casuariiformes: Emu (Dromaius novaehollandiae). The number of different animal and bird species of Zoological, Deer Parks and Tiger Safari are enlisted in table 1. Table 1: Distribution of species of animals and birds of different locations under study Location Species of birds Species of animals MC Zoological Park, Chhatbir, Punjab Tiger Safari, Ludhiana 7 (approx. 135 birds) 8 (37 animals) Bir Talab Deer Park, Bathinda Bir Motibagh Deer Park, Patiala and Patiala aviary 9 (approx. 208 birds) 7 (94 animals) 14 (approx. 146 birds) 9 (212 animals) Deer Park, Neelo 3 (approx. 125 birds) 4 (39 animals) 50

67 3.2 Assessment of gastrointestinal parasitism Faecal sample collection: The fresh faecal samples were collected randomly and pooled together from the animal enclosures in which the animals were kept in groups/ herds and individual faecal samples were taken from the animals kept individually. A total of 723 and 510 faecal samples of animals and birds, respectively were collected in 2013 and 735 and 886 faecal samples (animals and birds) were collected in 2014 in three different seasons (summer, winter and monsoon) from MC Zoological Park and other deer parks. The season wise distribution of total faecal samples collected from different locations has been depicted in Table 2. The samples were collected in polyethene bags of sizes 6" 4" and were taken to the zoo laboratory. The samples were kept at 4 C and were brought to the laboratory of the Department of Veterinary Parasitology, College of Veterinary Science, GADVASU, Ludhiana for further processing. The suspected samples of the zoo animals having zoonotic potential i.e. of lions for Toxascaris leonina, monkeys for Trichuris trichiura and bears for Baylisascaris transfuga were divided into two aliquots, one for classical parasitological studies and other for molecular assessment. Table 2: Season wise distribution of faecal samples collected from different locations Location Season MCZP Chhatbir, Punjab Tiger Safari, Ludhiana Bir Talab Deer Park, Bathinda Bir Motibagh Deer Park, Patiala and Patiala aviary Deer Park, Neelo Winter Summer Monsoon Winter Summer Monsoon Total

68 3.2.2 Processing of faecal samples: The faecal samples were subjected to detailed classical parasitological analysis for presence of parasitic eggs/ oocysts by direct smear examination, standard sedimentation and floatation techniques (Soulsby 1982). The samples found positive for parasitic eggs/ oocysts were subjected to quantitative technique (egg counting technique) to get the EPG (eggs per gram of faeces) (Soulsby 1982). The samples found positive for coccidian oocysts were subjected to sporulation for genus level identification of oocysts Direct smear examination: A pinch of faecal sample was placed on one end of a slide, mixed with a drop of water, then spread and covered with cover slip and examined directly at low power (10X) and then at high power (40X) objectives of the microscope. At least three slides from different parts of the faecal samples were examined Floatation concentration technique: About 2 g of faecal sample was taken into a pestle and mortar. A suspension of the sample just strainable with a tea strainer was made by adding water, followed by complete mixing. The strained material was taken into a beaker and well mixed with Sheather s sugar solution avoiding air bubble formation. The suspension was then transferred into a centrifuge tube and filled up to the brim until a convex meniscus was formed. A cover glass was placed on it avoiding air bubble formation. After about 30 minutes, the cover glass was gently lifted in a horizontal position and mounted on a glass slide and focused under microscope, firstly under low power (10X), followed by high power (40X) for detailed study Sedimentation technique: About 2 g of faecal sample was triturated with water in a pestle and mortar. The filtrate was filtered through a tea strainer into a beaker and was allowed to settle undisturbed for 25 minutes after which the supernatant was 52

69 discarded and sediment was examined on a glass slide for the presence of parasitic eggs under the low power (10X) and high power (40X) of the microscope McMaster egg counting technique: In this technique, 3 g of faecal samples was triturated in a pestle and mortar in 42 ml saturated salt solution. The triturated material was then filtered through a sieve into a beaker. Both the chambers of the McMaster egg counting chamber were charged and examined under low power (10X) of the microscope. The number of eggs/ oocysts was counted under the etched area and the calculation was done to get EPG as per the formula: No. of eggs/ oocysts per gram of faeces =(X/ 0.15) x 45/ 3 Where, X = Number of eggs/ oocysts in counting chamber 0.15 = Volume of sample in 1 sq. cm 45 = Total volume of final suspension Therefore, EPG was evaluated by multiplying the number of eggs counted in one chamber with Sporulation of oocysts: The faecal samples found positive for coccidian oocysts by direct and floatation concentration techniques were cultured for sporulation of oocysts. The positive faecal sample was triturated in Potassium dichromate solution (2.5%) in a pestle and mortar and was then transferred to a petri dish. The petri dish was filled up to the mark that it covered the faecal material. The petri dish was left as such at room temperature (preferably at 27 C) with periodic aeration and addition of Potassium dichromate solution (2.5%). The material was regularly checked for sporulation of the oocysts by taking a drop of the suspension on to a glass slide (or by floatation concentration method) and examined it under the low 53

70 power (10X) and later high power (40X) of the microscope to observe the detailed morphology Copro-culture: The positive samples were trichurated (if in the form of pellets) in pastle and mortar and consistency of the sample was maintained with the help of sprinkling the water in the dried faeces. The material was placed in a glass jar, covered with aluminium foil. The foil was pierced to maintain the air flow for proper aeration. The sample was kept at 27±1 C in the incubator and was examined regularly after 4 th day to harvest the infective third stage larvae (L 3 ). Regular sprinkling of water and stirring the faecal culture was carried out to avoid the growth of fungus and drying. The larvae were then recovered using Baermann s apparatus. The genus based identification of third stage larvae (L 3 ) of strongyles was done as per the key of Wyk and Mayhew (2013) Micrometry: The micrometry was performed using the stage and ocular micrometer. The length and breadth (micrometry) of the eggs was observed as per the method described by (Soulsby 1982, Bowman 1999, Sloss et al 1994). The genus based identification of the eggs/ oocysts was carried out as per Soulsby (1982) Embryonation: The faecal material of Melursus ursinus and Panthera leo persica was divided into two subsets to evaluate the embryonation pattern and interval required by ascarid eggs in faecal sample as such and in 0.4% formalin solution in distilled water at 27±1 C temperature (O lorcain 1995). For incubation in 0.4% formalin solution, approximately 500 eggs were harvested by floatation concentration technique using Sheather s sugar solution (Soulsby 1982) and then suspended in solution kept in petri-dishes (63.5 mm diameter). On the other hand, the faecal samples of both the animals were kept as such separately in petri-plates (with 50-70% relative humidity) and sprinkled with 1 ml of 2% formalin. The suspension material 54

71 was stirred daily to maintain the oxygenation of the eggs. A small subsample (by counting the number of eggs in one drop of the suspension material and by floatation concentration technique from faecal samples) of the material from each petri-dish containing approximately 100 eggs were examined thrice through light microscopy (10X) daily to study developmental stages. Thus, the sequences and intervals of embryonation were studied. The criteria adopted for evaluation of the developmental stages (Caceres et al 1987) has been explained in table 3. Table 3: Criteria to identify viability and stage of development of ascarid eggs by microscopy Nonviable egg Viable egg Poorly defined structures One to four cells: 1, 2, 3, or.4 cells within the egg Contraction, rupture, and loss of membrane continuity Early-morula: 5 to 10 cells within the developing embryo No larval movement observed by microscope light stimulation Vacuolization of cytoplasm and cellular condensation in unicellular stage with granulated and vacuolated cytoplasm Late-morula: 11 or more cells within the developing embryo Blastula: a spherical layer of cells surrounding a fluid-filled cavity Gastrula: a layer of cells surrounding the embryo plus a kidney shape invagination in one side of the embryo First-stage larva: a larval structure within the developing embryo without moult and with motility in response to light stimulation Second-stage larva: a larval structure within the developing embryo with moult and motility in response to light stimulation 55

72 After two weeks (14 days) of initiation of embryonation study, 100 eggs were examined microscopically (10X) for the assessment of viability as per Cruz et al (2012). The larval movement observed by microscope light stimulation was used to evaluate the viability of larval stages, infective for definitive as well as accidental and paratenic hosts. 3.3 Collection and preservation of adult worms The adult worm passed in faeces or recovered during post-mortem examination were collected in a glass vial and taken to the laboratory. In the laboratory the worms were separated from extraneous material i.e. from faeces and stomach or intestinal contents. The worms were then transferred to a petri dish and were fixed using hot (50 C) 10% formalin so that the worms were extended fully, the worms were then cleared in lacto-phenol and glycerin before examination by light microscopy. The worms used for molecular identification were kept in 70% ethanol instead of formalin. 3.4 Molecular detection of gastrointestinal parasites Genomic DNA extraction from eggs: The aliquot of the faecal material from monkeys, bears and lions suspected to be infected with Trichuris trichiura, Baylisascaris transfuga and Toxascaris leonina, respectively, kept for molecular analysis was introduced to floatation-concentration process as discussed above. Approximately 50 µl of the supernatant from the concentrated material was aspirated using micropipettes and was further introduced to 1.5 ml eppendorff vials (Traversa et al 2004). Two washings were given to the eggs with distilled water before introducing to the further process. The genomic DNA from the eggs was extracted by using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) by following 56

73 manufacturer s protocol. Finally, DNA was eluted by using 100 µl of elution (AE) buffer Genomic DNA extraction from adult parasite: The adult parasites retrieved from bears were subjected to genomic DNA extraction. The genomic DNA was extracted from individual adult worm with QIAamp tissue kit (Qiagen, Hilden, Germany) by following manufacturer s instruction with slight modification. The worm was kept at -80 C for 6 hours before starting the procedure. The worm was mechanically disrupted by using sterile pestle-mortar. The disrupted worm was kept in lysis (AL) buffer overnight at 56 C. The final elution of the genomic DNA was carried out by using 100 µl of elution (AE) buffer Primers used and PCR protocol: The PCR identification technique was opted for only three parasite species: Baylisascaris transfuga, Toxascaris leonina and Tichuris trichiura. For B. transfuga detection, the genomic DNA extracted from the adult nematodes and eggs were used in PCR to amplify the internal transcribed spacer regions (ITS-1 and ITS-2) (Testini et al 2011), by using the primers (forward: 5 -ACT GCT GTT TCG AGA CCT TTC GAG-3 and reverse: 5 -TAG CAC CTT CTT TGG ACT ATA GCC-3 ). The PCR protocol for amplification of the templates was performed in a 25 µl total reaction volume (containing 12.5 µl of premix, 1.5 µl of each forward and reverse primers, 4.5 µl of nuclease free water and 5 µl of DNA) with following conditions in sequential order: (i) Initial denaturation (94 C for 12 min.), (ii) Denaturation (30 cycles of 94 C for 30 sec.), (iii) Annealing (30 cycles of 58 C for 45 sec.), (iv) Extension (30 cycles of 72 C for 45 sec.), and (v) Final extension (72 C for 7 min.). For complete confirmation of the eggs belonging to the same parasite, the eggs retrieved from the faecal sample at day 1 post deworming were also used for DNA extraction and PCR amplification process. The faecal sample 57

74 of another bear, kept at Tiger safari, Ludhiana, India was screened twice and found negative for any parasite, was taken as negative control. The DNA from the eggs of Toxocara canis was also run simultaneously as control to rule out the presence of any other related species. For T. leonina, the PCR protocol of Li et al (2007) was followed for the detection, targeting ITS-1 and ITS-2 ribosomal DNA (rdna) genetic markers by using the forward (5 -ATA TCG GAA AAG GAC GCA CA-3 ) and reverse (5 -TTA GTT TCT TTT CCT CCG CT-3 ) primers. The PCR protocol for amplification of the templates was performed in a 25 µl total reaction volume (containing 12.5 µl of premix, 1.5 µl of each forward and reverse primers, 1 µl of MgCl 2, 3.5 µl of nuclease free water and 5 µl of DNA) with following conditions in sequential order: (i) Initial denaturation (94 C for 5 min.), (ii) Denaturation (35 cycles of 94 C for 30 sec.), (iii) Annealing (35 cycles of 57 C for 30 sec.), (iv) Extension (35 cycles of 72 C for 1 min.), and (v) Final extension (72 C for 5 min.). For complete confirmation of the eggs belonging to the same parasite, the eggs retrieved from the faecal sample at day 1 post deworming were also used for DNA extraction and PCR amplification process. The DNA from the eggs of T. canis was also run simultaneously as control to rule out the presence of any other related species. For T. trichiura detection, the protocol of Areekul et al (2010) was followed by targeting the ITS-1 and small subunit ribosomal RNA (ssr RNA) regions. The forward (5 -AGC GCT CCG CGG AGC ACC T-3 ) and reverse (5 -CTG TCC CAG TCA CGA GAA C-3 ) primers were used for the PCR amplification by preparing a total reaction volume of 25 µl (containing 12.5 µl of premix, 1.5 µl of each forward and reverse primers, 1 µl of MgCl 2, 3.5 µl of nuclease free water and 5 µl of DNA). The PCR conditions used were: Initial denaturation (94 C for 1 min.), (ii) Denaturation (30 58

75 cycles of 94 C for 30 sec.), (iii) Annealing (30 cycles of 62 C for 30 sec.), (iv) Extension (30 cycles of 72 C for 1 min.), and (v) Final extension (72 C for 5 min.). The identity of the sequences was confirmed after sequencing (Xcleris Genomics, Ahmadabad, Gujarat, India) and comparing with the sequences registered in GenBank, by using Basic Local Alignment Search Tool (BLAST ) (Testini et al 2011). The sequences thus obtained were analysed using MEGA 4.0 (Molecular Evolutionary Genetic Analysis) software (Tamura et al 2007) Visualization of PCR products by agarose gel electrophoresis: The resolution and purity of the DNA preparation was also analyzed by gel electrophoresis in a sub-marine horizontal electrophoresis unit (Power Pac Universal- Bio Rad). Agarose gel was prepared by dissolving and boiling ultra pure DNA grade agarose (1.5%) in TBE 1 1X buffer to dissolve it completely. After cooling to about 60 C, ethidium bromide (intercalating agent) was added to agarose solution to a final concentration of 0.5 µg/ ml. The gel platform was sealed with an adhesive tape by taping the open sides. Agarose in buffer was melted in a microwave oven. The melted agarose was then poured on to the gel casting platform and the gel comb was inserted making sure that no bubbles on the gel surface were present before it solidified. After the gel solidified, the comb was taken out and the adhesive tape was removed. The gel casting platform with the set gel inside was then submerged in the electrophoresis tank such that wells were at cathode end of tank. Now sufficient quantity of electrophoresis buffer (TBE 1X) was added to ensure about 1 mm deep buffer level on the surface of the gel. The test DNA sample (~ 5 µl) was mixed with 2 µl of 6X bromophenol blue (loading dye) and the sample was loaded into the well with a micropipette. Electrophoresis was performed at 5 V/ cm and the progress of mobility 1 TBE= 10X Tris Boric EDTA Buffer: 109g Tris Base, 55g Boric Acid, 4.65 g Ethylene diamine tetracetic acid; ph:

76 was monitored by migration of the dye in loading buffer. The DNA migration and resolution pattern was examined by UV trans-illuminator under gel documentation system. 3.5 Blood collection The blood samples were collected from coccygeal and ear veins of the wild felids (Royal Bengal tigers (n=11), Asiatic lions (n=3) and leopards (n=2) and elephants (n=4), respectively, kept at the MC Zoological Park, Chhatbir and Tiger Safari, Ludhiana in March 13 and August 13 separately. The blood samples were collected in the vacutainers with anticoagulant, which were stored at 4 C directly for haematological parameters. Sera were separated from the blood samples collected in the vacutainers with coagulation activators (Thrombin based) by centrifugation and stored at -20 C for further immunological and biochemical analysis Haemoparasite detection: Giemsa stained thin blood smear (GSTBS) examination was used as the conventional diagnostic technique for the detection of haemoparasites. Two thin blood smears were prepared immediately after the blood sample collection. Blood smears were fixed in methanol and stained with Giemsa stain according to the method of Kelly (1979) Acridine orange staining: Acridine Orange is a metachromatic dye which differentially stains double-stranded (ds) and single-stranded (ss) nucleic acids. When AO intercalates into dsdna, it emits green fluorescence upon excitation at nm. Thin blood smears were stained by AO as per the method described by Lauer et al (1981) and Ravindran et al (2007). The methanol fixed smears were flooded with 0.01% AO stain, allowed to act for two minutes and then washed slowly with tap water. The smears were mounted with a coverslip and examined when moist under a fluorescent microscope. 60

77 3.6 Molecular detection of haemoparasites DNA extraction: DNA was extracted using QIAamp blood kit (Qiagen, Hilden, Germany) as per the protocol of the manufacturer. Twenty µl of reconstituted proteinase K solution was added into 2.0 ml collection tubes containing 200 µl of fresh whole blood sample and was vortexed for sec. 20 µl of RNAse A solution was added to the collection tubes and again vortexed for seconds and incubated for 2 min at room temperature. Sample was then lysed with 200 µl of lysis solution and thoroughly vortexed for few seconds and incubated at 55 C for 10 min. 200 µl of ethanol was added with gentle pipetting and lysate was transferred into the column. The column was first centrifuged and then washed with 500 µl of pre-wash and wash buffer with subsequent centrifugation. Finally, DNA was eluted with 100 µl elution buffer. Amount of extracted DNA and its purity was measured at OD 260 and ratio of OD 260 to OD 280, respectively. The concentration and the purity of the isolated DNA were determined by UV spectroscopy using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). The DNA purity was estimated by determining the ratio of A260/ A280, which for pure sample should fall between 1.65 and Primers used and optimization of PCR conditions The DNA extracted from the blood of the zoo felids and elephants were subjected to the detection of Babesia species or Trypanosoma evansi. The primers used were: For Babesia species (Kledmanee et al 2009): Forward: Ba103F: 5 - CCAATCCTGACACAGGGAGGTAGTGACA- 3 Reverse: Ba721R: 5 - CCCCAGAACCCAAAGACTTTGATTTCTCTCAAG- 3 61

78 For Trypanosoma evansi (Chantra et al 1999): Forward: TR3F: 5 - GCGCGGATTCTTTGCAGACGA- 3 Reverse: TR4R: 5 - TGCAGACACTGGAATGTTACT- 3 PCR reaction for Babesia species and T. evansi were standardized as per the conditions given by Kledmanee et al (2009) and Chantra et al (1999), respectively with minor modifications. PCR amplification was carried out for a final reaction volume of 25 µl and the PCR conditions applied were: For Babesia species For Trypanosoma evansi Initial denaturation 95 C for 5 min. 95 C for 5 min. Denaturation 95 C for 1 94 C for 45 min. 35 cycles sec. 33 cycles Annealing 52 C for 90 sec. 57 C for 1 min. Extension 72 C for 90 sec. 72 C for 1 min. Final extension 72 C for 10 min. 72 C for 10 min. 3.7 Serological diagnosis of parasites The serological analysis was carried out for the detection of T. evansi by using Card agglutination test for trypanosomes (CATT/ T. evansi) and for Toxoplasma gondii and Neospora caninum by using commercial indirect enzyme linked immunosorbant assay (ielisa) kits. CATT/ T. evansi for antibody detection was originally described and converted into a test kit by the Institute of Tropical Medicine, Antwerp, Belgium (Bajyana and Hamers 1988) and was procured from the same institute for the present study. Briefly, 25 µl of diluted sera (1:3 dilutions with buffer) was thoroughly mixed with about 45 µl of well-homogenized CATT antigen. The card was agitated in a circular motion using electric rotator at rpm at room temperature for 5 min. 62

79 Samples having blue granular agglutination were considered positive. Samples were read in comparison with the control wells according to the supplied instructions. Agglutination patterns were scored as (negative) and +, ++ or +++ (positive). Two different commercial indirect ELISA (ielisa) kits from Cusabio (Wuhan Hi-tech Medical Devices Park, China) were used for the detection of T. gondii and N. caninum antibodies as per the manufacturer s protocol. The percent positivity (%P) of samples was obtained as, Percent positivity (%P) = Samples with %P value less than 90% of OD Critical control were considered negative and those with %P more than 110% of OD Critical control were considered positive, and the values in between were considered suspected. Thus, the animals were divided into three groups as per the infectivity pattern, viz. infected, suspected and non-infected and further analysis of haemato-biochemical alterations were carried out between these groups. 3.8 Haematological parameters The haematological parameters of the whole blood, viz. WBC (white blood cells), Hb (Haemoglobin), PCV (Packed cell volume and Plt (Platelets) were studied with fully automated analyzer, ADVIA 2120 Haematology System (Siemens Health Care Diagnostic Inc. Deerfield, IL, U.S.A). Differential leukocyte count (DLC; %) was performed manually under oil immersion of light microscope in blood smear stained by Giemsa stain (Jain 1986). Stained slides were air dried and examined under oil immersion to count 100 leucocytes per slide. 63

80 3.9 Biochemical parameters The sera from blood were separated after centrifugation at 5, 000 rpm for 10 min. and further stored at -20 C until use for estimation of biochemical parameters. The different serum biochemical parameters viz. Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total Bilirubin (TB), Glucose (GLU), Blood Urea Nitrogen (BUN), Total Protein (TP), Albumin (ALB), Creatinine (CRSC), Globulin (GLO), Gamma glutamyltransferase (GGT) and Iron (Fe) were analyzed using the manufacturer s protocols, given in the kits of Siemens Diagnostics Ltd. and Aggape Diagnostic Ltd. with semi automatic chemistry analyzer RA Histopathology Collection of tissue sample: The representative tissue samples of pigeons showing gross lesions were collected in 10% neutral buffered formalin at the time of necropsy for detailed histo-pathological studies Tissue processing and staining: The tissue samples (being fixed in 10% neutral buffered formalin) were cut to present the plain surfaces, required to prepare smooth sections. These tissues were then washed overnight in running tap water to remove the formalin absorbed in the tissues. It was then dehydrated by using ascending grades of alcohol starting from 30% to 90% ethanol and finally with dehydrated alcohol. Then cleared with benzene and lastly embedded in paraffin. The sections were cut to 5 µ thickness and stained with routine haematoxylin and eosin stain for the histopathological studies (Luna and Lee 1968) Therapeutic management The therapeutic management of the animals and birds of MC Zoological Park was carried out thrice during the study period. Twice in the year 2013 i.e. in March 2013 and October 2013 targeting post winter and post monsoon therapeutic management and once in 2014 i.e. March 2014 involving post winter therapeutic 64

81 management. All the animals and birds were provided with drugs during the study period considering therapeutic and prophylactic management. The birds and animals were treated with the drugs given in table 4. The herbivores found positive for Moneizia species infections were treated with liquid Nilzan (Oxyclozanide 150 mg + Tetramisole 150 mg/ ml) (Virbac). The individual samples of all the animals kept individually were taken at days zero, 1, 3, 7 and 21 post-treatment. On the other hand, random pooled samples were taken from the animals kept in groups/ herds and birds for the assessment of the efficacy of the drugs used and re-infection status of the birds and animals. Along with antiparasitic drugs multivitamin and mineral supplementation of the animals and birds were also performed to maintain the immunity status Drug dose rates: Fenbendazole (Bolus Fenomar, Virbac and Tablet Panacur, Intervet) were used for the treatment of herbivores (deer, elephants, hippopotamus, zebra, bisons and blue bulls) and was given at a dose rate of 5 mg/ kg body weight, once daily for three days and for primates, bears and large carnivores 30 mg/ kg body weight, once daily for 3 days during the first treatment period. For small carnivores, tablet Eazypet (Intas) was given at a dose rate of 1 tablet per 10 kg body weight during the first treatment. For birds, albendazole (Albomar, Virbac, 2.5% w/v) was given at a dosage of 30 ml/ 100 birds and Coccinil- plus (Vetspharma, containing Amprolium hydrochloride 20% w/w, sulphaquinoxaline 12% w/w, ethopabate 1% w/w and pyrimethamine 1% w/w) was given at a dosage of 12 g/ 20 litres of water for 5 days during the first treatment. During the second treatment schedule, similar drugs were used with same dose rate but for an extended period of time i.e. for 5 days in animals and for 7 days in birds. 65

82 Table 4: Description of the drugs used as per the animal and birds groups and seasons Deworming March 13 (Treatment I) October 13 (Treatment II) March 14 (Treatment III) Animal/ Birds species Drugs used Herbivores, omnivores and large carnivores Fenbendazole (Bolus Fenomar and Tablet Panacur) Small carnivores Fenbendazole and praziquantel combination (Tablet Eazypet) Birds Combination of albendazole (Liquid Albomar) and coccinilplus Similar drugs and supplements as were used in March 13 Herbivores and omnivores Ivermectin (Bolus Hitek and Tablet Neomec) Carnivores Combination of ivermectin and praziquantel (Tablet Ipraz Plus) Birds Combination of piperazine and coccinil plus Supplements Liv-52, Liquid Ascal gold, Liquid Proviboost and Liquid Brotone Liquid Provical pet Liquid Groviplex Liv-52, Liquid Ascal gold, Liquid Proviboost and Liquid Brotone Liquid Provical pet Liquid Groviplex 66

83 Ivermectin (Bolus Hitek, Virbac and Tablet Neomec, Intas) were given to the herbivores, omnivores and carnivores (Tablet Ipraz plus) at a dose rate of 100 µg/ kg body weight for 3 alternate days during the third treatment schedule. Piperazine (Piperazine liquid, Virbac, 45g/ 100ml) was given at a dosage of 20 ml/ 100 birds for 5 days. Liquid Nilzan (Virbac) was given to the bisons in water at a dosage of 1ml/ 5 kg body weight. Supplements including multivitamins and minerals were also administered to animals and birds. Liv. 52 Vet (Himalaya) was administered to elephants and zebra at the dosage of ml/ animal once daily for 5 days, Liquid Ascal gold (Alembic) was given to hippopotamus and bisons at a dosage of 100 ml/ animal for 5 days, Liquid Brotone (Virbac) to all the deer species at a dosage of 20 ml/ animal once daily for 4 alternate days, Liquid Proviboost (Virbac) and liquid Provical Pet (Vetcare) to all primates and carnivores. Liquid Groviplex (Virbac) was given at a dosage of 10 ml/ 100 birds daily for 10 days Assessment of drugs efficacy: The faecal egg count (FEC) is the most widely performed test to assess drug efficacy (Cabaret and Berrag 2004) and had been used in captive-wild animals (Goossens et al 2005, Young et al 2000). Thus, FEC was employed on the faecal samples collected from the animals and birds at day 1, 3, 7 and 21 post-treatment by using the formula of Young et al (2000) and Nalubamba and Mudenda (2012). Percent efficacy= (Pre-treatment mean EPG - Post-treatment mean EPG) 100 Pre-treatment mean EPG 3.12 Detection of ectoparasites The ectoparasites collected from the infested animals and birds were stored in screwed-vials containing 70% alcohol. These vials were then brought to the 67

84 laboratory of department of Veterinary Parasitology, College of Veterinary Science, GADVASU, Ludhiana for further processing. The ectoparasites were clarified in 10% potassium hydroxide (KOH) for hours. The ectoparasite samples were then dehydrated by introducing to the ascending grades of alcohol from 70% to absolute alcohol through 80, 90 and 95% alcohol concentrations each for 30 minutes. The specimens were then mounted on permanent slides with Canada balsam and were identified as per Soulsby (1982) Scanning Electron microscopy For scanning electron microscopy the ectoparasites were processed at Electron Microscopy Unit at Punjab Agricultural University, Ludhiana. The ectoparasite specimens were kept overnight in 2.5% glutaraldehyde for fixation. Thereafter, the specimens were given three washings with 0.1M rinsing buffer at 4 C for 15 minutes each. The rinsing buffer was then completely drained off. The samples were then kept in osmium tetraoxide for 2.5 hours at 4 C. Then again specimens were given three washings with 0.1M rinsing buffer at 4 C for 15 minutes each. The rinsing buffer was then completely drained off. The samples were then dehydrated gradually by subjecting them to serial dilutions of ethanol i.e. 30% ethanol for 1-20 min. at 4 C followed by 40, 50, 60, 70, 80, 90% at similar conditions and timings and finally three washings with 100% ethanol for 20 min. each at 4 C. After completely decanting the sampling containers, they were placed for overnight in dessicator. The dried specimens were mounted carefully on the brass stubs. The specimens on the stubs were then coated with very thin layer of gold ions with the help of ion-sputter. The prepared specimens were subjected to scanning electron microscopy (SEM). 68

85 3.12 Statistical analysis Chi-square test was employed to assess the association of prevalence of various gastrointestinal parasites in the animals (herbivores, carnivores and omnivores) and birds of various groups of different zoological/ deer parks. For the intensity of infection, the arithmetic mean was central tendency measure and standard deviation as a dispersion measure. One-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) 20.0 software was applied on various haematological and biochemical parameters to determine the variance of factors among different groups (infected, suspected and non-infected) of animals at 5% level of significance (P<0.05). To study the prevalence status of the gastrointestinal infections, the expected prevalence was considered to be 50% with confidence limits of 95%. ANOVA was also applied on eggs per gram values on different days (1, 4, 7 and21) post treatment for the assessment of drug efficacies. 69

86 CHAPTER IV RESULTS AND DISCUSSION The present study was conducted for an overall assessment of parasitic load of zoo animals and birds of Punjab state, India. To assess the intensity of gastrointestinal parasitism, 2854 fresh faecal samples were collected from animals and birds of MC Zoological Park, deer parks and safari of the state over a period of two years ( ). Molecular studies were conducted for ascarid infections in sloth bears and lions and trichurid infection in monkeys. The haemoprotozoan infection and serological detection of parasites was assessed in carnivores and elephants twice in the year Prevalence of gastrointestinal parasitism in animals and birds of MC Zoological Park, Chhatbir The prevalence of gastrointestinal parasites was observed in three different seasons (winter, summer and monsoon) of two years. Out of 1458 (909 animals birds) samples screened, 438 (232 animals birds) samples were found positive for one or other parasitic infection, showing an overall gastrointestinal parasitic prevalence of 25.52% (95% CI= %) in animals and 37.52% (95% CI= %) in birds, respectively (Table 5 and fig. 2). The overall prevalence was observed to be comparatively lower than the findings of Varadharajan et al (2001), Varadharajan and Subramaniam (2003), Cordon et al (2008) and Thawait et al (2014), who reported an overall prevalence of 68.05, 68.36, 72.5 and 46.20% in wild mammals of Thrissur zoo Kerala, Almunecar Zoological Garden, Spain and Nandan Van zoo, Raipur, Chhattisgarh, respectively. But, it was in concordance with and 33.22% prevalence observed in animals of Mysore zoo (Muraleedharan et al 1990) and in MCZP, Chhatbir, Punjab (Singh 2004), respectively. The comparatively lower prevalence of parasitic infections might be due to better management conditions 70

87 adopted at the zoological park involving regular screening of the captive animals and birds. The overall prevalence in animals (25.52%) (95% CI= %) was lesser than birds (37.52%) (95% CI= %) (Table 5 and fig. 2). Higher incidence of helminthic parasites in zoo birds as compared to the animals had also been previously reported (Dhoot et al 2002). Overcrowding of the birds in cages, could have lead to excessive stress, making them more vulnerable to endoparasitic and ectoparasitic infections. Both animals and birds individuallly showed maximum prevalence in monsoon 2013 i.e and 53.12%, respectively. Modi et al (1997a) and Kumar and Rao (2003) also reported maximum prevalence in the monsoon (rainy) season (51.90 and 46.59%, respectively) and minimum in the summers. This could be attributed to the favourable environmental conditions i.e. high humidity and suitable temperature for the development and prolonged survival of the infective parasitic stages in the monsoon season (Singh et al 2009b). 4.2 Captivity based prevalence The animals kept in herds showed higher prevalence (23.30%) (95% CI= %) in comparison to the individually enclosed animals (9.02%) (95% CI= %) (Table 6 and fig. 3) at the beginning of the study i.e. in winter But, birds showed highest prevalence (45.56%) (95% CI= %) at that time (Table 6 and fig. 3). Usually overcrowding, competition for feed and water results in stress and decreased immunity, leading to more vulnerability to parasitic infections (Dhoot et al 2002, Singh et al 2009a). 71

88 Table 5: Season wise prevalence of parasitic infection in animals and birds of MC Zoological Park, Chhatbir, Punjab S.N. Season Total Samples (Animal + Birds) Samples positive (animals) Prevalence (%) * Samples positive (birds) Prevalence (%) * 1. Winter 13 (January) 2. Summer 13 (June) 3. Monsoon 13 (Aug.-Sept.) 212 (133+79) 256 (162+94) 255 (159+96) ( ) ( ) ( ) 4. Winter 14 (January) 245 (148+97) ( ) 5. Summer (June) (154+94) ( ) 6. Monsoon (Aug.-Sept.) (153+89) ( ) Total 1458 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) χ * = 95% Confidence Interval 72

89 The individually enclosed animals showed a decline to 1.30% (95% CI= %) of prevalence at the end of the study i.e. in monsoon The gastrointestinal parasitic infection in the animals kept in herds and birds kept in flocks was also decreased to 16.99% (95% CI= %) and 29.21% (95% CI= %), respectively at the end of the study. The decline could be attributed to appropriate anthelmintic treatment of animals, which is discussed in the later part of the thesis. 4.3 Feeding behaviour based prevalence Based on their feeding habits wild animals were divided in three categories, viz. herbivores, omnivores and carnivores. The overall prevalence in wild herbivores was 40.41% (95% CI= %), which was in concordance with the observations of Singh et al (2009a) and Thawait et al (2014), who reported 38.17% and 45.6% prevalence, respectively. The moderate temperature range and more humidity conditions of the park might have induced the mat formation between the soil and the herbage, favourable to the survival of eggs and free-living stages of parasites (Singh et al 2009b). Further, grasslands may be one of the main sources of gastrointestinal parasitic diseases to animals especially herbivores (Mondal et al 2000). The overall parasitic prevalence recorded in omnivores and carnivores was 6.85% (95% CI= %) and 5.71% (95% CI= %), respectively (Table 7 and fig. 4). Low prevalence indicated better management conditions and may be due to the fact that most of the carnivores (especially wild felids) and omnivores (especially ursids) at zoo were kept invidually to prevent over crowding, infighting and stress conditions. An overall prevalence of 47.03% (95% CI= %) was observed in the birds and it ranged betwee % from the initiation to conclusion of the study period (Table 7 and Fig. 4). Parsani et al (2001, 2003) reported and 73

90 Table 6: Captivity based prevalence in animals and birds of MC Zoological Park, Chhatbir, Punjab Season Captivity Win. 13 Samples (Positive / Total) Prevalence (%) * Sum. 13 Samples (Positive / Total) Prevalence (%) * Mon. 13 Samples (Positive / Total) Prevalence (%) * Win. 14 Samples (Positive / Total) Prevalence (%) * Sum. 14 Samples (Positive / Total) Prevalence (%) * Mon. 14 Samples (Positive / Total) Prevalence (%) * χ 2 Individual enclosed 12/ ( ) 9/ ( ) 12/ ( ) 5/ ( ) 1/ ( ) 2/ ( ) Herds/ groups 31/ ( ) 40/ ( ) 48/ ( ) 27/ ( ) 19/ ( ) 26/ ( ) Birds 36/ ( ) 39/ ( ) 51/ ( ) 32/ ( ) 22/ ( ) 26/ ( ) * = 95% Confidence Interval 74

91 Table 7: Prevalence of parasitic infection in animals and birds based on feeding behaviour Animals and Birds Seasons Herbivores samples (Positive H / Positive T ) Prevalence (%) * Winter 13 30/ ( ) Summer 13 41/ ( ) Monsoon 13 46/ ( ) Winter 14 23/ ( ) Summer 14 16/ ( ) Monsoon 14 21/ ( ) Total 177/ ( ) Omnivores samples (Positive O / Positive T ) Prevalence (%) * 7/ ( ) 5/ ( ) 7/ ( ) 5/ ( ) 2/ ( ) 4/ ( ) 30/ ( ) Carnivores samples (Positive C / Positive T ) Prevalence (%) * 6/ ( ) 3/ ( ) 7/ ( ) 4/ ( ) 2/ ( ) 3/ ( ) 25/ ( ) Birds samples (Positive B / Positive T ) χ Prevalence (%) * 36/ ( ) 39/ ( ) 51/ ( ) 32/64 50 ( ) 22/ ( ) 26/ ( ) 206/ ( ) χ * = 95% Confidence Interval Positive H represent the samples positive for herbivores, Positive O = omnivores, Positive C = carnivores, Positive B = birds and Positive T = total posisitive samples 75

92 57.73% coproprevalence in the birds from Kankaria zoo, Ahmadabad and four zoos (Ahmadabad, Baroda, Junagadh and Rajkot) of Gujarat, respectively. Higher prevalence of the parasitic infection in captive birds was related to higher number of geo-helminths recorded. Recently, Parsani and Momin (2009) reported much higher prevalence (80.69%) in carnivorous birds of Baroda zoo. 4.4 Group wise copro-prevalence of endoparasites of animals Helminthic infections predominated in zoo animals of different categories, viz. herbivores (31.17%), omnivores (18.75%) and carnivores (7.62%) as compared to protozoans (Tables 8-10). This may be due to direct life cycle of the most of the parasites encountered in the zoological park (Panayotova-Pencheva 2013). The herbivores and carnivores although exhibited some protozoan parasitism and mixed infections as well, but only helminthic parasitism was reported in omnivores. The most common eggs of gastrointestinal parasites recorded in herbivores were of strongyles, trichurids, Strongyloides papillosus, ascarids and coccidian oocysts. The morphometric description of the eggs/ oocysts of different parasites recovered from herbivores, omnivores and carnivores had been given in the table 12, 13 and 14, respectively. 4.5 Group wise copro-prevalence of endoparasites of birds The birds showed higher helminth infection (overall prevalence = 20.04%) in all the seasons as compared to protozoan (overall prevalence = 6.01%) and mixed infections (overall prevalence = 11.47%) (Table 11). The helminths most commonly observed included Ascaridia sp. and Capillaria sp. and the reason could be their direct life cycle (Parsani et al 2001). The protozoan infection mainly involved coccidian infection of Eimeria species. The morphometric description of the eggs/ oocysts of different parasites recovered from birds had been provided in table

93 Table 8: Prevalence of endoparasitic infections in herbivores of MC Zoological Park, Chhatbir Win. 13 (n=76) Sum. 13 (n=102) Mon. 13 (n=95) Seasons of sampling Win. 14 (n=84) Sum. 14 (n=92) Mon. 14 (n=90) Total Positive (%) Groups Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Helminths 28 (36.84) 39 (38.23) 43 (45.27) 22 (26.19) 16 (17.39) 20 (22.22) 168 (31.17) Protozoa 2 (2.63) 2 (1.96) 2 (2.10) 1 (1.19) 0 1 (1.11) 8 (1.48) Mixed (1.05) (0.18) Total (%, 95% CI) 30 (39.47, ) 41 (40.19, ) 46 (48.42, ) 23 (27.38, ) 16 (17.39, ) 21 (23.33, ) 177 (32.83) n= total number of samples screened for endoparasitic infections in herbivores; CI= Confidence interval Table 9: Prevalence of endoparasitic infections in omnivores of MC Zoological Park, Chhatbir Seasons of sampling Groups Win. 13 (n=26) Sum. 13 (n=26) Mon. 13 (n=27) Win. 14 (n=27) Sum. 14 (n=27) Mon. 14 (n=27) Total Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Helminths 7 (26.92) 5 (19.23) 7 (25.92) 5 (18.52) 2 (7.41) 4 (14.81) 30 (18.75) Protozoa Mixed Total (%, 95% CI) 7 (26.92, ) 5 (19.23, ) 7 (25.92, ) 5 (18.52, ) 2 (7.41, ) 4 (14.81, ) 30 (18.75) n= total number of samples screened for endoparasitic infections in omnivores; CI= Confidence interval 77

94 Table 10: Prevalence of endoparasitic infections in carnivores of MC Zoological Park, Chhatbir Seasons of sampling Groups Win. 13 (n=31) Sum. 13 (n=34) Mon. 13 (n=37) Win. 14 (n=37) Sum. 14 (n=35) Mon. 14 (n=36) Total Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Helminths 5 (16.13) 2 (5.88) 4 (10.81) 3 (8.11) 1 (2.86) 1 (2.77) 16 (7.62) Protozoa 1 (3.22) 1 (2.94) 3 (8.11) 1 (2.70) 1 (2.86) 2 (5.56) 9 (4.28) Mixed Total (%, 95% CI) 6 (19.35, ) 3 (8.82, ) 7 (18.92, ) 4 (10.81, ) 2 (5.72, ) 3 (8.33, ) 25 (11.90) n= total number of samples screened for endoparasitic infections in omnivores; CI= Confidence interval Table 11: Prevalence of endoparasitic infections in birds of MC Zoological Park, Chhatbir Win. 13 (n=79) Sum. 13 (n=94) Mon. 13 (n=96) Seasons of sampling Win. 14 (n=97) Sum. 14 (n=94) Mon. 14 (n=89) Total Positive (%) Groups Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Positive (%) Helminths 23 (29.12) 22 (23.41) 23 (23.96) 18 (18.56) 12 (12.76) 12 (13.49) 110 (20.04) Protozoa 4 (5.06) 6 (6.38) 13 (13.54) 4 (4.12) 2 (2.13) 4 (15.38) 33 (6.01) Mixed 9 (11.39) 11 (11.70) 15 (15.62) 10 (10.31) 8 (8.51) 10 (11.24) 63 (11.47) Total (%, 95% CI) 36 (45.57, 39 (41.49, 51 (53.12, 32 (32.99, 22 (23.40, 26 (29.21, 206 (37.52) ) ) ) ) ) ) n= total number of samples screened for endoparasitic infections in omnivores; CI= Confidence interval 78

95 Table 12: Morphometric description of parasitic eggs/ oocysts recovered from herbivores S.N. Species of animal Parasitic egg/ oocysts Average (Length Breadth) in µm 1. Large and small Haemonchus ± 4.31 ruminants contortus (Fig. 5) 42.9 ± 4.23 Cooperia sp ± 2.42 (Fig. 6) ± 2.42 Trichostrongylus sp ± 7.03 (Fig. 7) ± Swamp deer Trichuris sp ± 4.23 (Fig. 8) ± Gaur Moniezia benedeni ± 1.85 (Fig. 9) ± Black buck, Strongyloides ± 5.85 barking deer, four papillosus ± 2.40 horned antelopes (Fig. 10) and Indian gazelle 5. Porcupines Ascaridia sp ± 7.02 (Fig. 11) ± 5.62 Trichuris sp ± 5.13 (Fig. 12) ± Elephants Strongyle ± 8.34 (Fig. 13) ± 5.53 Strongyloides sp ± 4.85 (Fig. 14) ± Gorals Eimeria pellita ± 2.04 (Fig. 15) ± 1.27 Eimeria kamoshika ± 4.24 (Fig. 16) ± Blue bull Eimeria cylindrica ± 5.05 (Fig. 17) ± 2.27 Range (Length Breadth) in µm

96 Table 13: Morphometric description of parasitic eggs/ oocysts recovered from omnivores S.N. Species of animal Parasitic egg/ oocysts 1. Sloth bear Ascarid eggs (Fig. 18) 2. Non human Trichuris sp. primates (Fig. 19) Strongyloides fuelleborni (Fig. 20) Average (Length Breadth) in µm ± ± ± ± ± ± 3.84 Range (Length Breadth) in µm Table 14: Morphometric description of parasitic eggs/ oocysts recovered from carnivores S.N. Species of animal Parasitic egg/ oocysts Average (Length Breadth) in µm 1. Jungle cat Isospora felis ± 5.07 (Fig. 21) 28.2 ± Asiatic lions Isospora felis ± ± 3.71 Toxascaris leonina ± 4.10 (Fig. 22) ± Leopard Isospora rivolta ± 2.43 (Fig. 23) ± Leopard cat Spirometra sp ± 4.11 and civet cat (Fig. 24) ± Hyaena Toxocara canis ± 4.67 (Fig. 25) ± 1.91 Range (Length Breadth) in µm

97 Table 15: Morphometric description of parasitic eggs/ oocysts recovered from birds S.N. Species of birds Parasitic egg/ oocysts Average (Length Breadth) in µm Range (Length Breadth) in µm 1. Galliformes (red jungle fowl, Indian peafowl, Ascaridia sp. (Fig. 26) ± ± golden pheasant, gray partridge, black partridge, Lady Amherst s pheasant) 2. Columbiformes (white pigeons, black pigeons Ascaridia sp ± ± and doves) 3. White pigeons, black pigeons, Capillaria sp. (Fig. 27) 51.4 ± ± Budgerigars, Indian peafowl, Lady Capillaria sp ± ± Amherst s pheasant, doves, cockatoo and cockatiel 5. Doves Syngamus trachea ± ± (Fig. 28) 6. White pigeons Ornithostrongylus quadriradiatus 72.4 ± ± (Fig. 29) 7. Mongolian pheasant Amidostomum sp. (Fig. 30) ± ± Indian peafowl Hymenolepis sp. (Fig. 31) ± ± Eimeria mayurai (Fig. 32) ± ±

98 9. Painted stork Echinostoma revolutum (Fig. 33) 10. Sarus crane Echinuria uncinata (Fig. 34) 11. White and black pigeons Eimeria labbeana (Fig. 35) 12. Red jungle fowl Eimeria sp. (Fig. 36) 13. Kalij pheasant Eimeria sp. (Fig. 37) ± ± ± ± ± ± ± ± ± Prevalence of gastrointestinal parasitic infections in Deer Parks Coprological study revealed an overall burden of gastrointestinal parasites to be 87.43% (95% CI= %) and (95% CI= %) (Table 16 and fig. 38) in the animals and birds of Bir Motibagh Deer Park, Patiala, respectively. Highest prevalence of endoparasites in animals was seen in monsoon 2014 (93.02%, 95% CI= %), whereas, highest prevalence in birds was recorded in summer 2014 (31.91%, 95% CI= %). The findings were in concordance with the findings of Gupta et al (2011), who revealed a comparative parasitic prevalence in wild ruminants (90% Sambar, 86.67% blue bull and 80% spotted deer). The birds of Patiala aviary exhibited an overall parasitic prevalence of 37.50% (95% CI= %) (Table 17 and fig. 39), where maximum prevalence was observed in Monsoon The comparatively lesser parasitism than the birds of MC Zoological Park, Chhatbir could be due to less stocking density of birds in Patiala aviary. 82

99 Table 16: Season wise prevalence of parasitic infection in animals and birds of Bir Motibagh Deer Park, Patiala S.N. Season Total Samples (Animal + Birds) Samples positive (animals) Prevalence (%) * Samples positive (birds) Prevalence (%) * 1. Summer 13 (June) 87 (40+47) ( ) ( ) 2. Monsoon 13 (Aug.-Sept.) 89 (42+47) ( ) ( ) 3. Winter 14 (January) 82 (38+44) ( ) ( ) 4. Summer 14 (June) 91 (44+47) ( ) ( ) 5. Monsoon 14 (Aug.-Sept.) 89 (43+46) ( ) ( ) Total 438 ( ) ( ) ( ) χ * = 95% Confidence Interval 83

100 Table 17: Season wise prevalence of parasitic infection in birds of Patiala aviary S.N. Seasons Total Samples Total positive Prevalence (%) * 1 Monsoon 13 (Aug.-Sept.) ( ) 2 Winter 14 (January) ( ) 3 Summer 14 (June) ( ) 4 Monsoon 14 (Aug.-Sept.) ( ) Total ( ) χ Table 18: Season wise prevalence of parasitic infection in animals and birds of Bir Talab Deer Park, Bathinda S.N. Season Total Samples (Animal + Birds) Prevalence (%) * Prevalence (%) * 1. Summer 13 (June) 65 (30+35) Samples positive (animals) ( ) Samples positive (birds) ( ) 2. Monsoon 13 (Aug.-Sept.) 68 (35+33) ( ) ( ) 3. Winter 14 (January) 72 (39+33) ( ) ( ) 4. Summer 14 (June) 64 (34+30) ( ) ( ) 5. Monsoon 14 (Aug.-Sept.) 68 (36+32) Total 337 ( ) ( ) ( ) ( ) ( ) χ * = 95% Confidence Interval 84

101 Table 19: Season wise prevalence of parasitic infection in animals and birds of Tiger Safari, Ludhiana S.N. Season Total Samples (Animal + Birds) Samples positive (animals) Prevalence (%) * Samples positive (birds) Prevalence (%) * 1. Summer 13 (June) 2. Monsoon 13 (Aug.-Sept.) 3. Winter 14 (January) 4. Summer 14 (June) 5. Monsoon 14 (Aug.-Sept.) 38 (22+16) 65 (23+42) 61 (24+37) 61 (25+36) 62 (23+39) Total 287 ( ) ( ) ( ) ( ) ( ) ( ) ( ) χ ( ) ( ) * = 95% Confidence Interval 85

102 Table 20: Season wise prevalence of parasitic infection in animals and birds of Deer Park Neelo S.N. Season Total Samples (Animal + Birds) Samples positive (animals) Prevalence (%) * Samples positive (birds) Prevalence (%) * 1. Summer 13 (June) 2. Monsoon 13 (Aug.-Sept.) 3. Winter 14 (January) 4. Summer 14 (June) 5. Monsoon 14 (Aug.-Sept.) 18 (12+6) 39 (21+18) 38 (21+17) 42 (24+18) 39 (21+18) Total 174 (97+77) (100) (100) ( ) ( ) ( ) ( ) χ ( ) ( ) ( ) * = 95% Confidence Interval 86

103 The gastrointestinal parasitic prevalence of animals and birds of Bir Talab Deer Park, Bathinda was recorded to be 66.67% (95% CI= %) and 45.39% (95% CI= %), respectively (Table 18 and fig. 40). The overall parasitic prevalence observed in animals and birds of Tiger Safari, Ludhiana was 13.67% (95% CI= %) and 67.64% ( %), respectively (Table 19 and fig. 41). Whereas, overall parasitic prevalence observed in animals and birds of Deer Park, Neelo was 94.84% (95% CI= %) and 16.88% (95% CI= %), respectively (Table 20 and fig. 42). The animals enclosed in Tiger Safari exhibited no gastrointestinal parasitic prevalence from summer 2013 to summer 2014, but in monsoon 2014, 69.56% (95% CI= %) parasitic infection was recorded in them. This may be attributed to the repeated dose administration of fenbendazole to the animals at a difference of three months, leading to the development of resistance against the drug. 4.7 Coproculture studies Almost all the deer parks showed mixed infection of the strongyles and the faecal culture revealed predominantly two most common parasitic larvae, Haemonchus contortus and Cooperia oncophora (Fig. 43 and 44). The larvae were identified based on the key of Wyk and Mayhew (2013). For the identification, different morphological characteristics of the larvae, viz. total length, sheath tail extension and number of intestinal cells were taken into consideration. The total length of H. contortus and C. oncophora was ±23.09 and 817±39.21 µm, respectively, with 16 intestinal cells each. The morphometric values for sheath tail extension for H. contortus and C. oncophora were 74.33±11.03 and 87±13.39 µm, respectively. There were some other larvae of strongyles as well which could not be identified. But, the strongyles eggs recovered after the deworming showed presence 87

104 of H. contortus after the coproculture. Similar to the present study, previously Nalubamba and Mudenda (2012) identified H. contortus larvae after deworming while observing the anthelmintic efficacy in antelopes. 4.8 Embryonation studies Detailed developmental studies associated with embryonation pattern of Baylisascaris transfuga and Toxascaris leonina had not been carried out earlier. But, descriptive developmental studies of ascarids of domestic animals, mainly dogs (Toxocara canis), cattle (Toxocara vitulorum) and pigs (Ascaris suum) has been carried out by O Lorcain (1995), Devi et al (2001) and Cruz et al (2012), respectively at different temperature conditions and observed maximum embryonation at 27±1 C. Thus, the present study was planned to evaluate developmental pattern of the ascarid eggs at reported optimum temperature i.e. 27±1 C. The complete description of embryonic development of eggs of B. transfuga and T. leonina in faecal sample as well as in 0.4% solution of formalin has been depicted in Fig. 45. In case of B. transfuga, the eggs remained in one cell stage for first two days and further development to two-cell stage was noticed at day 3 post incubation in both the subsets (faecal sample and 4% solution of formalin). Whereas, in case of eggs of T. leonina, development started on the very first day and hence, next stage was observed at day 1 post incubation in both the subsets (Fig. 45). At the end of the first week, majority of the eggs were in 1 st larval stage in case of T. leonina (approximately 48%) and at twocell and four-cell stage were observed in case of B. transfuga (approximately 32% each) in case of formalin solution. Similar observations were there in case of faecal samples as well. On the other hand, Cruz et al (2012) reported most of the embryos (72.5%) of A. suum eggs in late morula stage at the end of the first week at 28 C. The larvated egg stages containing 1 st stage larva in case of B. transfuga and T. leonina 88

105 were observed on days 9 and 5 post incubation in both formalin solution and faecal samples, respectively. Kim et al (2012) reported presence of 1 st stage larvae in the eggs of A. suum at day 19 post incubation but at a controlled temperature of 25 C in an environmental chamber. This observation reveals that a little variation in temperature may affect embryonation process of ascarid eggs. The final infective stages (i.e. all the larvated eggs of the subsamples were at 2 nd larval stages) along with other developmental stages, was observed on days 12 and 9 post incubation in 0.4% formalin solution and on days 14 and 9 post incubation in faecal samples in case of B. transfuga and T. leonina, respectively. The extended time interval required by the eggs of B. transfuga to acquire final infective stage could be attributed to its thicker cell wall as compared to T. leonina (Bauer 2013). The purpose of carrying out study in 0.4% formalin solution and in faecal samples simultaneously was to evaluate the variation in the time interval to reach the infective stage, which was not observed significantly different. Various developmental stages of ascarids have been presented in fig. 46. The viability of the eggs was assessed by evaluating the movements of the larvae inside the eggs after stimulation of light and approximately 86 and 84% of B. transfuga eggs were found viable in case of 0.4% formalin solution and faecal sample, respectively. Whereas, 88 and 87% of T. leonina eggs were found viable in case of 0.4% formalin solution and faecal sample, respectively. Cruz et al (2012) observed 87.5% viability of A. suum eggs at 21 days of incubation, which was in consonance of present study. 4.9 Identification of the adult parasites The parasites recovered in the faeces of the animals after deworming or at the time of post-mortem were identified after clearing in lactophenol, based on their 89

106 morphological characteristics and morphometry. The adult worms of B. transfuga recovered from sloth bear were identified on the basis of gross and microscopic morphological examination. Grossly, the size of the male worms (n=5) were measured 79.2±11.3 mm (64 94 mm) 1.5±0.3 mm ( mm) (length width), whereas, female parasites (n=5) were measured 162.8±18.7 mm ( mm) 2.3 ±0.5 mm ( mm) (length width), respectively (Fig. 47). The distinctive morphological features included three developed triradiate lips (one dorsal and two subventral) with cervical alae and filariform oesophagus (Fig. 48) in adult parasites of B. transfuga. The caudal ends of the male parasites were slightly rolled with presence of large number of precloacal papillae (48 70) (Fig. 49), with prominent cloacal opening and tail knob (Fig. 50), whereas female posterior end had a blunt tail. The morphological observations of adult male and female B. transfuga parasites were in corroboration with the observation of Testini et al. (2011), who recovered the same parasites from polar bears. The adult ascarid, capillarid and strongyle worms recovered at the necropsy of Columba livia domestica were identified on the basis of gross and microscopic morphological examination. Grossly, the length of the ascarid, capillarid and strongyle male and female worms (n = 5 each) were measured 63.2±4.7 mm (56 68 mm) and 70.9±4.9 mm (65 76 mm), 10.1±0.5 mm ( mm) and 12.6±0.2 mm ( mm) and 11.8±1.9 mm (9 14 mm) and 20±2.1 mm (18 23 mm), respectively. The distinctive morphological features observed in case of ascarid worms were developed triradiate lips (one dorsal and two subventral) with wide cephalic alae extending on both the lateral sides and filariform oesophagus (Fig. 51). Spicules in case of males were almost equal and there was presence of precloacal chitinous- 90

107 rimmed sucker (Fig. 52). The morphological characters of the ascarid worms depicted them to be Ascaridia columbae, which was in corroboration with the studies of Wehr and Hwang (1964) and Kajerova et al. (2004). The male of the Capillaria species was possessing oesophagus more than the half of the whole length of the parasite, whereas female possessed shorter oesophagus than males (Fig. 53). The vulva was slightly prominent in case of females and was observed slightly posterior to the intestinal-oesophageal union (Fig. 54). The morphological criteria about the adult worms of capillarid parasite in the present study were in agreement with the studies of Wakelin (1965), Soulsby (1982) and Park and Shin (2010), suggesting it to be Capillaria obsignata. In case of the strongyle worms, the cuticle at the anterior end was inflated forming a vesicular enlargement of about 94.9±6.1 µm ( µm). The mouth was lacking any visible papillae and was simple and unarmed (Fig. 55). The male parasites were comparatively smaller than the female counterparts. The vulva was situated at an average 4.6±0.9 mm (4-6 mm) from posterior end in female parasites and the body tapered to a blunt, narrow end with a short spine (Fig. 56). The characteristics features noticed about the adult strongyle worms were consistent with the study of Cuvillier (1937), who reported that Ornithostrongylus quadriradiatus was pathogenic for domestic pigeons. The morphology of female adult worms was distinguishably clear and could easily be differentiated on the basis of presence of spine at the posterior end Molecular detection of gastrointestinal parasites Identification of Baylisascaris transfuga An 1177 base pair product was obtained after amplification of the DNA extracted from the eggs and the adults. Similar size products were also obtained for 91

108 the DNA templates of the eggs retrieved after deworming, confirming the infection of sloth bear with B. transfuga only (Fig. 57). The similarity between the B. transfuga sequences obtained in the present study with the sequences available in GenBank targeting ITSs by Arizono et al (2010) (AB ), Testini et al (2011) (HM ) and Franssen et al (2013) (KC ) approached 99, 97 and 97 per cent, respectively (Fig. 58) by applying sequence diversity calculations targeting intra-population diversity. The genetic markers targeted ITSs, showed marked interspecific differences with B. procyonis species (JQ ), B. columnaris species (KC , KC , KC , KC , KC ) and B. schroederi species (JN , JN ) reported from Norway, the Netherlands and China, respectively (Fig. 58) on application of sequence diversity calculations. All other 12 samples found negative by classical parasitological technique were also confirmed negative by PCR. The primers were not able to amplify the DNA retrieved from the eggs of Toxocara canis Identification of Toxascaris leonina With the optimized cycling conditions, the DNA extracted from the faecal samples of the Asiatic lions before and after deworming, amplified a product of approximately 380 base pairs (Fig. 59) confirming the infection of Asiatic lions with T. leonina only, which was almost in concordance with the findings of Li et al (2007). No product was amplified from DNA extracted from Toxocara canis eggs, determining the specificity of the primers Identification of Trichuris trichiura The DNA retrieved from the faecal sample of the non human primates did not amplify and hence indicated the absence of T. trichiura. 92

109 4.11 Detection of haemoparasites All the blood samples of captive wild carnivores and elephants, retrieved during the study period were found negative by classical (thin smear and acridine orange staining) and molecular techniques for the presence of haemoparasites Serological detection of parasites Detection of Toxoplasma gondii The indirect ELISA revealed one animals positive ( lion) and four suspected (3 tigers and 1 lion) for T. gondii at the beginning of the study period (winter), which by the end (monsoon) turned out to be four positive and one suspected (Fig. 60). The antibody titres of two tigers and one lion exceeded the positive threshold value at the last observation. The anti-toxoplasma antibody titres showed only a slight increase in antibody titres with the change of seasons, while the animals in negative titre range at the beginning of the study remained in the same range. All the leopards and elephants were seronegative in both the seasons (Fig. 60), with percent optical density (OD) values ranging between % and % for leopards and elephants, respectively. Thus, haemato-biochemical alterations were studied in detail only for tigers and Asiatic lions, as the levels of all the haematological and biochemical parameters fell in normal range in case of leopards and elephants. The sero-detection of toxoplasmosis has been carried out in domestic ruminants (sheep, cattle and buffalo) in India (Selvaraj et al 2007, Sharma et al 2008), but no such study has been stipulated in wild felids. This investigation indicated the first serological evidence of T. gondii exposure of wild felids especially RB tigers and Asiatic lions in India, in which sero-occurrence increased from winter to monsoon. This may be due to the fact that parasites were in incubation stage during the winter 93

110 with no/ less detectable anti-toxoplasma gondii antibodies. These levels increased in monsoon. Further, this increase in number of infected individuals could be attributed to the stress conditions encountered by the animals during the summer and monsoon seasons, resulting in immunosuppression of the animals and hence, increased number of infected cases (Salant and Spira 2004). Another factor could be the plentiful moisture of monsoon that helps in prolonged survival of oocysts in the cool, moist conditions and hence aggravating the infection in these animals (Bisson et al 2000). The most frequent cause of the infection of toxoplasmosis is the ingestion of infected intermediate hosts. Thus, consumption of raw buffalo meat, raw chicken or intruding rodents could be held responsible for the infection in wild felids. The other routes of transmission might have involved mechanical transmission by flies, cockroaches or dung beetles into the living area of these wild felids (Thiangtum et al 2006). Although the elephants and leopards displayed negative antibody titres, but comparatively lower titre values of the elephants could be attributed to herbivorous nature of the elephants. The serum biochemical studies in the infected, suspected and non-infected groups including tigers and lions revealed no significant difference in the values of TBIL, AST, ALT, ALKP and TP and values of all the parameters were in the normal range in both the seasons irrespective of the group (Table 21). The values of ALB recorded in the infected and suspected group were significantly lower (P<0.05) than the values of non-infected group (Table 21). Globulin level showed significant increase in case of infected and suspected groups as compared to the non-infected group. The values of BUN in both the seasons in case of infected and suspected groups were significantly higher than that of non-infected group (P<0.05). The 94

111 creatinine levels of infected and suspected group animals were significantly higher than the non-infected group animals. The glucose levels of the infected and suspected groups were significantly lower (P<0.05) than the non-infected group. Individually, the values of all the suspected and infected tigers and lions were below the normal range, whereas the values of non-infected individuals were within the range (Table 21). The values of UA, K and Cl did not vary significantly among three groups. The values of Ca, Na and Fe in suspected and infected groups were significantly lower than the non-infected group (P<0.05), whereas the levels of P and CK were significantly higher in infected and suspected group animals than the values of noninfected animals (Fig. 61 and 62). All the haematological parameters varied non-significantly except for TLC and neutrophils percentage, where the values of infected and suspected group were significantly higher than the values of non-infected group (P<0.05) (Table 22). Further the values of all the groups in case of TLC were higher than the normal range. Although the values of the serum biochemical parameters (ALT, AST, TBIL and ALKP) were within the normal range, which were in concordance with the findings of Mosallanejad et al (2007), but, the increased levels of the globulins in both the infected and suspected groups insinuated the presence of immunoglobulins generated against the ongoing chronic infection (Sedlack and Bartova 2006). Higher creatinine level was suggestive of T. gondii infection in the definitive hosts (Lappin 1996). The decreased blood glucose levels in the present study could be correlated with the lethargy in T. gondii infected felids (Elmore et al 2010). The significantly increased creatine kinase levels in infected and suspected animals as compared to the noninfected group animals is an indicative of brain or muscle damage. The decreased 95

112 Table 21: Serum biochemical alterations in Infected, Suspected and Non-infected Royal Bengal tigers and Asiatic lions Biochemical TBIL AST ALT ALKP TP ALB GLO BUN CRE GLU Parameters (mg/dl) (U/L) (U/L) (U/L) (g/dl) (g/dl) (g/dl) (mg/dl) (mg/dl) (mg/dl) Groups Group I (Infected) Group II (Suspected) Group III (Noninfected animals) 0.74± 41± 51.2± 33.4± 7.56± 3.26± 4.3± 69.6± 3.26± 29.8± 0.50 a a 7.79 a 6.91 a 0.53 a 0.26 a 0.43 a a 0.50 a 7.01 a 0.66± 45.4± 46.4± 32.8± 7.38± 3.26± 4.12± 64.2± 3.06± 23± 0.34 a a a 4.21 a 0.19 a 0.24 a 0.18 a 8.67 a 0.39 a 3.74 a 0.96± 51.5± 46.11± 33.28± 7.36± 4.03± 3.33± 50.61± 2.08± 68.72± 0.38 a a a 6.73 a 0.46 a 0.44 b 0.36 b 5.19 b 0.29 b 12.5 b Normal range (NR) Shrivastav et al., 2011 (Tigers) and Jani and Sabapara, 2010 (Lions) Values indicated as Mean± Standard deviation Values with different superscripts differ significantly at P<

113 Table 22: Haematological values of Infected, Suspected and Non-infected Royal Bengal tigers and Asiatic lions Haematological Parameters Hb (g/dl) TLC (x10 3 / µl) TEC (x10 6 / µl) PCV (%) Platelets (10 4 cells/ µl) N (%) L (%) E (%) M (%) Groups Group I 14.02± 18840± 8.53± 41.08± 204.2± 76.2± 20.6± 2± 0.8± (Infected) 1.39 a a 0.69 a 4.86 a a 7.22 a 5.55 a 2.34 a 1.79 a Group II 13.04± 17652± 8.46± 38.64± 274± 76.4± 22.4± 1.2± 0 a (Suspected) 2.46 a a 1.26 a 6.11 a a 9.20 a 9.21 a 1.79 a Group III 14.72± 14418± 8.14± 42.98± ± 68.94± 29.28± 1.11± 0.5± (Non-infected) 1.03 a b 0.70 a 7.62 a a 2.62 b 2.99 a 1.41 a 1.15 a Shrivastav et al., Values indicated as Mean± Standard deviation Values with different superscripts differ significantly at P<

114 Table 23: Serum biochemical observations of elephants Biochem. AST ALT AKP TB TP Alb Glo A:G BUN Cre Fe G GT Na Cl Parameters (U/L) (U/L) (U/L) (mg/dl) (g/dl) (g/dl) (g/dl) (mg/dl) (mg/dl) (µg/dl) (U/L) Infected animals (n=2) 29.5± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.41 Noninfected animals (n=4) ± ± ± ± ± 0.16** 3.32 ± ± 0.21* 0.74 ± 0.06* 17 ± ± ± ± ± 3.09** ± 2.5** Normal range (Silva and Kuruwita 1993) Values expressed as Mean ± Standard deviation n indicates number of animal in a group Superscript * and ** indicated values varying significantly at P<0.05 and P<

115 Table 24: Haematological assessment of the elephants Haematological Hb TLC TEC PCV Platelets N L E M Parameters (g/dl) (x10 3 / µl) (x10 6 / µl) (%) (10 4 cells/ µl) (%) (%) (%) (%) Infected animals ± ± 2.52 ± ± ± 17 ± 33 ± 3.5 ± 45 ± 4.24 (n=2) Non-infected 13.32± ± 2.86 ± ± ± 29.75± 37.5± 2± 21.5± animals (n=4) * * Normal range (Silva and Kuruwita 1993) Values expressed as Mean ± Standard deviation n indicates number of animal in a group Superscript * indicated values varying significantly at P<

116 serum Ca levels revealed the involvement of pancreas in chronic toxoplasmosis leading to pancreatitis in definitive hosts (Advincula et al 2010). The neutrophilic leucocytosis observed in the present study accede with the findings of Mosallanejad et al (2007), who considered it as a major finding in the toxoplasmosis. Although, the other important attributes of the T. gondii infection, viz. anaemia, jaundice etc., were not observed due to the chronic nature of the infection in these animals (Advincula et al 2010) Detection of Neospora caninum All the samples were found negative for N. caninum antibodies Detection of Trypanosoma evansi All the serum samples were found negative for the presence of T. evansi antibodies except for two samples. One sample from female elephant was found strongly positive with +++ agglutination pattern (Fig. 63), whereas one sample of male elephant was slightly positive (+). Although the molecular diagnosis revealed no sample positive for T. evansi infection, thus, serological detection reveals prior exposure of the animals to the parasite. The T. evansi infection had earlier been reported in Indian elephants (Tresamol 2002). The haemato-biochemical studies also revealed alterations in the biochemical parameters of the infected as compared to the non-infected individuals (Table 23). Hyperproteinemia and hyperglobulinemia in the infected as compared to non-infected elephants could be related to the exposure of the individuals to the parasite, stimulating the immune system to secrete immunoglobulins (Ahmadi-hamedani et al 2014). With hyperglobulinemia in trypanosomosis, hypoalbuminemia could take place as a compensatory mechanism to maintain osmolarity (Da Silva et al 2010). The significant decrease in the concentration of the sodium in T. evansi infected animals had already been described 100

117 by Da Silva et al (2011), who carried out the estimation of the alteration in the serum minerals level in T. evansi infected rabbits and reported that the alteration in sodium levels could only be recorded in chronic cases. All the haematological parameters of the infected and non-infected elephants were non- significantly different, except leucocytosis (Table 24). Leucocytosis had earlier been reported in the camels infected with T. evansi infection (Gutierrez et al 2006, Ahmadi-hamedani et al 2014), which could be attributed to increased activity of the mononuclear phagocytic system during trypanosomosis (Ahmadi-hamedani et al 2014). The percentage of monocytes was also recorded significantly higher in the infected than non- infected individuals Histopathological studies A detailed microscopic study on the tissues retrieved from intestines, gizzard, liver, lungs, heart and kidneys of two pigeons, which were found infected with gastrointestinal parasites at the time of necropsy was carried out, where marked lesions with cut section of parasites were observed in intestines and kidneys. Although, no parasites were detected in heart, lungs, liver and gizzard, but certain microscopic alterations were there in some organs viz. hyperplasia of lining epithelium with sloughing at places was observed in gizzard. Most of the lesion in case of intestine was observed in the anterior part of the small intestine i.e. duodenum, from where most of the parasites were recovered. At places, cut sections of adult Capillaria obsignata, larvae of Ascaridia columbae and adult of Ornithostrongylus quadriradiatus with sloughing and hyperplasia of lining epithelium was observed (Fig ). The histopathological findings depicting concurrent inflictions to the intestinal tract were in agreement with the findings of Adang et al (2010) and Park and Shin (2010), which carried out the histopathological studies to adjudge the 101

118 adverse effect of Ascaridia galli and concurrent infection of Heterakis gallinarum and Capillaria obsignata in domestic pigeons and rock partridges, respectively. The histopathological study of the kidneys revealed the presence of a digenetic trematode parasite, consistent with Paratanaisia bragai. The brown coloured eggs, some of which containing fully developed embryos were also observed (Fig. 66). Some of the parasites and their eggs were found trapped in the glomeruli, resulting in their destruction (Fig. 66). The major lesions were characterized by haemorrhages and infiltration of mononuclear inflammatory cells around the cut sections of the parasites in the collecting tubules (Fig. 67). The renal tubules exhibited degeneration and presence of brown hemosiderin pigment within the lining epithelial cells. Some of the eggs and hatched miracidium were found free within parenchyma thereby causing damage to the glomeruli (Fig. 67) tubules along with infiltration of inflammatory cells. The Paratanaisia species had previously been reported to parasitize the kidneys of domestic pigeons (Borah et al 2009), cattle egret (Abdo and Sultan 2013), guinea fowl (Menezes et al 2001), whistling ducks (Fedynich et al 1996), red bird of paradise (Unwin et al 2013), ruddy ground dove (Pinto et al 2004), turkey (Brener et al 2006) and ring- necked pheasant (Gomes et al 2005) and the parasite had been incriminated as a cause of death of the heavily infected birds (Arnizaut et al 1992). The detailed examination revealed that tubular and glomerular damage was primarily due to the parasites as well as by the invading eggs. However, Abdo and Sultan (2013) incriminated the renal damage primarily due to the invading eggs. All other observations in the present study were in corroboration with the findings of Abdo and Sultan (2013), who reported the presence of P. bragai in cattle egret. The microscopic 102

119 changes were also in agreement with the observations of Pinto et al (2004) and Gomes et al (2005) Identification of ectoparasites Flea: The fleas recovered from the jungle cat were identified to be Ctenocephalides felis felis based on the morphological characteristics i.e. based on the shape of the head, first spine of the genal comb and number of bristles in lateral metanotal area. The length of the head was found to be twice the height (Fig. 68) and first spine of the genal comb was approximately equal to the second one (Fig. 69). There was only one spine on lateral metanotal area (Fig. 70). All the observations were in corroboration with findings of Linardi and Santos (2012) and were suggesting the flea to be C. felis felis. Louse: The lice recovered from a pigeon (Columba livia domestica) were delineated as Afrimenopon waar based on the observations of three lice specimen targeting head length (310 ± µm), head width (370 ± µm), thorax length (350 ± 10 µm), thorax width (320 ± µm), abdomen length (676.7 ± µm), abdomen width (400 ± 5.77 µm) and total length ( ± µm) (Fig ). The observations were in concordance with the report of Dik (2010) who coined this ectoparasite on a budgerigar in Turkey and identified as A. waar based on the morphometric observations seen in the present study Therapeutic management and drug efficacy assessment The therapeutic management had been carried out thrice during the study period, twice in the year 2013 and once in 2014, but with different drug dosage schedules or with different drugs. During the first treatment schedule in animals, fenbendazole and its combinations were used singly for three days, whereas, during the second treatment fenbendazole and its combinations were used for 5 days. During the third 103

120 treatment, ivermectin and its combinations were used singly on three alternate days. The fenbedazole and its combinations used during the first treatment showed failure in reducing the faecal egg count in almost all the infected species of animals. The observation was in concordance with the finding of Nalubamba and Mudenda (2014), which carried out an anthelmintic efficacy assessment study on antelopes. Liquid Nilzan proved effective for the treatment of anoplocephalid infection in wild herbivores. The drug efficacy studies were carried out against most prevalent parasitic infections and where different infected groups/ individuals were available. In the large and small ruminants, drug efficacy studies were conducted against strongyles, in lions against Toxascaris leonina and in primates against Trichuris sp. infection, whereas, the drug efficacy assessment against Ascaridia sp. infection was carried out in galliformes and collumbiformes birds. Drug efficacy assessment against strongyles in large ruminants: Six different groups of large ruminants including blue bull, guar and four horned antelopes were considered for the assessment of anthelmintic efficacy of different drugs. The fenbendazole and its combinations used during the first treatment period showed highest faecal egg count reduction (i.e.79%) at day 3 post treatments (Table 25). But when the same drug was used for an increased period i.e. for 5 days, a maximum faecal egg count reduction (96.41%) was observed at day 7 post treatments (Table 25). The resistance against benzimidazoles develop in stages and in the initial phase the individual parasites respond to repeated doses of the drug (Coles et al 2001), as it had been observed in the present study. But, in case of treatment II, an increase in the egg per gram values at day 21 post treatments indicated the temporary sterilizing/ knock down effect of the fenbendazole on helminth parasites (Coles et al 2006), depicting the development of resistance against fenbendazole in large wild ruminants. 104

121 On the other hand, ivermectin used for three alternate days proved highly effective and showed highest egg count reduction to up to 100% at day 7 post treatments (Table 25). The faecal egg count reduction remained around 91.42% at day 21 post treatments, demonstrating higher efficacy of ivermectin. Thus, alteration of drugs could prove highly beneficial for the control of gastrointestinal parasites in captive wild ruminants (Nalubamba and Mudenda 2014), because if resistant genotypes have a reduced ecological fitness compared to susceptible genotypes, then some reversion towards susceptibility would be expected in those periods when an alternate drug class was used (Coles and Roush 1992). But, a combination of anthelmintics (e.g. benzimidazoles and avermectins) could also be tried to reduce the probabilities of developing resistance against a single anthelmintic (Leathwick et al 2012) and also could help to expand the spectrum of efficacy of drugs against a wider range of helminths. Some authors (Dobson et al 2011, Leathwick 2011) also suggested the use of an active (presently in-use) class of anthelmintic with an older one to hinder or slow down the development of resistance against both new and older class(es) of anthelmintics. Drug efficacy assessment against strongyles in small ruminants: For the assessment of drug efficacy, 20 groups of small ruminants including goral, hog deer, swamp deer, black buck, white buck, Indian gazelle, barking deer, Manipuri deer and spotted deer were observed. Fenbendazole used during the first treatment period proved little effective and showed a maximum faecal egg count reduction of 37.41% at day 3 post treatment (Table 26). In this case, the egg per gram values of strongyles were observed even higher at day 21 post treatment ( ± ) than day 1 post treatment (972.5 ± ), depicting the resistance developed against fenbendazole. When fenbendazole was used for an increased period during the second treatment 105

122 schedule, it again proved slightly resistant at day 7 post treatment with 94.46% faecal egg count reduction (Table 26). The repeatitive indiscriminate use of routine anthelmintics with a high frequency of administration in captive wild ungulates runs the risk of encouraging the development of anthelmintic resistance as described in other livestock (Isaza et al 1995). During third treatment schedule, oral ivermectin started showing its effect even at day 3 post treatment (84.09% of faecal egg count reduction) and proved highly effective at day 7 post treatment with 96.21% faecal egg count reduction, which remained around 92.04% even at day 21 post treatments (Table 26). Although, Mackintosh et al (2014) reported the resistance of abamectin against the strongyles in a deer farm in New Zealand, but better efficacy of ivermectin used in the present study could be attributed to the alteration of drug and use of ivermectin for the first time against the deer species. Drug efficacy assessment in Asiatic lions: Anthelmintic efficacy against Toxascaris leonina in three lions was evaluated using fenbendazole and ivermectin. Fenbendazole used for three days schedule was proved ineffective to eliminate the infection in lions and showed a maximum egg reduction to 69.35% at day 3 post treatments (Table 27). The reason could be inappropriate drug dosage given to the animals due to unclear picture of the actual body weights of the animals under study. Fenbendazole used for extended period of time during second treatment period and ivermectin used during third treatment period proved effective with and 95.74% of egg reduction in lions, respectively (Table 27). Ivermectin had earlier been also used by Singla et al (2003) to evade piperazine resistant toxocarosis in lions at MCZP, Chhatbir, Punjab. But, in the present study the egg per gram values again increased significantly to ± and 400± (P<0.05), respectively after treatment II and III on day 21 post treatments (Table 27). This could be attributed to 106

123 the housing conditions of the zoo animals, especially the wooden floors where the faecal material remained clogged in the wooden planks. The eggs survived for a longer time even under harsh conditions, contributing to generation of a perpetual source for the transmission of infection (Bowman 1999, Singh et al 2006). Due to the direct life cycle of T. leonina and development of infective stages inside the eggs within a shorter period (around 9 days) favour the earlier completion of the life cycle (Moudgil et al 2014). This could be considered as a reason for persistence of T. leonina infection in well sanitized cages (Bowman 1999, Singh et al 2006). The similar findings were observed by Singh et al (2006a) and Dehuri et al (2013) while carrying out the assessment of efficacy of pyrental pamoate and ivermectin, respectively, against T. leonina infection in lions. Drug efficacy assessment against Trichuris sp. in non human primates: The complete care was taken while administering the drugs to the non human primates (kept in groups) so as to avoid the wastage or overconsumption of the drugs by providing the food containing the drug to the individual animal specifically. There were six groups of non human primates including hamadryas baboon, rhesus macaque, bonnet macaque, common/ gray langoor targeted in the present study. The drug (fenbendazole) proved ineffective for the first treatment period by depicting a maximum egg reduction of 58% at day 3 post treatments (Table 28) and the reason could be inappropriate drug dose due to variation in the body weights of the animals. An increased dosage schedule during the second treatment proved effective and showed a maximum faecal egg reduction of 99.20% at day 7 post treatment (Table 28). On the other hand, ivermectin proved completely effective and depicted 100% reduction in egg count at day 7 post treatments (Table 28). A combination of benzimidazole (albendazole) and ivermectin used by Kagira et al (2011) to treat 107

124 gastrointestinal parasites of non human primates showed 100% faecal egg count reduction till day 28 post treatments. But, individually used ivermectin did not prove effective after day 7 post treatments, which is in consonance with the present study as faecal egg per gram values increased significantly to ± (P<0.05) at day 21 post treatments from zero (at day 7 post treatments). The finding could be attributed to temporary sterilizing effect of ivermectin on nematode parasites (Coles et al 2006). Drug efficacy assessment against ascarids in Galliformes and Columbiformes: The study for the assessment of anthelmintic efficacy of albendazole and piperazine against six flocks each of galliformes (red Jungle fowl, Indian Peafowl, golden pheasant, gray partridge, black partridge, Lady Amherst s pheasant) and columbiformes (white pigeons, black pigeons and doves) was carried out (Tables 29 and 30, respectively). The albendazole used for five days during the first treatment period proved ineffective to reduce ascarid egg burden in galliformes and columbiformes with a maximum egg reduction of and 55.71%, respectively at day 7 post treatments. Albendazole used for an extended period of time proved effective in galliformes with a reduction of 99% faecal egg count at day 7 post treatments but not in case of columbiformes (94.28% faecal egg count reduction at day 7 post treatments). Albendazole used by Parsani et al (2007) for the treatment of nematodiasis in zoo pigeons also showed maximum faecal egg count reduction (88.92%) at day 7 post treatments. But, the egg per gram values increased afterwards showing decreased efficacy of albendazole in columbiformes. On the other hand, in the present study piperazine proved effective in columbiformes with a faecal egg count reduction of 95.87% at day 7 post treatments but not in case of galliformes, where 94.28% egg count reduction was observed. 108

125 Table 25: Drug efficacy assessment against strongyles in large ruminants (n=6) Animals EPG Pre treatment Post treatments FECR (%) I II III IV I II III IV Treatment I 500± a 465± a 105± b 160± b 375± a Treatment II 1115± a 550± b 220± c 40± d 235± c Treatment III 525± a 410± b 85± c 0 45± d Post treatments EPG: I, II, III, IV- days 1, 3, 7 and 21 post treatments, respectively FECR (%): I, II, III, IV- faecal egg count reduction at days 1, 3, 7 and 21 post treatments, respectively Large ruminants: Blue bull, Guar, Four horned antelopes Values expressed as Mean ± Standard deviation n indicates number of animal in a group Different superscripts indicate values varying significantly at P<

126 Table 26: Drug efficacy assessment against strongyles in small ruminants (n=20) Animals EPG Pre treatment Post treatments FECR (%) I II III IV I II III IV Treatment I ± a 972.5± ab 727.5± a 955± ab ± a Treatment II ± a ± a 680± b 92.5± d 297.5± c Treatment III 660± a 470± b 105±72.36 c 25± d 52.5± d Post treatments EPG: I, II, III, IV- days 1, 3, 7 and 21 post treatments, respectively FECR (%): I, II, III, IV- faecal egg count reduction at days 1, 3, 7 and 21 post treatments, respectively Small ruminants: Goral, Hog deer, Swamp deer, Black buck, White buck, Indian gazelle, Barking deer, Manipuri deer, Spotted deer, Fallow deer Values expressed as Mean ± Standard deviation n indicates number of animal in a group Different superscripts indicate values varying significantly at P<

127 Table 27: Drug efficacy assessment against toxascariosis in lions (n=3) EPG Pre treatment Post treatments FECR (%) Animals I II III IV I II III IV Treatment I ± a ± b ± b ± b 500± 100 b Treatment II ± a ± b ± bc 66.67± c ± b Treatment III ± a ± b ± bc 33.33± c 400± b Post treatments EPG: I, II, III, IV- days 1, 3, 7 and 21 post treatments, respectively FECR (%): I, II, III, IV- faecal egg count reduction at days 1, 3, 7 and 21 post treatments, respectively Values expressed as Mean ± Standard deviation n indicates number of animal in a group Different superscripts indicate values varying significantly at P<

128 Table 28: Drug efficacy assessment against Trichuris species in non human primates (n=6) Animals EPG Pre treatment Post treatments FECR (%) I II III IV I II III IV Treatment I ± a ± ab 175± c 200± bc 250± bc Treatment II ± a 450± b ± c 8.33± c 66.67± c Treatment III 225± a ± a 41.67± b ±58.45 b Post treatments EPG: I, II, III, IV- days 1, 3, 7 and 21 post treatments, respectively FECR (%): I, II, III, IV- faecal egg count reduction at days 1, 3, 7 and 21 post treatments, respectively Non human primates- Hamadryas baboon, Rhesus macaque, Bonnet macaque, Common/ Gray langoor Values expressed as Mean ± Standard deviation n indicates number of animal in a group Different superscripts indicated values varying significantly at P<

129 Table 29: Drug efficacy assessment against ascarids in Galliformes (n=6) EPG Pre treatment Post treatments FECR (%) Birds I II III IV I II III IV Treatment I ±97.03 ab ±81.65 ab ±25.82 a 175± a 400± b Treatment II ± a ± b ± c 8.33± d 66.67± cd Treatment III ± a ± a ± b 16.67± c 83.33± b Post treatments EPG: I, II, III, IV- days 1, 3, 7 and 21 post treatments, respectively FECR (%): I, II, III, IV- faecal egg count reduction at days 1, 3, 7 and 21 post treatments, respectively Galliformes- Red Jungle fowl, Indian Peafowl, Golden pheasant, Gray partridge, Black partridge, Lady Amherst s pheasant Values expressed as Mean ± Standard deviation and n indicates number of animal in a group Different superscripts indicated values varying significantly at P<

130 Table 30: Drug efficacy assessment against ascarids in Columbiformes (n=6) EPG Pre treatment Post treatments FECR (%) Birds I II III IV I II III IV Treatment I 1750± a ± ab 775± b ± b ±97.03 ab Treatment II ± a 1125± b ±97.03 c ± c 500± c Treatment III ± a ± b 200± c 33.33± c ± c Post treatments EPG: I, II, III, IV- days 1, 3, 7 and 21 post treatments, respectively FECR (%): I, II, III, IV- faecal egg count reduction at days 1, 3, 7 and 21 post treatments, respectively Columbiformes- White pigeons, Black pigeons and doves Values expressed as Mean ± Standard deviation and n indicates number of animal in a group Different superscripts indicated values varying significantly at P<

131 Fig. 1: Map of Punjab depicting the Zoological and Deer Parks targeted under the study 115

132 Fig. 2: Season wise prevalence of parasitic infection in animals and birds of MC Zoological Park, Chhatbir, Punjab 116

133 Prevalence 20 Individually enclosed animals Herds/Groups Birds Win. 13 Sum. 13 Mon. 13 Win. 14 Sum. 14 Mon. 14 Seasons Fig. 3: Captivity based prevalence in animals and birds of MC Zoological Park, Chhatbir, Punjab 117

134 Prevalence 60 Herbivores Omnivores Carnivores Birds Win. 13 Sum. 13 Mon. 13 Win. 14 Sum. 14 Mon. 14 Seasons Fig. 4: Season wise prevalence of parasitic infection in animals and birds based on feeding behaviour 118

135 Fig. 5: Photomicrograph of Haemonchus contortus egg from Indian gazelle Fig. 6: Photomicrograph of Cooperia sp. egg from black buck Fig. 7: Photomicrograph of Trichostrongylus sp. egg from blue bull Fig. 8: Photomicrograph of Trichuris sp. egg from swamp deer Fig. 9: Photomicrograph of Moniezia benedeni egg from gaur Fig. 10: Photomicrograph of Strongyloides papillosus egg from black buck 119

136 Fig. 11: Photomicrograph of ascarid egg from porcupine Fig. 12: Photomicrograph of Trichuris sp. egg from porcupine Fig. 13: Photomicrograph of strongyle egg from elephant Fig. 14: Photomicrograph of Strongyloides sp. egg from elephant Fig. 15: Photomicrograph of Eimeria pellita oocyst from goral Fig. 16: Photomicrograph of Eimeria kamoshika oocyst from goral 120

137 Fig. 17: Photomicrograph of Eimeria cylindrica oocyst from blue bull Fig. 18: Photomicrograph of ascarid egg from sloth bear Fig. 19: Photomicrograph of Trichuris sp. egg from non human primate Fig. 20: Photomicrograph of Strongyloides fuelleborni egg from non human primate Fig. 21: Photomicrograph of Isospora felis oocyst from jungle cat Fig. 22: Photomicrograph of Toxascaris leonina egg from Asiatic lion 121

138 Fig. 23: Photomicrograph of Isospora revolta oocyst from leopard Fig. 24: Photomicrograph of Spirometra sp. egg from leopard cat Fig. 25: Photomicrograph of Toxocara canis egg from hyaena Fig. 26: Photomicrograph of Ascaridia sp. egg from black pigeon Fig. 27: Photomicrograph of Capillaria sp. egg from white pigeon Fig. 28: Photomicrograph of Syngamus trachea egg from dove 122

139 Fig. 29: Photomicrograph of Ornithostrongylus quadriradiatus egg d from white pigeon Fig. 30: Photomicrograph of Amidostomum sp. egg from Mongolian pheasant Fig. 31: Photomicrograph of Hymenolepis sp. egg from Indian peafowl Fig. 32: Photomicrograph of Eimeria mayurai oocyst from Indian peafowl Fig. 33: Photomicrograph of Echinostoma revolutum egg from painted stork Fig. 34: Photomicrograph of Echinuria uncinata egg from sarus crane 123

140 Fig. 35: Photomicrograph of Eimeria labbeana oocyst from sarus crane Fig. 36: Photomicrograph of Eimeria sp. oocyst from red jungle fowl Fig. 37: Photomicrograph of Eimeria sp. oocyst from kalij pheasant 124

141 Fig. 38: Season wise prevalence of parasitic infection in animals and birds of Bir Motibagh Deer Park, Patiala 125

142 Prevalence Mon. 13 Win. 14 Sum. 14 Mon. 14 Seasons Fig. 39: Season wise prevalence of parasitic infection in birds of Patiala Aviary 126

143 Fig. 40: Season wise prevalence of parasitic infection in animals and birds of Bir Talab Deer Park, Bathinda 127

144 Fig. 41: Season wise prevalence of parasitic infection in animals and birds of Tiger Safari, Ludhiana 128

145 Fig. 42: Season wise prevalence of parasitic infection in animals and birds of Deer Park, Neelo 129

146 Fig. 43: Microphotograph of larva of Haemonchus contortus (10X) Fig. 44: Microphotograph of larva of Cooperia oncophora (40X) Fig. 45: Embryonic developmental stages of Baylisascaris transfuga and Toxascaris leonina in 0.4% formalin solution and faecal sample at 27±1 C temperature 130

147 One- cell Two- cell Four- cell Larvated egg Baylisascaris transfuga Toxascaris leonina Fig. 46: Photomicrograhs of various developmental stages of ascarids Fig. 47: The adult male and female Baylisascaris transfuga worms retrieved from sloth bear Fig. 48: Photomicrograph of anterior end Baylisascaris transfuga showing triradiate lips (A), cervical alae (B) and oesophagus (C) Fig. 49: Photomicrograph of the curved caudal end of male Baylisascaris transfuga with precloacal papillae (P) Fig. 50: Microphotograph of posterior end of male Baylisascaris transfuga: cloacal opening (C) and tail knob (T) 131

148 Fig. 51: Photomicrograph depicting anterior end of adult Ascaridia columbae with triradiate lips (black arrow), filariform oesophagus (yellow arrow) and wide cephalic alae (white arrow) Fig. 52: Photomicrograph depicting posterior end of adult male Ascaridia columbae with precloacal sucker (white arrow) and spicules (black arrow) Fig. 53: Photomicrograph of female Capillaria obsignata Fig. 54: Female C. obsignata characterized by slightly prominent vulva (white arrow), just posterior to the union of the oesophagus and the intestine (black arrow) Fig. 55: Anterior end of Ornithostrongylus quadriradiatus with vesicular enlargement (white arrow) Fig. 56: Posterior end of female O. quadriradiatus with vulva (white arrow) and spine (black arrow) 132

149 Fig. 57: PCR amplification for identification of Baylisascaris transfuga targeting ITSs: M- 100bp plus Gene Marker, 1- PCR product of adult worm, 2- PCR product of eggs retrieved before deworming, 3- PCR product of eggs retrieved after deworming, 4- Negative control, 5- Negative control containing PCR products of Toxocara canis eggs, 6- Non template control (without amplicon) Fig. 58: Phylogenetic relationship of Baylisascaris transfuga recovered from Melursus ursinus with related species of Baylisascaris genus (The values/ numbers at the nodes of the branches depict confidence level) 133

150 Fig. 59: PCR amplification for identification of Toxascaris leonina: 1 & bp plus Gene marker, 2- Non template control, 3- Negative control containing PCR products of Toxocara canis eggs, 4-7- PCR products of the eggs Fig. 60: Seasonal distribution of the antibody titres of the wild felids and their segregation into infected, suspected and non-infected groups, where T represented Tigers, L=Lions, E=Elephants and LEOP=Leopards. 134

151 Fig. 61: Biochemical parameters of the tigers depicting alterations in uric acid (UA), potassium (K), calcium (CA) and phosphorus (P) values (Superscript '*' and ** indicated values differing significantly at P<0.05) Fig. 62: Biochemical parameters of tigers depicting alterations in creatinine kinase (CK), sodium (Na), chloride (Cl) and iron (Fe) values (Superscript '*', ** and *** indicated values differing significantly at P<0.05) 135

152 Fig. 63: Card agglutination test for trypanosomosis: Positive titre (+++): Elephant 2 (Hema); Negative titre (-): Elephant 1(Rajkali), lion, tiger, leopard 136

153 Fig. 64: Photomicrographs depicting cut section of Capillaria obsignata (white arrows) and Ornithostrongylus quadriradiatus (black arrow) in the lumen of the intestine (10X) Fig. 65: Photomicrographs depicting cut section of C. obsignata (black arrows), Ascaridia columbae (brown arrow) and O. quadriradiatus (red arrow) in the lumen of the intestine (10X) Fig. 66: Photomicrograph of pigeon s kidney showing presence of brownish parasitic eggs (white arrows) with tubular damage (10X) Fig. 67: Photomicrograph depicting cut sections of the parasites trapped in renal glomeruli (red arrow) of pigeon, damaging renal tubules (black arrows) and causing marked haemorrhages and infiltration of mononuclear inflammatory cells (yellow arrow) (4X) 137

154 Fig. 68: Photomicrograph of the anterior end of Ctenocephalides felis felis Fig. 69: Photomicrograph of C. felis felis depicting first spine of the genal comb approximately equal to the second one Fig. 70: Photomicrograph of C. felis felis depicting only one spine on lateral metanotal area Fig. 71: Photomicrograph of the louse recovered from pigeon Fig. 72: Photomicrograph depicting morphometry of the head of the louse recovered from pigeon Fig. 73: Photomicrograph of the posterior end of the louse 138