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Threats of Disease Spillover from Domestic Dogsto Wild Carnivores in the Kanha Tiger Reserve,IndiaVratika ChaudharyClemson University, vchaudh@g.clemson.edu

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Recommended CitationChaudhary, Vratika, "Threats of Disease Spillover from Domestic Dogs to Wild Carnivores in the Kanha Tiger Reserve, India" (2016).All Theses. 2352.https://tigerprints.clemson.edu/all_theses/2352

THREATS OF DISEASE SPILLOVER FROM DOMESTIC DOGS TO WILD CARNIVORES IN THE KANHA TIGER RESERVE, INDIA

A Thesis Presented to

the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree

Master of Science Biological Sciences

by Vratika Chaudhary

May 2016

Accepted by: Dr. David W. Tonkyn, Committee Chair

Dr. Charles Rice Dr. A. B. Shrivastav

ii

ABSTRACT

Many mammalian carnivore species persist in small, isolated populations as a

result of habitat destruction, fragmentation, poaching, and human conflict. Their small

numbers, limited genetic variability, and increased exposure to domestic animals such as

dogs place them at risk of further losses due to infectious diseases. In India, dogs ranging

from domestic to feral are associated with villages in and around protected areas, and

may serve as reservoirs and vectors of pathogens to the carnivores within. India’s Kanha

Tiger Reserve (KTR) is home to a number of threatened and endangered mammalian

carnivores including tigers (Panthera tigris), leopards (Panthera pardus), wolves (Canis

lupus), and dhole (Cuon alpinus). It also contains hundreds of small villages with

associated dog populations, and my goal was to determine whether these dogs pose a

disease threat to KTR’s wild carnivores. In the summer of 2014 and again in the winter

of 2015 I estimated the density of dogs in villages of varying sizes and distances from

KTR’s core zone, and the exposure of these dogs to four pathogens that could threaten

wild carnivores: rabies, canine parvovirus (CPV), canine distemper (CDV), and canine

adenovirus (CAV). Dog population densities ranged from 3.7 to 23.7/km2 (14 to 45

dogs/village), and showed no systematic variation with village area or human population

size. These dog populations grew in all villages between the summer of 2014 and winter

of 2015, primarily through reproduction. No dog tested positive for rabies but I found

high levels of seroprevalence to the other three pathogens: CPV (83.6% in summer 2014,

68.4% in winter 2015), CDV (50.7% in summer 2014, 30.4% in winter 2015) and CAV

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(41.8% in summer 2014, 30.9% in winter 2015). The declines in seroprevalence between

summer and winter were primarily due to births in the population, of animals not exposed

to the viruses. I opportunistically documented interactions between the dogs and wild

carnivores that might allow disease transmission. I measured these interactions as the

presence of wild carnivores in surveyed villages. In this study I document the existence

of a large population of unvaccinated dogs in and around KTR, with high levels of

seroprevalence to pathogens with broad host ranges. These dogs also have frequent

contact with wild carnivores. I conclude that these dogs pose a high risk of disease

spillover to wild carnivores in the region.

I also tested for CPV and CDV in wild carnivore samples obtained from the KTR

Forest Department from 2010 to 2015. While one tiger blood sample was seropositive

for CPV antibodies, the reverse transcriptase polymerase chain reaction found no

evidence of CPV in tissue samples from five tigers, one leopard and one palm civet

(Paradoxurus hermaphroditus), and no CPV or CDV in the three blood samples of tigers.

Despite these results, I argue for continued surveillance in KTR, given the ubiquity of

village dogs in the area with high seroprevalence of CDV and CPV and the contact

between dogs and endangered carnivores in KTR.

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ACKNOWLEDGMENTS

I acknowledge Clemson University and the National Wildlife Refuge Association

for providing funds for the study, the Madhya Pradesh Forest Department particularly

Mr. Narendra Kumar and Dr. Suhas Kumar for issuing permits, and the Kanha Tiger

Reserve Forest Department particularly Mr. J. S. Chauhan, Dr. Sandeep Agrawal, Dr.

Rakesh Shukla and Mr. Rajneesh Singh for providing samples and support. I thank Dr.

Kajal Jadhav, Dr. Himanshu Joshi, Dr. K. P. Singh, Dr. Amitod, Dr. Amol Rokade and

Dr. Sunil Goyal of the Centre for Wildlife Forensic and Health in Jabalpur, India and Dr.

Dharmaveer Shetty of the University of California, Davis, USA for their help in the

laboratory and field. I thank Mr. Amit Sankhala and Mr. Tarun Bhati for logistical

support.

Surveys and sample collection for this study were conducted in accordance with

Institutional Animal Care and Use Committee number 2014-025 of Clemson University and

Madhya Pradesh Forest Department’s research permit number I/3023.

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TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i

ABSTRACT ..................................................................................................................... ii

ACKNOWLEDGMENTS .............................................................................................. iv

LIST OF TABLES .......................................................................................................... vi

LIST OF FIGURES……………………………………………………………………..viii

CHAPTER

1. ESTIMATION OF DOG ABUNDANCE AND SURVEY OF DISEASEEXPOSURE IN VILLAGE DOGS OF KANHA TIGER RESERVE, INDIA AND ESTIMATION OF POTENTIAL CONTACT RATE OF THESE DOGS WITH WILD CARNIVORES OF KANHA TIGER RESERVE, INDIA……………..........................1

Introduction……………………………………………………………...1 Study site and methods…………………………………………………..7 Results ………………………………………………………………….16Discussion………………………………………………………………31 References……………………………………………………………....44

II. A SURVEY FOR CANINE PARVOVIRUS AND CANINE DISTEMPERVIRUS IN WILD CARNIVORES OF KANHA TIGER RESERVE, INDIA, USING REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ...................... 51

Introduction ............................................................................................ 51 Study site and methods .......................................................................... 53 Results………………………………………………………………… 56Discussion……………………………………………………………...57 References………………………………………………………….......59

Appendices…………………………………………………………………………….62 Appendix A……………………………………………………………………………63 Appendix B……………………………………………………………………………64

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LIST OF TABLES

Tables Page

1.1 Examples of rabies CPV, CDV and CAV infections in wild carnivores that were contracted from domestic dogs………………………………………..3

1.2 Primer base pairs for RT-PCR. Primer base pair sequence (forward (F) and reverse (R)) used in RT-PCR for each of the viruses along with the nucleotide positions and length of the band in base pairs……………………………..14

1.3 Seroprevalence in various categories of dogs in summer 2014 (S) and winter 2015 (W). This lists the number of dogs sampled in each season in each category: males, females, adults, juveniles, female adults (FA), female juveniles (FJ), male adults (MA) and male juveniles (MJ), and the percent of each that were seropositive for CPV, CDV and CAV..................................23

1.4 Seroprevalence of CPV, CDV and CAV in dogs of near and far villages in winter 2015, as classified in various ways: males, females, adults, juveniles, female adults (FA), female juveniles (FJ), male adults (MA) and male juveniles (MJ).…………………………………………………………..…24

1.5 Antibody titer of CPV, CDV and CAV in the dogs. Number of dogs and proportion of dogs that tested positive with antibody titer range of S5 (high antibody titer), S3 (moderately high antibody titer) and, S1 (low antibody titer) are listed for categories of total number of dogs, adult and juvenile dogs, male and female dogs, over the summer 2014 and winter 2015.……….…25

1.6 Antibody titer proportion in dogs of KTR of near and far villages in winter 2015. Listed is proportion of dogs in pooled category as total and adults and juveniles with S5, S3 and S1 titer for near villages (NS5, NS3, NS1) and far villages (FS5, FS3 and FS1) …………………………………………………………………………….26

1.7 Co-infection of pathogens. P value and odds ratio (95% confidence limits) for the relation between seroprevalence for each pathogen (outcome) and seroprevalence of one or both of the other two pathogen (condition) listed for categories: S (summer 2014) total (near and far villages combined), W (winter 2015) total (near and far villages combined), winter 2015 (only near villages) and winter 2015 (only far villages)……………………………………… 27

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List of Tables (contd.)

Tables Page

1.8 Signs of wild carnivores in surveyed villages. Number of signs of wild carnivore presence noted in surveyed near and far villages in summer 2014 (S) and winter 2015 (W)………………………………………………………..30

2.1 Animals tested for CPV and CDV, with their sex (male (M), female (F)), age (in years), tissue used, preservative used to store tissue and year of collection of sample…………………………………………………………………....55

2.2 Primer base pair sequence (forward (F) and reverse (R)) used in RT-PCR for each of the viruses along with the nucleotide positions and length of the band, (in base pairs)…………………………………………………………….... 55

viii

LIST OF FIGURES

Figures Page

1.1 Study site. Map of Kanha Tiger Reserve (KTR) with core (shaded) and buffer zones, and with villages that were sampled in summer 2014 and winter 2015. The inset shows the location of KTR in India………………………………. 9

1.2 Estimated number of dogs in various categories. (a) Estimated number of dogs in four near villages (N1, N2, N3, N4) and four far villages (F1, F2, F3, F4) in summer 2014 (blue) and winter 2015 (red), (b) actual counts of juveniles (blue) and adults (red) in the near and far villages in summer (S) and winter (W), (c) actual counts of females (blue) and males (red) in near and far villages in summer (S) and winter (W)………………………………….. …17

1.3 Seroprevalence of a. CPV, b. CDV and c. CAV in dogs for summer 2014 and winter 2015. The number of dogs sampled in each category is listed below the corresponding bar …………………………………………………………..20

1.4 Seroprevalence of a. CPV, b. CDV and c. CAV in dogs from near and far villages. The seroprevalence is listed as the percentage of dogs and adults and juvenile categories only for winter 2015. The numbers of dogs tested are listed on the x axis for each category……………………………………… .21

1.5 Potential numbers of contacts between wild carnivores and dogs. Number of individual events of wild carnivore presence in surveyed villages of KTR noted over two field seasons (60 days per seasons) and categorized for near and far villages………………………………………………………………29

1

CHAPTER ONE

ESTIMATION OF DOG ABUNDANCE AND SURVEY OF DISEASE

EXPOSURE IN VILLAGE DOGS OF KANHA TIGER RESERVE, INDIA

AND ESTIMATION OF POTENTIAL CONTACT RATE OF THESE DOGS

WITH WILD CARNIVORES OF KANHA TIGER RESERVE, INDIA.

1.1 INTRODUCTION

Large mammalian carnivores, henceforth carnivores, around the world are

threatened by habitat destruction and fragmentation, poaching, and prey depletion (Di

Marco et al., 2014). Many now persist in small, fragmented populations that are also

vulnerable to infectious diseases, including those caused by pathogens with broad

geographic and host ranges (Altizer et al., 2003; Smith, Acevedo-Whitehouse &

Pedersen, 2009; Thorne & Williams, 1988; Young, 1994). In fact, carnivores are

threatened by infectious diseases to a greater degree than other mammalian taxa

(Pedersen et al., 2007), with transmission occurring from livestock (De Vos et al., 2001)

and domestic carnivores (Alexander et al., 2010; Cleaveland et al., 1999; Cleaveland et

al., 2001). Examples include Mycobacterium bovis infection in African lions (Panthera

leo: Viljoen, Van Helden & Millar, 2014), canine distemper virus (CDV) in Amur tigers

(Panthera tigris altaica: Seimon et al., 2013) and African lions (Roelke-Parker et al.,

1996), and rabies virus in Ethiopian wolves (Canis simensis: Laurenson et al., 1998).

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The small and isolated populations of wild carnivores do not sustain many pathogen

species on their own (Lafferty & Gerber, 2002). However, large and geographically

extensive populations of domestic carnivores can serve as reservoirs for pathogens that

threaten many wild carnivores (Carpenter et al., 1998; Funk et al., 2001; Woodroffe,

1999), and contact between domestic and wild carnivores can facilitate disease

transmission (Cleaveland et al., 1999; Cleaveland et al., 2000; Lafferty & Gerber, 2002;

SilleroZubiri, King & MacDonald, 1996). In particular, dogs (Canis familiaris) are

present globally in large numbers (Gompper, 2014) and pose multiple threats to wild

carnivores (Fiorello et al., 2006; Young et al., 2011). These threats include competing

for food or other resources (Glen & Dickman, 2005) and preying on wild carnivore

young, but also the transmission of diseases through a variety of interactions:

interspecific hybridization (Bohling & Waits, 2011), scavenging on the same carcasses,

or being preyed upon by wild carnivores (Butler, 2000; Butler et al., 2004).

Of the 13 pathogens known to threaten wild carnivore health and survival, seven

(56%) are viruses (Pedersen et al., 2007) and dogs are reservoirs for all of them. Four

viruses that are carried by dogs and affect many carnivore species worldwide are the

rabies virus, canine parvovirus (CPV), CDV (Pedersen et al., 2007), and canine adeno

virus (CAV) (Belsare, Vanak & Gompper, 2014) (Table 1.1). These pathogens may pose

a greater threat to endangered carnivores in countries such as India, where large human

and associated dog populations live in and around protected areas. Recent reports of

CDV in captive Bengal tigers (P. tigris tigris) in India

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(http://www.huffingtonpost.com/2014/01/13/india-tigers-virus_n_4588004.html), CPV

seropositivity in one wild tiger (Chaudhary et al., unpublished data, chapter 2) and CDV

and CAV induced mortality in Bengal foxes (Vulpes bengalensis) (Belsare, Vanak &

Gompper, 2014) have raised concerns about infectious disease threats to the wild

carnivores in India. This thesis describes my study of the risk of transmission of these

four viruses from dogs to wild carnivores in one of India’s premier national parks.

The viruses that I studied spread in various ways. Rabies virus is shed from the

salivary glands and usually transmitted when an infected animal bites or licks a

susceptible animal, transferring the virus into a wound or mucosa. Rabies can also be

contracted when a susceptible animal consumes virus-infected tissue (Wandeler et al.,

1993). CPV is transmitted through the feco-oral route and can persist in the feces and

other organic substrate such as soil for months. Thus the habitation of wild carnivores

and dogs in the same area, even in the absence of direct contact, may be sufficient for

CPV transmission from dogs to wild carnivores. CDV and CAV are excreted as airborne

aerosoled virus and can also be contracted through inhalation, contact with body fluids

such as urine and/or by consuming an infected animal (Alexander et al., 1993b).

Table 1.1 Examples of rabies, CPV, CDV and CAV infections in wild carnivores that

were contracted from domestic dogs.

Pathogen Examples

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Rabies Ethiopian wolf (Canis simensis: Randall et al., 2004), African wild dogs (Lycaon pictus: Alexander et al., 1993a)

CPV Maned wolf (Chrysoyon brachyurus: Cubas, 1996; Deem & Emmons, 2005)

CDV Siberian tigers (Panthera tigris altaica: Gilbert et al., 2015), Maned wolf (Chrysocyon brachyurus: De Almeida et al., 2012), Black-footed ferrets (Mustela nigripes: Thorne & Williams, 1988; Williams et al., 1988)

CAV Crab-eating fox (Cerdocyon thous: Monteiro et al., 2015), Indian fox (Vulpes bengalensis: Belsare, Vanak & Gompper, 2014)

Here I assess the threat of disease spillover that dogs pose to wild carnivores in a

tiger conservation priority area in central India - Kanha Tiger Reserve (KTR). KTR

supports 13 wild carnivores (DeFries, Karanth & Pareeth, 2010): tigers, leopards

(Panthera pardus), jungle cats (Felis chaus kutas), dhole (Cuon alpinus), wolves (Canis

lupus pallipes), jackals (Canis aureus indicus), honey badger (Melivora capensis),

pangolins (Manis crassicaudata), common mongoose (Herpestes edwardsii), ruddy

mongoose (H. smithii), striped hyaena (Hyaena hyaena), sloth bear (Melursus ursinus)

and Bengal fox. It also contains approximately 150 villages in the buffer zone (1,134

km2) and 8 villages in the core zone (940 km2), which support populations of dogs that

may interact with wild carnivores in various ways.

The dogs in KTR may be classified as feral, semi-owned or owned. Feral dogs do

not associate with any household while the few owned dogs are kept on a leash. The rest

are semi-owned, and associate with households and may accompany their owners in the

5

fields and forest but are unrestrained. None of these dogs is vaccinated against pathogens

(pers. comm. with park authorities and owners) hence, depending on their numbers,

exposure to diseases, and interactions with wild carnivores, they may pose a disease

spillover risk to wild carnivores. Wild carnivores in KTR frequent the villages to prey on

livestock and dogs (Miller et al., 2015), while dogs may enter the KTR core zone with or

without their owners. As a result, dogs can interact with wild carnivores directly

(scavenging on carcasses, predation) or indirectly (scats, spray marks) on a regular basis

both in the core and buffer areas. These interactions may be more frequent in villages

that are closer to the core of the reserve as found in a study in Chile (Torres & Prado,

2010). These interactions may also be more frequent and significant in villages that are

larger, if they support more dogs and higher level of infection. Finally, it is worth noting

that carnivores infected by one of these viruses may be at a greater risk of co-infection by

the others, due to common routes of transmission and/or immune suppression. Co-

infection leads to poorer host health and enhanced pathogen abundance, thereby

amplifying the threat of any one viral disease (Griffiths et al., 2011).

Over two field seasons, I estimated the abundance of dogs in villages of varying

sizes and distances from the core, using photographic mark-recapture techniques. I also

measured the exposure status of the dogs to rabies, CPV, CDV and CAV by the

seroprevalence of antibodies in the dogs to these viruses. I analyzed both datasets for

trends with the age and sex of the dogs, size and distance of the village from the core, and

seasons, to identify factors associated with higher threats. I also recorded

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opportunistically the occurrence of wild carnivores in the villages, and compared these by

season and with distance to the core zone. This should provide a minimum estimate of

the contact rate with dogs, since many wild carnivores are solitary and nocturnal and

could enter villages undetected, and it does not account for additional interactions in the

surrounding farmlands and forests. In a separate study (chapter 2), I analyze wild

carnivores from KTR directly for two of these viruses, CPV and CDV.

This is the first study in this region to investigate seasonal and spatial variation in

dog population density, pathogen seroprevalence, and minimum contact rate with wild

carnivores. Our results will aid the KTR Forest Department in assessing and mitigating

the threats of disease transmission from dogs to wild carnivores. These results may also

extend to protected areas across India and elsewhere where villages with unvaccinated

dogs are in close proximity, given the wide geographic and host ranges of the viruses.

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1.2 STUDY SITE AND METHODS

Kanha (2207‘– 22027‘N, 80026‘– 8103’E) (Figure 1.1), was established as a

National Park in 1955 to protect endangered swamp deer (Cervus duavelli), and was

brought under ‘Project Tiger’ in 1973 (Damodaran, 2009) to protect one of the last tiger

populations in India. The park ranges in elevation from about 600 m to 870 m above sea

level. The winter season from October to January has a mean temperature of 17 0 C,

spring is from February to March, summer season from April to June has a mean

temperature of 32.5 0 C. July and August is the monsoon season and the area has a mean

annual rainfall of 1800 mm. The vegetation of KTR is composed of dry deciduous forest

(51%) mainly in the highlands, moist deciduous forest (27%) mainly in the lowlands, and

former agricultural fields that are now maintained as grasslands. The dry deciduous

forest is characterized by the trees Angoiessus latifolia, Gardenia latifolia, Buchanania

lanzan and Sterulica urens, while the moist deciduous forest is dominated by Shorea

robusta, Tectonia grandis, Terminalia chebula and Terminalia tomentos. The bamboo

Dendrocalamus strictus (Newton, 1988) dominates the understory in both forests.

There are about 100,000 people in the KTR core and especially buffer zones, in

about 150 villages (DeFries, Karanth & Pareeth, 2010). Their primary economic

activities are cotton and rice farming, management of cattle, goats and buffalos, and

employment in wildlife tourism. Villagers in the buffer zone can use it for farming,

sustainable firewood collection and livestock grazing, while villagers in the core zone are

8

allowed some restricted farming and livestock grazing there. The villagers suffer losses

from crop raids by herbivores and livestock depredation by wild carnivores (Karanth et

al., 2013). In a recent survey, livestock depredations were reported by 13% of the

households, and attributed to tigers, leopards, hyaenas, jackals, and dhole (Karanth et al.,

2013). More generally, leopards were identified as the most damaging species to

livestock in Indian protected areas (Karanth et al., 2013) including KTR. Since wild

carnivores frequent the villages to predate on livestock and dogs, their interaction with

dogs is inevitable.

Dog abundance and demography

I censused dog populations and measured their seroprevalence against four

viruses over two field seasons, from May 15, 2014 to July 20, 2014 (here after called

summer 2014) and from January 15, 2015 to March 30, 2015 (winter 2015). I conducted

these surveys in five randomly selected villages that are near to the core boundary

(village center < 2 km from core) and four villages (summer 2014), and then five (winter

2015), that are far (village center > 6 km) (Figure 1.1), in a stratified random sample.

Most villages were sampled in both seasons with the exception of Arandi, which was

replaced by Ranwahi in winter 2015 among near villages, and Chiraidongri, which was

added in winter 2015 as the fifth far village. I used the population finder feature of the

Census India website to obtain the human population size of each village from the 2011

census (http://censusindia.gov.in/), and calculated the area of each village by tracking the

9

outermost boundary of the village on a motorcycle, using a Garmin GPS (model Montana

650, Olathe, Kansas, USA).

Figure 1.1 Study site. Map of Kanha Tiger Reserve (KTR) with core (shaded) and buffer

zones, and with villages that were sampled in summer 2014 and winter 2015. The inset

shows the location of KTR in India.

Legend Buffer boundary Core boundary Near villages Far villages

10

I used mark-recapture statistics on photographic samples to estimate the number

of dogs in each village. I found with pilot surveys that the dogs were most active from

6:00-9:00 am and 5:00-6:30 pm in summer 2014, and from 7:00-9:00 am and 4:00-5:30

pm in winter 2015. I surveyed for dogs at these times while driving on a motorcycle on

roads and alleys and by walking in areas that were otherwise inaccessible. I surveyed for

two consecutive days (a “mark” and “recapture” in each village) in summer 2014 and for

three consecutive days (a “mark”, 2 morning and 2 afternoon “recaptures” in each

village) in winter 2015. I photographed all dogs encountered with a Nikon D3000 digital

camera and 80-200 mm lens, and noted sex (males by descended testicles), age category

(by asking the villagers who said they owned the dog and/or estimation), and natural

color and coat patterns. This allowed me to identify individuals and recaptures without

handling the animals.

I used CAPTURE (Otis et al., 1978; White & Burnham, 1999; White, 2008), an

extension of MARK (White, 2008) for closed populations, to estimate the number of

dogs in each village. I assumed that these populations were closed for the 2-3

consecutive survey days for each village because the dogs are territorial (Pal, 2003) and

unlikely to enter or leave during that time. Village population sizes were estimated

separately for each season, as the assumption of closure would not likely hold. I assumed

that there was no loss of marks, since the animals were identified by their natural color

and coat pattern. CAPTURE uses Akaike information criterion (AIC) values to compare

seven models that differ in their assumed source of variation in capture probability. They

11

vary from assuming that the capture (and recapture) probability is constant (M0), to

assuming that it varies among individuals (Mh), with time (Mt), and with behavior after

first capture (Mb), and all possible combination of these (Mbh, Mth, Mtb, Mtbh). As I did not

physically trap or handle the animals, I ruled out model Mb and the related models Mtb,

Mtbh and only compared models that assumed a constant probability of capture and

recapture M0, and that it varies among individuals Mh, with time Mt, or both Mth. I

selected the model that consistently had the lowest AIC value for all population

estimates, and divided these estimates by the village areas to obtain the density of dogs.

Blood and tissue sample collection

After the population censuses were completed, I returned to the same villages to

sample dogs for exposure to rabies, CPV, CDV and CAV. In summer 2014, with

veterinarians including Dr. Nidhi Rajput from the Centre for Wildlife Forensic and

Health (CWFH), Jabalpur, India I collected blood opportunistically from 67 dogs (42

males and 25 females) including some from all nine villages surveyed. In winter 2015, I

collected blood from 5 male adults and 5 female adult dogs (> 1 year old) and 4 male

juveniles and 4 female juveniles (4 months to < 1 year old) from each of the villages

censused, except from 3 near and 2 far villages in which I could only capture 3 male and

3 female juveniles. 35 dogs were sampled in both seasons, providing information on

changes in seroprevalence. I selected a minimum age for juvenile dogs of 4 months to

decrease the likelihood of sampling maternal antibodies (Greene, 1994). I was unable to

take blood samples from feral dogs, as they were aggressive and wary of humans. Each

12

animal was held gently while a veterinarian collected about 4 ml of blood from the

saphenous vein and immediately transferred it to a 4 ml vacuette tube kept on ice. Each

day’s samples were transferred to the CWFH laboratory at the end of the day and stored

overnight at 4 0C. On the next day, the serum was separated by centrifugation (REMI

cooling centrifuge, model number: CM-24, Goregaon E, Mumbai 400063, India) at 3,000

g for 15 minutes and immediately stored at -40 0C, for analysis.

Seroprevalence survey and molecular diagnosis

In the summer of 2014, I tested the serum of all 67 animals and one known

vaccinated dog sample as a positive control for rabies antibodies with Bio-Rad’s Platelia

II test kit (Bio-Rad, Hercules, California, USA). This immune-enzymatic kit uses solid

phase inactivated rabies glycoprotein G. Since no sample tested positive except the

positive control, I did not repeat this test in the winter of 2015. However, I tested all

samples from both seasons for antibodies against CPV, CDV and CAV with BioGal’s

Immunocomb canine vaccichek solid phase immunoassay kit (Bio Galed lab, Kibbutz

Galed, Israel, 1924000). This has been used to detect antibodies in vaccinated and

unvaccinated dogs (Belsare & Gompper, 2013; Waner et al., 2003). It semi-quantifies

antibodies and results can be measured by comparison with a color comb scale provided

in the kit. The results on the comb are in the form of ‘S’ units in the manufacturer’s

guidelines, which vary from 0 to 6 on the scale. S0 indicates that antibodies were not

detected; S1 and S2 suggest that the antibodies are present but with insufficient

immunity; S3, S4, S5 and S6 suggests that antibodies are present with minimum 1:16 titer

13

of virus neutralization for CAV, 1:80 titers by hemagglutination inhibition test for CPV,

and 1:32 virus neutralization test for CDV. I considered all dogs above the titer of S1 to

be seropositive for the specific antibodies. For the purpose of this study S1 (S1 and S2

combined) refers to antibodies present in low titers, S3 refers to antibodies present in

medium titers and S5 (S4, S5 and S6 combined) refers to antibodies present in high titers.

Samples from dogs that were seropositive for CPV and CDV were tested for the

actual pathogens using the polymerase chain reaction (PCR). I did not do this for CAV

as I lacked a positive control. I extracted DNA from CPV seropositive blood samples

using Qiagen’s DNA extraction kit (Qiagen Inc., 171 Forbes Blvd. Suite 1000,

Mansfield, MA 02048, USA). I extracted RNA from CDV seropositive blood samples

using INVITROGEN life sciences RNA extraction kit (Fisher Scientific, Bishop Road,

Leicestershire, United Kingdom, LE115RG) (Deng et al., 2005) and immediately

converted it to cDNA using the Thermo Scientific cDNA conversion kit (Thermo-Fisher

Scientific, Bishop Road, Leicestershire, United Kingdom, LE115RG). I tested the purity

of all the extracted cDNA and DNA samples in a Thermo Scientific’s Nanodrops 3300

spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA) (1.7 -2.0 at OD

260/280) and immediately stored them at -80 o C. I used Reverse Transcriptase-PCR

(Frisk et al., 1999) for detection of CPV and CDV. The primer base pairs used, along

with the position of the nucleotides and lengths of the expected amplicon bands are listed

in table 1.2. I used the most sensitive primer pair (Table 1.2) for CDV detection (Frisk et

al., 1999) and broad-based primers sensitive to detect all strains of CPV-2 and FPV for

14

CPV detection (Table 1.2) (Park et al., 2007). I used blood from two clinically and

serologically positive dogs for CDV and fecal swabs from two clinically and

serologically positive dogs for CPV (exhibiting hemorrhagic gastroenteritis) as positive

controls. I used nuclease-free water for the negative controls. All PCR products were

analyzed using 2% agarose gel electrophoresis.

Table 1.2 Primer base pairs for RT-PCR. Primer base pair sequence (forward (F) and

reverse (R)) used in RT-PCR for each of the viruses along with the nucleotide positions

and length of the band in base pairs.

Virus Primer pair Sequence Position/ length

CPV 1F ACT ATG CCA TTT ACT CCA GCT 3330-3350/248 CPV 1R TCC TGT AGC AAA TTC ATC ACC 3557-3578/248 CPV 2F GTA CAT TTA AAT ATG CCA GA 3029-3048/451 CPV 2R ATT AAT GTT CTA TCC CAT TG 3461-3480/451 CDV 1F ACA GGA TTG CTG AGG ACC TAT 769-789/281CDV 1R CAA GAT AAC CAT GTA CGG TGC 1055-1035/281

Contact rate

I estimated the potential minimum contact rate between wild carnivores and dogs

in the surveyed villages of KTR. In each field season over the period of 60 days, I

opportunistically documented signs of wild carnivore presence in surveyed villages,

including direct sightings, photographic captures, sound recordings, scats and footprints.

Naturalists from the Kanha Tiger Reserve confirmed the identification of carnivores

based on scats. Whenever I had permission from the KTR Forest Department, I installed

15

camera traps (Capture IR, 5 MP camera, Cuddeback, Greenbay, WI, USA, 54115) on

carcasses of livestock killed by tigers and leopards in the surveyed villages to record

predator presence. I recorded the geographical location of each sign (e.g., scat and

footprints) in GPS and collected only once from the particular coordinates to avoid any

replication. Sighted animals and their calls were recorded only once during each day to

avoid replication.

Statistical analysis

I analyzed my data using SAS software (SAS Studio 3.4, SAS Institute Inc., Cary,

NC, USA) and R studio (http://www.R-project.org/). I conducted linear regressions to

test for correlations between estimated dog density and human density, dog counts and

human population size, and dog abundance and area of the village in the surveyed

villages. I used Fisher’s two-tailed exact test and binomial regression to examine the

dependence between seroprevalence (positive or negative) of each pathogen and sex and

age category of the dogs, and the seroprevalence of other pathogens in both summer 2014

and winter 2015. I used binomial regression to examine the dependence of

seroprevalence on distance of the village from the core, only in data of winter 2015. I

calculated odds ratio (OR) for all significant relationships, which tells the likelihood of

seroprevalence given the presence or absence of other conditions, here, the sex and age

class of the dogs, and the presence of other pathogens.

16

1.3 RESULTS

Dog abundance

I captured (photographed) 85 unique dogs in the 4 near villages and 70 in the 4 far

villages in the summer of 2014, and 119 and 110 respectively in these same villages in

the following winter. Over both field seasons combined, 21% of the dogs were

categorized as feral, 77% were categorized as semiowned, and the rest were owned dogs.

In analyzing the recapture data, MARK gave the lowest AIC scores to model M0 for all

villages in both seasons except two, for which Mt had a slightly lower score (Appendix

A). Therefore, I used M0 for all population estimates, and these estimates ranged from

21- 45 dogs per village (Appendix B) and in all 8 villages increased from summer 2014

to winter 2015 (Figure 1.2a).

Demographic shifts from summer to winter

Juveniles formed 29% of the total dog population in summer 2014 and 37.6% in

winter 2015 (Figure 1.2b). Females constituted 32% of the total dog population in

summer 2014 and 37.6% in winter 2015 (Figure 1.2c). In the 8 villages that were

surveyed in both seasons, 84 dogs that were captured (photographed and identified) in the

summer of 2014 (54.2 % of the 155 dogs) were recaptured the following winter. This

recapture rate was slightly lower for juveniles (40% of juveniles were recaptured in

winter 2015), and all were then classified as adults. The proportion of dogs classified as

feral declined from 25% (of 155) in summer 2014 to 17% (of 229) in winter 2015.

17

Changes with proximity to KTR core

The proportion of dogs that were juveniles was 23% in near and 28.5% in far

villages in summer 2014, rising to 36.1% and 34% respectively in winter 2015 (Figure

1.2a). There was no statistically significant relationship between the number of juveniles

in near and far villages over the two seasons (P = 0.36, R2 = 0.34). Females constituted

32.67% of the population in near villages and 30% in far villages in summer 2014,

dropping slightly to 28.5% and 26.2% respectively in winter 2015 (Figure 1.2b). There

was no statistically significant relationship between number of females in near and far

villages over the two seasons (P= 0.66, R2 = 0.62). Out of 84 dogs captured in both field

seasons, 49 were from near villages and 35 were from far villages. Feral dogs formed

21% of the total dogs in near villages and 26% of the total dogs in far villages in summer

2014 and they formed 17% of the total dogs in near villages and 14% of the dogs in far

villages in winter 2015. There was no statistically significant effect of distance on the

number of feral dogs over the two field seasons (P= 0.1, R2 = 0.58).

Figure 1.2 Estimated number of dogs in various categories. (a) Estimated number of dogs

in four near villages (N1, N2, N3, N4) and four far villages (F1, F2, F3, F4) in summer

2014 (blue) and winter 2015 (red), (b) actual counts of juveniles (blue) and adults (red) in

the near and far villages in summer (S) and winter (W), (c) actual counts of females

(blue) and males (red) in near and far villages in summer (S) and winter (W).

a.

18

b.

34

2623

2123

19 20

25

45

2630

28

34

27 27

32

0

5

10

15

20

25

30

35

40

45

50

N1 N2 N3 N4 F1 F2 F3 F4

Summer2014 Winter2015

1117

49

6 84

96

13

48

4 4 612

19

24

14

17

14

18

13

17

14

17

10

17

1420

12

16

0

5

10

15

20

25

30

35

40

45

SN1

WN1

SN2

WN2

SN3

WN3

SN4

WN4

SF1

WF2

SF2

WF2

SF3

WF3

SF4

WF4

Juveniles Adults

19

c.

Dog density

The villages surveyed ranged in area from 1.52 - 8.87 km2 and in human

population size from 142 to 1,324. There was no systematic difference in either area (P =

0.36, R2 = 0.28) or human population size (P = 0.22, R2 = 0.47) between near and far

villages. The estimated density of dogs in summer 2014 was 10.3/km2 in near villages

and 12.3/km2 in far villages while the corresponding values in winter 2015 were 12.2

dogs/km2 and 14.3/km2. There was no significant difference between density of dogs in

near and far villages in either season (summer: P= 0.17, R2 =0.59), winter 2015 (P = 0.34,

R2 = 0.52). There was no significant relationship between the number of dogs and

humans per village in summer 2014 (P= 0.09, R2 = 0.39) and winter 2015 (P = 0.47, R2 =

9 11 5 7 7 8 5 8 5 8 5 7 7 6 4 7

21

30

1319

1318

12

1815

22

9

1811

1814

21

05

1015202530354045

SN1

WN1

SN2

WN2

SN3

WN3

SN4

WN4

SF1

WF1

SF2

WF2

SF3

WF3

SF4

WF4

Females Males

20

0.35) nor after dividing by area, their densities in summer 2014 (P= 0.55, R2 = 0.45) and

in winter 2015 (P = 0.24, R2 = 0.16).

Seroprevalence status of dogs in KTR

Seroprevalence of CPV, CDV and CAV were high in summer 2014 and declined

in the winter 2015: CPV decreased from 83.6% to 68.4% (Figure 1.3a), CDV decreased

from 50.7% to 30.4% (Figure: 1.3b) and CAV decreased from 41.8% to 30.9% (Table

1.3b). There was no statistically significant relationship between any of the pathogens

with age and/or sex of the dogs in summer 2014 (Table 1.3) and in near villages in winter

2015 (Table 1.4). However, in villages of far category that were sampled in winter 2015

(Table 1.4), adults were more likely to be seropositive than juveniles for CPV (P = 0.003,

Odds Ratio or OR = 4.16), CDV (P = 0.0038, OR = 1.02) and CAV (P = 0.01, OR =

3.70). I found no relationship between sex and seroprevalence of CPV (P = 0.54) , CDV

(P = 0.27) or CAV (P = 0.63) in far villages of winter 2015.

Figure 1.3 Seroprevalence of a. CPV, b. CDV and c. CAV in dogs for summer 2014 and

winter 2015. The number of dogs sampled in each category is listed below the

corresponding bar.

a.

21

b.

c.

83.6 87.872.268.4

77.9

56.2

020406080100

Total(67/168) Adult(49/95) Juveniles(18/73)

CPV

Ser

opre

vale

nce

(%)

Number of dogs tested in each category

Summer Winter

50.7 46.961.1

30.440

17.8

0

20

40

60

80

Total(67/168) Adult(49/95) Juveniles(18/73)

CD

V S

erop

reva

lenc

e (%

)

Number of dogs tested in each category

Summer Winter

22

Figure 1.4 Seroprevalence of a. CPV, b. CDV and c. CAV in dogs from near and far

villages. The seroprevalence is listed as the percentage of dogs and adults and juvenile

categories only for winter 2015. The numbers of dogs tested are listed on the x axis for

each category.

a.

41.846.9

27.830.9

43.2

15.1

01020304050

Total(67/168) Adult(49/95) Juveniles(18/73)CAVSeroprevalen

ce(%

)

Number of dogs tested in each category

Summer Winter

74.1 78.768.4

62.6

76.6

44.4

0

20

40

60

80

100

Total(85/83) Adult(50/47) Juveniles(34/36)

CPV

sero

prev

alen

ce (%

)

Numberofdogsthatweresampled

Near Far

23

b.

c.

Table 1.3 Seroprevalence in various categories of dogs in summer 2014 (S) and winter

2015 (W). This lists the number of dogs sampled in each season in each category: males,

females, adults, juveniles, female adults (FA), female juveniles (FJ), male adults (MA)

17.723.4

10.5

42.9

57.4

25

010203040506070

Total(85/83) Adult(50/47) Juveniles(34/36)CD

V S

erop

reva

lenc

e (%

)

Numberofdogsthatweresampled.

Near Far

23.5

36.2

7.9

38.6

51.1

22.2

0102030405060

Total(85/83) Adult(50/47) Juveniles(34/36)CAV

Ser

opre

vale

nce

(%)

Number of dogs that were sampled

Near Far

24

and male juveniles (MJ), and the percent of each that were seropositive for CPV, CDV

and CAV.

Table 1.4: Seroprevalence of CPV, CDV and CAV in dogs of near and far villages in

winter 2015, as classified in various ways: males, females, adults, juveniles, female

adults (FA), female juveniles (FJ), male adults (MA) and male juveniles (MJ).

Category Sample N F

CPV (%) N F

CDV (%) N F

CAV (%) N F

Total 85 83 74.1 62.6 17.7 42.9 23.5 38.6 Males 46 44 71.7 56.8 17.4 43.2 28.3 36.4 Females 39 39 76.9 69.2 18.0 43.6 18.0 41.0 Adults 50 47 78.7 76.6 23.4 57.4 36.2 51.1 Juveniles 34 36 68.4 44.4 10.5 25.0 7.9 22.2 FA 23 22 82.6 86.4 27.1 47.1 27.1 59.1 FJ 16 17 68.7 47.0 18.8 23.5 18.8 17.6 MA 24 25 75.0 68.0 28.2 56.0 28.2 44.0 MJ 22 19 68.2 42.1 25.9 26.3 25.9 26.3

Category Sample size S W

CPV (%) S W

CDV (%) S W

CAV (%) S W

Total 67 168 83.6 68.4 50.7 30.4 41.8 30.9 Males 42 89 83.3 62.9 52.4 29.2 40.5 33.7 Females 25 79 84.0 74.7 48.0 31.6 44.0 27.8 Adults 49 95 87.8 77.9 46.9 40.0 46.9 43.2 Juveniles 18 73 72.2 56.2 61.1 17.8 27.8 15.1 FA 18 45 83.3 86.7 44.4 26.8 44.4 40.0 FJ 7 34 85.7 58.8 57.1 20.2 42.9 11.8 MA 31 50 90.3 70.0 48.4 29.8 48.4 46.0 MJ 11 39 63.6 53.8 63.6 23.2 7.1 17.9

25

Antibody titers in dogs of near and far villages over the two field seasons

The proportion of the dogs with antibody titer categories S5, S3 and S1 varied

with the pathogen (Table 1.5). The largest proportion of dogs that tested positive for

CPV antibodies were in the highest titer range (S4, S5 and S6) in both seasons, whereas

dogs that tested positive for CDV and CAV were predominantly in the moderately high

titer range (S3) in both seasons. In winter 2015, dogs in near villages had antibody titer

of S5 in greater proportion as compared to far villages (Table 1.6).

Table 1.5. Antibody titer of CPV, CDV and CAV in the dogs. Number of dogs

and proportion (Pr) of dogs that tested positive with antibody titer range of S5 (high

antibody titer), S3 (moderately high antibody titer) and, S1 (low antibody titer) are listed

for categories of total number of dogs, adult and juvenile dogs, male and female dogs,

over the summer 2014 and winter 2015.

Season Disease Category Antibody titer S5 Pr. S5 S3 Pr.S3 S1 Pr. S1

Summer CPV Total 40 0.71 14 0.25 2 0.04 A 32 0.76 10 0.24 0 0.00 J 8 0.57 4 0.29 2 0.14 M 25 0.71 8 0.23 2 0.06 F 15 0.71 6 0.29 0 0.00

Winter CPV Total 60 0.52 25 0.22 30 0.26 A 56 0.77 14 0.19 3 0.04 J 4 0.10 11 0.26 27 0.64 M 38 0.69 9 0.16 8 0.15 F 22 0.37 16 0.27 22 0.37

Summer CDV Total 5 0.15 25 0.74 4 0.12

26

A 3 0.12 21 0.84 1 0.04 J 2 0.22 4 0.44 3 0.33 M 4 0.18 16 0.73 2 0.09 F 1 0.08 9 0.75 2 0.17

Winter CDV Total 19 0.36 31 0.58 3 0.06 A 13 0.33 25 0.64 1 0.03 J 6 0.43 6 0.43 2 0.14 M 11 0.39 15 0.54 2 0.07 F 8 0.32 16 0.64 1 0.04

Summer CAV Total 8 0.24 17 0.52 8 0.24 A 5 0.23 13 0.59 4 0.18 J 3 0.27 4 0.36 4 0.36 M 5 0.29 7 0.41 5 0.29 F 3 0.19 10 0.63 3 0.19

Winter CAV Total 13 0.25 27 0.53 11 0.22 A 11 0.28 23 0.58 6 0.15 J 2 0.18 4 0.36 5 0.45 M 7 0.27 14 0.54 5 0.19 F 6 0.24 13 0.52 6 0.24

Table 1.6 Antibody titer proportion in dogs of KTR of near and far villages in winter

2015. Listed is proportion of dogs in pooled category as total and adults and juveniles

with S5, S3 and S1 titer for near villages (NS5, NS3, NS1) and far villages (FS5, FS3 and

FS1)

Disease Category NS5 NS3 NS1 FS5 FS3 FS1

CPV Total 0.31 0.11 0.12 0.21 0.10 0.14 A 0.42 0.08 0.01 0.32 0.12 0.04 J 0.12 0.17 0.31 0.02 0.07 0.31

CDV Total 0.17 0.09 0.04 0.21 0.47 0.02

27

A 0.18 0.10 0.03 0.15 0.54 0.00 J 0.14 0.07 0.07 0.36 0.29 0.07

CAV Total 0.12 0.24 0.06 0.14 0.29 0.16 A 0.13 0.23 0.08 0.15 0.35 0.08 J 0.09 0.27 0.00 0.09 0.09 0.45

Dogs that were seropositive for any one pathogen in winter 2015 tended to be

seropositive for one or both of the others (Table 1.7). This trend was statistically

significant in far villages and near and far villages combined, but not in near villages

alone, in winter 2015. For example, in far villages, dogs that were seropositive for either

CPV and/or CDV were more likely to be seropositive for CAV (P = 0.001, OR = 13.72)

Table 1.7 Co-infection of pathogens. P value and odds ratio (95% confidence limits) for

the relation between seroprevalence for each pathogen (outcome) and seroprevalence of

one or both of the other two pathogen (condition) listed for categories: S (summer 2014)

total (near and far villages combined), W (winter 2015) total (near and far villages

combined), winter 2015 (only near villages) and winter 2015 (only far villages).

Pathogen Condition P value OR OR (95% confidence limit)

S/total CPV CDV and CAV 0.304 2.29 0.52 - 10.07 CDV CAV and CPV 0.182 2.53 0.68 - 9.41 CAV CDV and CPV 0.22 4.90 0.55 - 43.05 W/total CPV CDV and CAV 0.001* 13.72 3.15 - 59.28 CDV CAV and CPV 0.001* 4.16 1.95 - 8.88

28

CAV CDV and CPV 0.006* 3.12 1.34 - 7.25 W/ near CPV CDV and CAV 0.117 4.40 0.53 - 36.01 CDV CAV and CPV 0.296 2.04 0.66 - 6.24 CAV CDV and CPV 0.769 1.38 0.40 - 4.73 W/ far CPV CDV and CAV 0.001* 39.77 5.02 - 314.7 CDV CAV and CPV 0.001* 9.37 3.22 - 27.27 CAV CDV and CPV 0.0008* 6.73 2.06 - 21.96

* Statistically significant relation.

Thirty-five dogs were sampled in both summer 2014 and winter 2015. Of these,

all 19 dogs that were seropositive for CPV antibodies in summer 2014 remained

seropositive in winter 2015, while 7 (43.7%) of the remaining 16 became seropositive by

winter 2015 (6-8 months later). Similarly, all 13 dogs that were seropositive for CDV in

summer 2014 remained so in winter 2015, and 3 of the remaining 22 showed positive

seroconversion for CDV (13%). For CAV, out of 9 dogs that were seropositive in

summer 2014, 8 remained seropositive in winter 2015 and 1 dog showed positive

seroconversion for CAV (3.9% of previously seronegative). A single case showed

negative seroconversion for CAV, which is possible if the individual animal has not been

exposed to the pathogen in the recent past.

In the RT-PCR tests, the positive controls formed bands at the 281 base pair

location for CDV and 451 base pair location for CPV as expected, while there were no

bands visible for the negative controls. I observed a positive band at the 451 base pair

29

location indicating an active CPV infection in an 8 month old male dog, and positive

bands at the 281 base pair location indicating active CDV infections for a 2 year old

female and a 6 year old male.

Contact rate

Wild carnivores were present in surveyed villages during the period of study and

ones that were heard or seen, or that left signs such as scats or tracks that were seen, were

noted in this study. Evidence of wild carnivores was greater in near villages and during

the winter field period (Figure 1.3, Table 1.6).

Figure 1.5 Potential numbers of contacts between wild carnivores and dogs. Number of

individual events of wild carnivore presence in surveyed villages of KTR noted over two

field seasons (60 days per seasons) and categorized for near and far villages.

16

6

18

4

0

5

10

15

20

Near Far

Summer2014 Winter2015

30

Table 1.8 Signs of wild carnivores in surveyed villages. Number of signs of wild

carnivore presence noted in surveyed near and far villages in summer 2014 (S) and winter

2015 (W).

Village (type/season)

Type (number)

Near/S Animals sighted (2 jackals, 1 leopard, 1 tiger); footprints and drag marks (3 tigers, 1 jackal); scat (6 jackals, 1 leopard), distinctive call (2 jackals)

Near/W Camera trap (1 wild dog, 1jackal), livestock predation in front of the owner’s house (4 tigers), footprints (3 tigers, 1 leopard, 1 jackal), animal sighted (2 tigers), scat (1 tiger, 2 jackals), distinctive call (1 fox, 1 jackal)

Far/S Animal scat (3 jackals), animal sighted (2 jackals), photographic report (1 fox)

Far/W Animal sighted (3 jackals), footprints (1 jackal)

31

1.4 DISCUSSION

Large numbers of semi-owned and feral dogs are present in both the core and

buffer zones of KTR. Their numbers per village do not vary with the area, human

population size, or distance of the villages from the core of the reserve, but they did show

a rapid increase in numbers from summer 2014 to winter 2015, mainly through births.

These dogs had a high seroprevalence of CPV in the summer of 2014 (83.6%) and lower

but still high seroprevalence of CDV (50.7%) and CAV (41.8), indicating that all three

viral pathogens are circulating in the population. The seroprevalence of all three declined

in the winter 2015, respectively to 68.4%, 30.4% and 30.9%, primarily due to the influx

of new juvenile dogs. The seroprevalence to CPV in these new juveniles in winter 2015

was already 56.2%, compared with 77.9% for adults, indicating that CPV transmission

rates are high. However, the seroprevalence of CDV and CAV in these same juveniles

was much lower, 17.8% and 15.1%, suggesting either that transmission rates for these

pathogens are lower or that mortality rates are higher. Distinguishing between these

possibilities will require different or more finely resolved data though, in either case,

there is a clear risk of transmission to wild carnivores in KTR. The seroprevalence of

CPV and CDV was higher in far than near villages, which might reduce the risk slightly,

though wild carnivores are found even in the far villages and, besides, the lower rates of

infected animals in near villages may indicate that they are preferentially eaten by

leopards or other carnivores, increasing the risk of transmission. My results clearly show

that there is a risk of spillover of three viral pathogens of great conservation concern from

abundant village dogs to the less common and in some cases endangered carnivores in

32

KTR. Furthermore, any such spillover brings the risks of large-scale outbreaks and

mortality in the wild carnivores.

Density of dogs in KTR

Photographic mark-recapture methods have been used successfully to estimate the

abundance of dogs and other species in which individuals have unique markings (Belsare

& Gompper, 2013; Lubow & Ransom, 2009; Mancini, Elsadek & Madon, 2015). My

surveys had a single recapture in summer 2014 and four recaptures in 2015, and yielded

slightly higher estimates than the actual number of animals recorded in each village, with

small standard errors. This indicates that most dogs in these villages were outside and

visible, and identified in my surveys. The estimated density of dogs in the villages

ranged from 3.7 to 17.1 dogs/km2 in summer 2014 and from 5.4 to 23.7 dogs/km2 in

winter 2015. The values are slightly higher than the estimated density of dogs, 5/km2,

near a protected area in the Russian Far East – the Sikhote-Alin Biosphere Zapovednik

(Gilbert et al., 2015). However, a similar study of villages near a smaller protected area

in India, the Great Indian Bustard Sanctuary (GIB), yielded a much higher estimate of

719 dogs/km2 (Belsare & Gompper, 2013). This difference may be due partly to the

larger human populations in the six surveyed villages there (2,973 - 7,448 compared to

171 - 1,321 in KTR) though dog density was not correlated with human population size

in my study. Perhaps more importantly, the areas of the villages were calculated

differently in GIB, around the clusters of houses themselves and excluding nearby

farmlands (Belsare & Gompper, 2013). In KTR, the farmlands are situated between the

33

houses and the areas over which the dog populations are assumed to roam are therefore

larger. In addition, there are leopards in KTR but not in GIB (Belsare & Gompper,

2013), which might exert predatory control on dog numbers only in KTR (Athreya et al.,

2016).

Categorization of dogs and new births:

Dog populations increased in winter 2015 mainly through births, as the 45

juveniles in summer 2014 were replaced by 80 new juveniles in winter 2015. My field

seasons were 6 months apart, with juvenile dogs averaging 9 months of age in summer

and 5 months of age in winter. The gestation period is about two months so I conclude

that August/September are the primary whelping months for dogs in KTR. In winter

2015, I saw only 40% of juveniles that were seen in summer 2014 and all these dogs were

categorized as adults in winter. This may be because of high mortality in the first year or

that they were present but not seen. Dog abundance did not systematically vary with

distance of the village from the core. Male dogs were more abundant than female dogs in

both summer 2014 and winter 2015; this is suggestive of greater survival or activity of

male dogs.

Infection status of dogs in KTR

Seroprevalence is the proportion of individuals exposed to a pathogen during their

life (Greiner & Gardner 2000a, b), but it does not give any information on current disease

status. Seroprevalence may vary with sex, because of sexual selection of the pathogen or

34

gender-specific anatomy or behavior. It may also vary with host community interactions,

for generalist pathogens such as these, as well as with co-infection by other pathogens,

and the general health of the population. High seroprevalence may indicate high

transmission rates of the pathogen and/or high post-exposure survival rates. Similarly,

low seroprevalence may indicate low transmission or low survival rates of the pathogen.

To distinguish between these possibilities, we would need to monitor individual infected

dogs or populations at higher sampling frequency.

I found no dogs to be seropositive for rabies using the ELISA test. This may be

because of the high and rapid mortality induced by the disease, as most dogs die within 9

days of infection (Tepsumethanon et al., 2004), rather than the absence of the disease. In

fact, canine rabies is a serious public health problem in India, and dog bites are

responsible for 91.5 % of the 15 million animal bites in India. In India 20,000 deaths

occur per year due to canine rabies (36% of the world total, WHO 2015,

http://www.who.int/rabies/resources/en/). It is likely that canine rabies is also a serious

threat to wild carnivores, even though infections are short-lived and not easily detected.

A large fraction of the dogs in KTR had been exposed to CPV (83.6%

seropositive in summer 2014 and 68.4% in winter 2015), which is comparable to reports

from GIB (88% of dogs seropositive: Belsare & Gommper, 2013), Chile (74%: Acosta-

Jamett et al., 2015) and Uganda (83%: Millan et al., 2013).. These high values suggest

that the virus is endemic in the dogs and they may serve as a reservoir for it; it also

35

reflects the hardiness of the virus which can survive in the soil for months and

transmission can occur through feces, contaminated soil, inanimate objects and vectors

such as flies (Bagshaw et al., 2014). High seroprevalence may also indicate that the

mortality of dogs caused by CPV is not high (McCallum & Dobson, 1995). Only 56.2%

of juveniles were seropositive for CPV in winter 2015, whereas 72% of juveniles were

seropositive in summer 2014. Adults were 2.32 times more likely to be infected than

were juveniles in winter 2015, whereas there was no significant difference between the

age classes in summer 2014. This indicates that new juveniles are more susceptible to

catching the infection and dying once exposed to it. CPV seroprevalence was higher in

near villages, where wild carnivores enter frequently (Miller et al., 2015) and may get

infected. Of the dogs that tested positive for CPV, the greatest number had high antibody

titers suggesting that they have suffered from mild disease with complete recovery. This

also suggests that these dogs may have had repeated exposure to the virus and have

recovered.

CDV seroprevalence in dogs was 50.7% in summer 2014 and 30.4% in winter

2015. These values are both lower than in GIB (73%: Belsare & Gommper, 2013) and

Uganda (100%: Millan et al., 2013) but comparable to other regions such as in Chile

(47%: Acosta-Jamett et al., 2015). They are also low compared to CPV in KTR. This

could mean either that the transmission of the CDV is low in the region or the resulting

mortality is high. In winter 2015, CDV seroprevalence in near villages (17.7%) was

much lower than that in far villages (42.9%), which indicates that dogs and wild

36

carnivores in near villages are not exposed to CDV, and are at risk of introduced

infection. Pathogens such as CDV have complex relationships with the host and their

pathogenesis differs significantly based on the region. CDV infections have resulted in

mortality of lions in the Serengeti region (Roelke-Parker et al., 1996), however in

southern Africa, lions have existed with CDV without any significant impact despite high

exposure (Alexander et al., 2010). The complex disease dynamic of generalist pathogens

such as CDV make further research essential for understanding the disease ecology of

domestic and wild carnivores in KTR.

Seroprevalence of CAV was 41.8% in summer 2014 and 30.9% in winter 2015,

both of which are low in comparison to GIB (68%: Belsare & Gompper, 2013). CAV is

stable in the environment for days, and infected dogs can excrete the virus in urine for at

least 6 months (Greene, 1994). In winter 2015, dogs of far villages had higher

seroprevalence to CAV than those of near villages. Seroprevalence of both CDV and

CAV were higher in summer 2014, primarily because of higher seroprevalence in

juveniles in summer 2014 (average age 9 months) and uninfected status of juveniles in

winter 2015 (average age 6 months). Odds of adults being seropositive when compared

to juveniles were higher for both CDV (1.12) and CAV (4.29) in winter 2015, but there

was no significant relationship between age and seroprevalence in summer 2015. This

suggests that like CPV, new juveniles are more susceptible to infection of CDV and CAV

and dying once exposed.

37

I observed high seroprevalence for CPV, CDV and CAV in a number of cases,

however, the PCR results detected active infection of CPV and CDV in just four animals.

In my other study, I tested the presence of CPV and CDV in wild carnivore samples

opportunistically (Chaudhary et al., unpublished, chapter 2). In those samples, one tiger

blood sample tested positive for CPV and FPV antibodies in KTR in 2015, but it was

negative for the viruses in PCR tests.

Co-infection can be an important factor in any mass die-offs (Goller et al., 2010).

The mortality of lions attributed to CDV in Serengeti population was possibly the result

of co-infection with Babaesia (Munson et al., 2008). I observed a statistically significant

association between the seroprevalence of each of the three pathogens and the other two

in the dogs of far villages in winter 2015 but not in near villages then or in either category

of village in summer 2014. This could result from any of several reasons; for example,

all three viruses share transmission routes through feces and body fluids. In addition,

CDV can cause immunosuppression (Sykes, 2010), thus facilitating secondary infections

by other pathogens (Holzman, Conroy & Davidson, 1992). Finally, since the far villages

had a higher seroprevalence of CDV and CAV than near villages, the chances of a dog

being infected with all the three diseases are higher. The pathological mechanism of co-

infection is beyond the scope of this study.

Contact rate between dogs and wild carnivores

38

My results confirm that carnivores occur in the villages surrounding KTR and at

higher rates in villages near to the KTR core. This supports another study of livestock

predation in KTR, which found that wild carnivores prey on livestock close to the

villages (Miller et al., 2015). My observed rates of contact are minimum values, as they

do not include unobserved entries of carnivores to the villages, or contacts with dogs in

the surrounding lands. In fact, I also have photographic evidence of human habitants and

their dogs illegally going in the restricted part of core zone of the KTR to collect

firewood and fruits, where direct or indirect transmission of pathogens might also occur.

This is true of other protected areas in India as well, where wild carnivores are

surrounded by humans and their associated dogs. However, in regions with low dog

densities such as the Sikhote-Alin Biosphere Zapovednik, home to endangered Amur

tigers, dogs are not considered to pose disease spillover risk to wild carnivores because of

their low contact rate with wild animals (Gilbert et al., 2015). This is suggested due to

the sparse human population in villages that are located far apart (2.59/ km2), and the lack

of feral dog populations because of severe weather (Gilbert et al., 2015).

Implications of dog disease exposure status in wild carnivore conservation

Dogs, free of human restraint, irrespective of ownership status, form about 75%

of the global dog population (WSPA, 2011, http://www.fao.org/3/a-i4081e.pdf). Feral

and semiowned dogs in KTR are free roaming. Feral dogs are prone to suffer from high

mortality, malnutrition, disease and parasitism (Sowemimo, 2009). In winter 2015, I

recaptured only 11% of the feral dogs that were captured in summer 2014, suggesting

39

high mortality and/or that they travel further distances and are less likely to stay within

the confines of village. Feral dogs are more ferocious and form packs to hunt wild and

domestic herbivores. This study does not incorporate any seroprevalence data from feral

dogs, which may be higher and associated mortality may be higher too. Feral dogs

probably have a more intense interaction with wild carnivores, and there is no available

seroprevalence data for them from the region, making them a greater unknown threat to

wild carnivores.

Disease transmission of a generalist pathogen in a multi-host carnivore

community may vary with the contact pattern, social behavior and spatial distribution of

different host species (Dobson, 2004). Simulation studies based on serological data from

the Serengeti have shown that multi-host systems have more susceptible hosts with

increased disease transmission than single-species systems (Craft et al., 2008). This has

serious implications for a carnivore community such as that of KTR, where more

numerous host species such as dogs and jackals can act as reservoirs for the pathogens.

A pathogen may not survive in a single species system where the hosts experience low

intraspecific contact such as with tigers, but may well persist in in a well-mixed

interspecific carnivore community. The decline of vulture populations (Accipitridae &

Cathridae family) in the last decade in Asia and Africa may have exacerbated the

problem in two ways. Vultures rapidly scavenge carcasses and limit the spread of

infectious diseases they may carry (DeVault et al., 2003). Without vultures, infected

carcasses will persist longer on the landscape where they might be fed upon and infect

40

wild carnivores, perpetuating their transmission. In addition, these carnivores may spend

more time on the carcasses and be more likely to encounter other species of carnivores,

increasing the rates of both the initial uptake and subsequent transmission of infectious

pathogens (Ogada & Bujl, 2011).

Infectious disease exposure in dogs can have implications for public health as

well. Dogs share at least 60 pathogen species with humans (MacPherson, 2005), making

unvaccinated dog populations a public health concern. There have been concerns over

human health from zoonotic pathogens, after fatal infections of CDV in crab-eating

macaques (Macaca fascicularis: Sakai et al., 2013a) in Japan in 2008. This strain can

readily adapt to human receptors (Sakai et al., 2013b). Dog bites are responsible for 99%

of the 55,000 human deaths from rabies in Asia and Africa each year (Knobel et al.,

2005). In India, canine rabies kills approximately 20,000 people per year (Sudarshan et

al., 2007). Dogs are not only the reservoirs of these pathogens, but they also form

important link for pathogen exchange between wild carnivores, humans and livestock

(MacPherson, 2005). All three coexist in the villages in and around KTR, and the threat

caused by the abundant dogs in sustaining pathogen populations that threaten wildlife,

livestock and humans should not be underestimated.

Conclusion

The expansion of human activities including habitations, agriculture, and

deforestation leads to fragmentation of carnivore habitats and populations to the point

41

where they are vulnerable to many threats. Habitat fragmentation also leads to greater

and more intense interactions between humans, domestic carnivores and wild carnivores

(Holmes, 1996; Thorne & Williams, 1988). These intense interactions may be in the

form of competition, predation or cohabitation but may also result in the spread of

infectious diseases from domestic to wild carnivores. The risk from diseases can be

amplified by drought, parasitic infection, prey depletion, inbreeding (Evermann, Roelke

& Briggs, 1986), climate change (Munson et al., 2008) and other factors, and will

become more prevalent as human settlements continue to expand with their associated

dog populations. Disease spillover from dogs to wild carnivores is especially likely when

there are large unvaccinated dog populations with high exposure to the pathogens and

where the dogs have frequent and intense contact with wild carnivores.

As a general trend, population declines of wild carnivores caused by disease are

due to generalist pathogens such as rabies (Ethiopian wolves: Randall et al., 2006), CPV

(Wolves: Mech & Goyal, 1995) and CDV (Lynx canadensis: Origgi et al., 2012; Amur

tigers: Seimon et al., 2013). Small populations are more susceptible to extinction from

diseases (Gilpin & Soule, 1986) and a high number of domestic carnivores constitute a

spillover risk to less abundant wild carnivores. My results document that dogs in KTR

are present in high densities and are exposed to highly infective generalist pathogens such

as CPV, CDV and CAV. Dogs in KTR have rapid turnover and despite the relatively low

density, the constant presence of new susceptible hosts is sufficient to maintain the

pathogens in the system. Therefore, there is a significant potential of disease spillover

42

from relatively dense dog population to less dense wild carnivore population, which

could result in disease epidemics and mortality of wild carnivores (Gascoyne et al., 1993;

Roelke-Parker et al., 1996). Such episodes depend on the immunological status and

exposure of the pathogen to the wild carnivores. I detected exposure of CPV and FPV in

one tiger in KTR (Chaudhary et al., unpublished, Chapter 2), and I strongly recommend

further surveillance of wild carnivore disease exposure status. I also recommend that we

conduct detailed studies of dog ecology there, including their movement ecology, home

ranges, coinfection and disease recovery rates, as these are important factors in

developing epidemiological models of disease transmission in the region. A full model

would include the other carnivore species present, especially the more abundant ones

such as jackals that might also serve important roles in sustaining and propagating

disease outbreaks.

I hope this initial study alerts wildlife managers to disease threats in natural areas

surrounded by human habitations, and can be used in population viability analyses to

explore management options. The two main methods to curb transmission of the

pathogens in wild and domestic carnivore populations are culling and vaccination.

Culling has proved to be beneficial in disease control in wild populations such as rabies

in foxes (Barlow, 1996). However, culling is often carried out without considering the

altered demography and compensatory recruitment that will follow. Culling is also not

practical in areas such as India with religious and ethical opposition to lethal control. The

other commonly used method is to vaccinate the reservoir population. Well-planned

43

mass vaccination programs for abundant and wide-ranging hosts of generalist pathogens,

such as dogs, may benefit wild carnivore and human health. Such programs have

resulted in the elimination of rabies in the Serengeti ecosystem (Lembo et al., 2010).

Oral baiting of dogs with vaccines for rabies virus has been used successfully for mass

vaccinations (Cliquet & Aubert, 2004). As vaccination methods improve and become

cheaper, such methods may also be used in KTR. Studies have also shown that

combination of vaccination and contraception in dogs may reduce disease spread and

population control (Carroll et al., 2010) and I recommend the use of oral baiting to

deliver both kinds of compounds for disease control in dogs in KTR. I also recommend

that there be regular monitoring of domestic and wild carnivores in the region for wildlife

diseases, and more research on the dynamics of these diseases in both. These results also

suggest the need for further research on multi pathogen-host systems that combine field

studies with epidemiological modeling: the success of any mitigations such as

vaccination and population control will depend on better understanding of disease

dynamics in the multi-host community of KTR.

44

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51

CHAPTER TWO

A SURVEY FOR CANINE PARVOVIRUS AND CANINE DISTEMPER VIRUS IN

WILD CARNIVORES OF THE KANHA TIGER RESERVE, INDIA, USING

REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION

2.1 INTRODUCTION

Many wild carnivores around the world have become endangered due to habitat

fragmentation, poaching, intolerance, and other factors, and now survive in small

scattered populations (Di Marco et al., 2014). Even if these populations and their

habitats are protected from direct threats, they remain vulnerable to infectious pathogens

with broad geographic and host ranges such as canine parvovirus (CPV), canine

distemper virus (CDV), and rabies (Deem et al., 2000). The magnitude of this threat has

been revealed by several epidemics associated with crashes of endangered populations,

including lions (Panthera leo) in Kenya’s Serengeti National Park (CDV), gray wolves

(Canis lupus) in the USA’s Yellowstone National Park (Almberg et al., 2009) (CDV,

CPV), and Ethiopian wolves (Canis simensis) in Ethiopia’s Bale Mountains (CDV,

rabies). Immunological surveys of Amur tigers (Panthera tigris altaica: Goodrich et al.,

2012) and recent deaths of some of these tigers raise the prospect of epidemics in these

endangered cats, and have led India’s National Tiger Conservation Authority to call for

increased surveillance

52

(http://projecttiger.nic.in/WriteReadData/userfiles/file/THREAT%20TO%20TIGERS.jpg

/).

CPV of the family Parvoviridae is a close relative of feline panleukopenia virus

(FPV), which infects both domestic and wild felid species and has been modified by

mutations to become CPV-2a and CPV-2b strains which have a broader host range and

can spread from domestic dogs to wild carnivores (Steinel et al., 2000). CDV is a virus

of the Morbillivirus family that can spill over from feral and domestic dogs to a wide

range of wild carnivores (Deem et al., 2000; Vianna et al., 2015) leading to the

suggestion that it be renamed carnivore distemper virus. As part of my study on the

disease spillover threat from domestic dogs to wild carnivores Chaudhary et al.,

unpublished, chapter 1) in Kanha Tiger Reserve, India, I used an enzyme linked

immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-

PCR) to test for CPV and CDV in wild carnivores there.

53

2.2 STUDY SITE AND METHODS

Kanha Tiger Reserve (KTR) in the central Indian state of Madhya Pradesh is

home to several endangered mammalian carnivores: Bengal fox (Vulpes bengalensis),

dhole (Cuon alpinus), leopard (Panthera pardus), sloth bear (Melursus ursinus), and tiger

(P. tigris tigris). The reserve is divided into a 950 km2 core area where human activity is

severely restricted and an 1,134 km2 buffer area that contains about 150 villages with

large livestock and dog populations. The Forest Department of KTR collects blood and

tissue samples opportunistically from wild animals and provides them to the Centre for

Wildlife Forensic and Health (CWFH) in Jabalpur, India for analyses. CWFH is a state-

run institute that monitors disease and clinical health of captive and wild animals and

conducts forensic analyses for unexplained wild animal deaths.

All samples from wild carnivores at CWFH that had been obtained from the

Kanha Forest Department between 2010 and 2015 and could be analyzed for CDV and

CPV were identified. These consisted of blood samples from three tigers provided for

routine hematological examination, as well as lung, spleen and tissue samples from two

tigers, one leopard and one common palm civet (Paradoxurus hermaphroditus). Details

of the animals and samples tested are in Table 2 1. The three tiger blood samples were

analyzed for the presence of antibodies using Biogal’s immunocomb canine and feline

vaccichek kits (Bio Galed lab, Kibbutz Galed, Israel, 1924000). They use solid phase

immunoassay technology to detect antibodies against CPV, CDV, and canine adenovirus

54

(canine kit), as well as feline panleukopenia virus (FPV), herpes virus, and feline

calicivirus (feline kit).

Table 2.1 Animals tested for CPV and CDV, with their sex (male (M), female (F)), age

(in years), tissue used, preservative used to store tissue and year of collection of sample.

Unk (unknown)

Genetic tests for the viruses themselves were also run on the blood and tissue

samples. RNA was extracted from the blood samples using Invitrogen (Fisher Scientific,

Bishop Road, Leicestershire, United Kingdom, LE115RG) RNA extraction kit (Deng et

al., 2005) and immediately converted to cDNA using the ThermoScientific cDNA

conversion kit (Thermo-Fisher Scientific, Bishop Road, Leicestershire, United Kingdom,

LE115RG). DNA was extracted from the blood samples using Qiagen’s DNA extraction

kit (Qiagen, 171 Forbes Blvd. Suite 1000, Mansfield, MA 02048, USA). DNA from the

tissue samples was extracted using the Qiagen FFPE DNA extraction kit (Qiagen, 171

Forbes Blvd. Suite 1000, Mansfield, MA 02048, USA). This kit has been designed for

Sample Sex Age Tissues Preserved Year

Tiger F 6 Blood EDTA 2014 Tiger F 7 Blood EDTA 2014 Tiger M 3 Blood EDTA 2015 Tiger M 8 Spleen, lungs, kidneys Formalin 2012 Tiger M unk Spleen, lungs, kidneys Formalin 2013 Leopard unk unk Spleen, lungs, kidneys Formalin 2012 Palm civet cat unk unk Spleen, lungs, kidneys Formalin 2012

55

optimal extraction of the DNA from formalin-preserved samples (Sam et al., 2012). All

cDNA and DNA samples were tested for purity in a Thermo Scientific’s Nanodrop 3300

spectrophotometer (1.7 -2.0 at OD 260/280) and immediately stored at -80o C.

RT-PCR was used to test for CPV in DNA from blood and tissue samples and for

CDV in the cDNA extracted from blood samples. The most sensitive primer pair was

used for CDV detection (Yoshida et al., 1998) (Table 2.2) and broad-based primers

sensitive to all strains of CPV-2 and FPV were used for CPV detection (Park et al., 2007)

(Table 2.2).

Table 2.2: Primer base pair sequence (forward (F) and reverse (R)) used in RT-PCR for

each of the viruses along with the nucleotide positions and length of the band, (in base

pairs).

Virus Primer pair Sequence Position/ length

CPV 1F ACT ATG CCA TTT ACT CCA GCT 3330-3350/248 CPV 1R TCC TGT AGC AAA TTC ATC ACC 3557-3578/248 CPV 2F GTA CAT TTA AAT ATG CCA GA 3029-3048/451 CPV 2R ATT AAT GTT CTA TCC CAT TG 3461-3480/451 CDV 1F ACA GGA TTG CTG AGG ACC TAT 769-789/281CDV 1R CAA GAT AAC CAT GTA CGG TGC 1055-1035/281

56

2.3 RESULTS

Fecal swabs from two dogs clinically diagnosed with CPV (exhibiting

hemorrhagic gastroenteritis) and blood samples from two dogs clinically diagnosed with

CDV, were used as positive controls for PCR. All these cases were serologically

confirmed to be suffering from the respective diseases. Nuclease-free water was used as

the negative controls for PCR. All PCR products were analyzed using 2% agarose gel

electrophoresis.

One tiger sample tested positive for both CPV and FPV in the ELISA test (male/3

years). CPV and FPV are viruses of the same family and seropositive results may also

indicate cross-reactivity of antibodies with both antigens. In the RT-PCR test, positive

controls formed bands at the 451 base pair location for CPV and 281 base pair location

for CDV, while there were no bands visible for the negative controls. None of the

carnivore samples produced bands on gel electrophoresis of the PCR product, suggesting

that none of the seven wild carnivores was infected by CPV, and none of the three tigers

was infected with CDV.

57

2.4 DISCUSSION

A variety of methods are used for ante-mortem diagnoses of CPV and CDV in

animals, including clinical diagnosis and postmortem histopathology. These methods are

laborious and not always suited for live wild carnivores or specific tissues. Molecular

techniques such as RT-PCR (Frisk et al., 1999) have been tested in a number of studies,

and were the preferred method for formalin-preserved samples, including in the

successful detection of CDV (Sam et al., 2012; Stanton et al., 2002). With these

techniques, I did not find CPV or CDV in any of my wild carnivore samples.

One tiger blood sample tested positive for CPV and FPV antibodies, indicating

that the tiger has been infected in the past; however, it was not infected at the time of

sample collection as shown by the negative PCR results (Shender et al., 2014). Despite

the lack of evidence for active CPV and CDV in any of the carnivores tested, these

viruses may still pose a threat to the wild carnivores of KTR. They have wide geographic

and host ranges (Deem et al., 2000), so that many domestic and wild species might serve

as reservoirs or vectors. All the tiger reserves in India are surrounded by human

habitations, and my research has found that, village dogs near KTR had high levels of

seropositivity in 2015 to CPV (68.4%), CDV (30.4%), and canine adeno virus (30.9%)

(Chaudhary et al., unpublished, chapter 1), posing a high risk of spillover to wild

carnivores. Endangered carnivore populations will be particularly vulnerable to

infectious diseases because of their small sizes and limited genetic diversity. Finally, the

small number of samples that were analyzed had been collected opportunistically and

58

may not be representative, since infected animals may die quickly or be less likely to be

sampled.

The long-term plan for tiger conservation in central India is to connect tiger

reserves such Kanha, Satpura and Pench with corridors to facilitate gene flow and create

a larger, more robust metapopulation. However, corridors may also increase the risk that

a pathogen that enters one reserve can spread to another (Hess, 1996). This makes

regular surveillance of domestic and wild carnivore population of all the connected

protected areas even more necessary. Periodical surveillance in wild carnivores for

antibody seroprevalence is strongly recommended, and that all wild carnivore carcasses

should be analyzed for CPV and CDV. Mitigation steps including vaccination programs

and removal of infected individuals should be considered in both domestic and wild

carnivores. I am heartened that these two diseases did not turn up in my samples, but

broader and long-term sampling will be required to determine if the threats they pose are

small.

59

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62

APPENDICES

63

Appendix A

AIC value of the models used to estimate the abundance of dogs for near and far village

in winter

2015. Villages that got Mt as the most appropriate model are in bold text.

Village M0 Mh Mt Mth

N1 62.20 64.31 59.98 62.38 N2 57.98 60.08 62.37 64.62 N3 53.87 57.31 51.63 59.29 N4 61.49 65.81 63.59 68.59 N5 49.72 51.98 56.11 54.03 N6 60.73 62.84 66.13 68.39 F1 61.41 63.50 63.12 65.35 F2 61.16 63.26 66.19 68.43 F3 58.28 60.35 63.91 66.14 F4 62.19 64.28 65.29 67.50 F5 61.37 63.30 63.82 67.76

64

Appendix B

Observed and estimated number of dogs for near and far villages for summer 2014 (S)

and winter 2015 (W), listed as: observed/estimated and standard error (SE) using M0

model with lowest AIC value, density of dogs for both field seasons, human population

density (HD) calculated using data from census on India website and percentages of

juveniles and females. The near and far villages that were sampled in both field seasons,

have been allotted acronyms: N1, N2, N3, N4 and F1, F2, F3, F4

Village

Near

Observed/ estimated (SE)

S W

Dog density

S/W

HD Juvenile (%)

S W

Female (%)

S W

Bhilwani (N1) 30/ 34(3.5) 41/45(3.9) 11.6/17.4 141.4 38 42.3 30.4 35.6

Lagma (N2) 18/ 26(7.3) 26/26(0.6) 3.7/5.4 273.5 24.8 35 27.4 38.5

Samnapur (N3) 20/23 (2.0) 26/30(3.1) 5.1/7.7 253.8 30 34.7 36 42.4

Lapti (N4) 17/ 21(3.6) 26/28(1.7) 9.5/12.6 64.2 23.9 30.8 31.7 46.2

65

*Chiraidongri

Arandi (N5) 21 /29(6.0) NA 9.7/NA 56.6 28 NA 42.8 NA

Ranwahi (N6) NA 24/29(3.7) NA/19.1 98 NA 33.4 NA 37.5

Village Far

Rajma (F1) 20/23 (1.3) 30/34(2.9) 5.6/8.2 205.5 35.4 43.3 27 40

Parasmau (F2) 14/19(4.5) 25/27(1.3) 2.1/2.8 61.3 31.7 44.6 32 38

Chartola (F3)

F3

18/20(1.1) 24/27(1.9) 17.1/23.7 147 24.2 37.1 40 44.5

Ghana (F4)

F4

18/25(5.7) 28/32(2.7) 11.4/14.3 129.9 35.2 42.9 24 39.9

Chiraid* (F5) NA 29/34 (3.3) NA/23.1 192.2 NA 31.1 NA 44.9