Clemson UniversityTigerPrints
All Theses Theses
5-2016
Threats of Disease Spillover from Domestic Dogsto Wild Carnivores in the Kanha Tiger Reserve,IndiaVratika ChaudharyClemson University, [email protected]
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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
vi
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
vii
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).
2
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
6
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.
7
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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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