Interactions and Pathogen Transmission Between Carnivores

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Interactions and Pathogen Transmission BetweenCarnivores in MadagascarFidisoa [email protected]

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Interactions and Pathogen Transmission Between Carnivores in Madagascar

Fidisoa Rasambainarivo

Student Education/Degrees DVM, University of Antananarivo, Madagascar, 2008

MSc, EpidemiologyUniversite de Montreal, Canada, 2013

A Thesis Submitted to The Graduate School at the University of Missouri-St. Louis in partial fulfillment of the requirements for the degree

Doctor of Philosophy in Biology

May 2018

Advisory Committee

Patricia Parker, Ph.D. Chairperson

Matthew Gompper, Ph.D.

Robert Marquis, Ph.D.

Robert Ricklefs, Ph.D.

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Abstract

Introduced carnivores exert considerable pressure on native predators through

predation, competition and disease transmission. Improved understanding of

determinant factors of interactions and pathogen transmission between

introduced and endemic wildlife may help to predict disease emergence, avoid

pathogen spillover and help control outbreaks. Using non-invasive camera traps,

I identified areas where transmission of pathogens might happen through records

of shared space-use within a protected area in Eastern Madagascar. I showed

that indirect interactions between animals were more likely to occur near the

research station which may constitute a disease transmission hotspot for

carnivores in the landscape. Secondly, I investigated the associations between

individual and spatial variables with the exposure to pathogens in multiple

sympatric endemic carnivores. I showed that individual characteristics such as

age, sex and species are associated with exposure to Toxoplasma gondii, but

not Leptospira spp. or Canine Parvovirus. Finally, I revealed where exchange of

microbes has already occurred by using microbial genetics of Escherichia coli.

Specifically, DNA fingerprinting methods were used to construct a microbial-

sharing network between carnivores in the Betampona natural reserve

ecosystem. Collectively, the results that are presented here may help the

conservation efforts of the unique Malagasy carnivores by highlighting the need

for disease monitoring and mitigation at the domestic animal and wildlife interface

of Madagascar.

i

Acknowledgments

Firstly, I would like to express my most sincere gratitude to my advisor

Dr. Patricia Parker for giving me the opportunity to conduct a project that I am

passionate about. Patty, I am extremely grateful for your support, patience and

guidance throughout the course of this project. Thank you for inspiring me to

believe in my abilities to “do good” in science and conservation, and for modeling

excellence in teaching. I am proud to have you as a mentor.

I would like to thank the rest of my thesis committee: Dr Matthew Gompper,

Dr Robert Marquis, Dr.Robert Ricklefs, Dr Eric Miller and Ingrid Porton for their

insightful comments and encouragement, but also for the hard questions which

incented me to widen my research from various perspectives. Particularly, I am

thankful to Ingrid Porton for her hospitality, support and friendship. Thank you for

believing in me.

I am thankful to all the members of the Parkerlab and of the BGSA

(Biology Graduate Student Association) of UMSL for their friendship. Thank you

for helping me keep my sanity during these years.

I am thankful to all the staff and faculty at the UMSL biology department for their

assistance during the past five years. You have made graduate school an

enjoyable experience.

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I would like to thank my friends and colleagues in Madagascar without whom this

work would not have been possible. Torisy, Flavien, Dr Andrianizah Hertz,

Dr Mamy Navalona Andriamihajarivo and Dr Natacha Rasolozaka, I am grateful

for your hard work and assistance in collecting the data and samples for this

project.

I would like to acknowledge the incredible institutional support I have received

from the Saint Louis Zoo Wildcare Institute, Harris World Ecology Center at

UMSL, Madagascar Fauna and Flora Group, the Department of Veterinary

Sciences and Medicine of the University of Antananarivo and Madagascar

National Parks.

I would like to thank my funding sources, Saint Louis Zoo Wildcare Institute,

Rufford Small Grants Foundation, DesLee Collaborative Vision Initiative at UMSL

and Madagascar Fauna and Flora Group.

Last but not least, thank you to my family. Liz, Jhon, Eliane, Haja, Dominique,

Mathias and Loic. Words cannot express my gratitude for your continued support

throughout this adventure and my life in general. Thank you for encouraging me

in all of my pursuits and inspiring me to follow my dreams. Your love pushed me

forward.

Table of Contents

INTRODUCTION ................................................................................................................................... 1

CHAPTER 1: INTERACTIONS BETWEEN CARNIVORES IN MADAGASCAR AND THE RISK

OF DISEASE TRANSMISSION ........................................................................................................... 7

INTRODUCTION .................................................................................................................................... 8

METHODS .......................................................................................................................................... 11

Study area ................................................................................................................................... 11

Camera trap survey .................................................................................................................... 12

Geographical Information System data ..................................................................................... 13

Data analysis ............................................................................................................................... 13

RESULTS ........................................................................................................................................... 18

DISCUSSION....................................................................................................................................... 20

LITERATURE CITED ............................................................................................................................. 43

CHAPTER 2: PREVALENCE OF ANTIBODIES TO SELECTED VIRUSES AND PARASITES IN

INTRODUCED AND ENDEMIC CARNIVORES IN WESTERN MADAGASCAR ......................... 55

INTRODUCTION ............................................................................................................................. 56

MATERIALS AND METHODS ....................................................................................................... 58

Study sites ................................................................................................................................... 58

Sample collection ........................................................................................................................ 59

Laboratory analysis..................................................................................................................... 60

Statistical analysis ...................................................................................................................... 61

RESULTS ........................................................................................................................................ 62

DISCUSSION .................................................................................................................................. 64

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LITERATURE CITED ...................................................................................................................... 70

CHAPTER 3: PATTERNS OF EXPOSURE OF CARNIVORES TO SELECTED PATHOGENS IN

THE BETAMPONA NATURAL RESERVE LANDSCAPE, MADAGASCAR. ............................... 86

INTRODUCTION .................................................................................................................................. 87

MATERIALS AND METHODS ................................................................................................................. 88

RESULTS ........................................................................................................................................... 90

DISCUSSION....................................................................................................................................... 90

LITERATURE CITED ............................................................................................................................ 94

CHAPTER 4: SHARING IS NOT CARING, MICROBIAL TRANSMISSION NETWORK AMONG

CARNIVORANS IN BETAMPONA, MADAGASCAR. ....................................................................104

INTRODUCTION .................................................................................................................................105

MATERIALS AND METHODS ................................................................................................................109

RESULTS ..........................................................................................................................................113

DISCUSSION......................................................................................................................................116

REFERENCES ....................................................................................................................................121

Introduction

In different areas of our planet, human activities such as deforestation and

hunting are threatening the survival of many wildlife species. Furthermore,

accidental or intentional introduction of different organisms are exerting additional

pressures on native animals, and in certain cases the former becomes invasive

(Lowe, 2000). Introduced animals have been shown to exclude endemic species

either directly by predation or by reducing available resources (Vanak and

Gompper, 2009, 2010; Krauze-Gryz et al., 2012; Hughes and Macdonald, 2013).

This increase in native-exotic animal interactions also presents potential for

“pathogen pollution,” the introduction of a pathogen into a new geographic area

or a host species (Cunningham, 2003). Invaders may also act as an additional

competent host for native pathogens, thereby increasing infection rates of native

species via spillback mechanisms. Domestic carnivores are often cited as a

source for pathogens such as canine distemper (CDV), canine parvovirus (CPV)

and rabies, whereas cats may transmit feline immunodeficiency virus (FIV) and

Toxoplasma gondii for example (Roelke-Parker et al., 1996; Fiorello et al., 2007;

Belsare et al., 2014). Some of these pathogens, such as canine parvovirus and

Leptospira sp. can survive for an extended period of time in the environment and

be transmitted indirectly through the shared use of the habitat.

Madagascar is a well-known biodiversity hotspot with approximately 90%

of plant and animal species being endemic, including all non-human primates

(superfamily: Lemuroidea), all native rodents (subfamily Nesomyinae), a radiation

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of insectivore-like animals (family Tenrecidae), and seven of its eight

wild carnivorans (family Eupleridae) (Myers et al., 2000). Through a series of

colonization events, probably during the Tertiary Period via some sort of over-

water rafting, the ancestor for each of the four groups of terrestrial mammals

occurring on the island (primates, rodents, insectivores

and carnivorans) established initial populations and their subsequent adaptive

radiations produced the high diversity and 100% endemism rate observed today

(Poux et al., 2005). Among the native families, the Eupleridae are arguably one

of the least known and most threatened families of carnivorans in the world

(Yoder et al. 2003, Brooke et al. 2014). Today, the Eupleridae comprise seven

monospecific genera and seven subspecies that are threatened mainly by habitat

loss (Veron et al. 2017).

Madagascar’s fauna is not only remarkable by the diversity of endemic

taxa found on the island, but also because of the absence of entire families that

are otherwise present in nearby continental Africa. For instance, no member of

the families Canidae, Felidae, and Bovidae have naturally crossed the

Mozambique Channel separating mainland Africa from Madagascar. The

different dispersal filters and subsequent isolation of these animals that were

able to colonize the island successfully may have important implications for

the sensitivity of Malagasy animals to diseases carried by exotic species. In fact,

species inhabiting island ecosystems tend to be more heavily affected by

invasive species because they lack appropriate behavioral traits or

immunological capacity, making them particularly vulnerable to the threat of

3

introduced species and/or their associated pathogens. The high rate of bird

extinction on Hawaii due to the introduction of arthropod vectors and the

transmission of avian malaria and Avipoxvirus are cases in point (Van Riper et al.

1986, 2002).

Research on carnivores inhabiting several regions of Madagascar have

shown an alarming correlation between increasing numbers of introduced cats

and dogs and decreasing detection of endemic euplerids and lemurs (Gerber et

al., 2012; Farris et al., 2014).As in other ecosystems, the reason for the observed

decline is likely a combination of factors including resource competition,

predation and disease (Vanak and Gompper, 2010; Medina et al., 2014).

Improved understanding of determinant factors of interactions and pathogen

transmission between introduced and endemic wildlife may help to predict

disease emergence, avoid pathogen spillover and help control outbreaks.

The Betampona Natural Reserve (BNR) is considered a biodiversity

hotspot within Madagascar and covers 2,228 ha of lowland rainforest completely

isolated from other forest patches. Betampona harbors a rich diversity of plant

and animal species and is home to 11 lemur species, all of which are

endangered, 4 species of endemic carnivores, as well as various rodents and

insectivores. The BNR, established in 1927, is one of the last protected remnants

of lowland rainforest in Madagascar, but recent studies show that half of its

surface is degraded and invasive plants now occupy about 17.5% of its area

(Ghulam et al., 2011; Ghulam et al., 2014). The BNR is also surrounded by

human habitation. At least eight villages, each constituted by 20-50 households

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are located within 1km of the reserve border. As in many rural areas of

Madagascar, domestic dogs and cats are common, unrestrained and receive

little food supplementation from their owners. This may facilitate the incursion of

invasive animal species into the reserve and the pathogens they may host.

The goals of this thesis are to study the patterns of interactions and

assess the risks of disease transmission between introduced and endemic

carnivorans in the Betampona Natural Reserve landscape in Madagascar. In the

first chapter, I identified areas where transmission of microbes might happen

through records of shared space-use within the protected area in Eastern

Madagascar. In the second chapter, we used serum samples collected from

dogs, cats and fosa (Cryptoprocta ferox) collected in and around two protected

areas of Western Madagascar in order to estimate the exposure of carnivoran

species to selected pathogens. Third, using similar laboratory procedures, I

investigated the associations between individual and spatial variables associated

with the exposure to pathogens in multiple sympatric endemic carnivoran species

in the BNR landscape. In the fourth part of the study, I revealed where exchange

of microbes has already occurred by using microbial genetics of Escherichia coli.

Specifically, DNA fingerprinting methods were used to construct a microbial-

sharing network between carnivores in the Betampona natural reserve

ecosystem. Taking heterogeneities in transmission dynamics into account will

increase our ability to understand and predict the dynamics of infectious diseases

and ultimately limit their spread between species of exotic and endemic

carnivores. Collectively, the results that are presented here may help the

5

conservation efforts of the unique Malagasy carnivores by highlighting the need

for disease monitoring and mitigation at the domestic animal and wildlife interface

of Madagascar.

Literature cited

Cunningham A, Daszak P, Rodriguez J. Pathogen pollution: Defining a parasitological

threat to biodiversity conservation. J Parasitol 2003;89:S78-S83.

Farris ZJ, Golden CD, Karpanty S, et al. Hunting, exotic carnivores, and habitat loss:

Anthropogenic effects on a native carnivore community, madagascar. PloS one

2015:e0136456.

Lowe S, Browne M, Boudjelas S, et al. 100 of the world's worst invasive alien species: A

selection from the global invasive species database. The Invasive Species Specialist

Group (ISSG) of the Species Survival Commission (SSC) of the World Conservation Union

(IUCN), 2000.

Knobel DL, Butler JR, Lembo T, et al. Dogs, disease, and wildlife.In Gompper ME, Ed.,

Free-Ranging Dogs and Wildlife Conservation, Oxford: Oxford University Press, 2014:

144–169

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Mooney HA, Cleland EE. The evolutionary impact of invasive species. Proc Natl Acad Sci

USA 2001;98:5446-5451.

Myers N, Mittermeier RA, Mittermeier CG, et al. Biodiversity hotspots for conservation

priorities. Nature 2000;403:853-858.

Poux C, Madsen O, Marquard E, et al. Asynchronous colonization of madagascar by the

four endemic clades of primates, tenrecs, carnivores, and rodents as inferred from

nuclear genes. Syst Biol 2005;54:719-730

Vanak AT, Gompper ME. Interference competition at the landscape level: The effect of

free�ranging dogs on a native mesocarnivore. J Appl Ecol 2010;47:1225-1232.

van Riper C, van Riper SG, Goff ML, et al. The epizootiology and ecological significance of

malaria in hawaiian land birds. Ecol Monogr 1986;56:327-344.

van Riper C, van Riper SG, Hansen WR, et al. Epizootiology and effect of avian pox on

hawaiian forest birds. Auk 2002;119:929-942.

Chapter 1: Interactions between carnivores in Madagascar and the

risk of disease transmission

Published in Ecohealth as : “Rasambainarivo, F., Farris, Z. J., Andrianalizah, H.,

& Parker, P. G. (2017). Interactions between carnivores in Madagascar and the

risk of disease transmission. EcoHealth, 14(4), 691-703.”

Abstract

Introduced carnivores exert considerable pressure on native predators through

predation, competition and disease transmission. Recent research shows that

exotic carnivores negatively affect the distribution and abundance of the native

and endangered carnivores of Madagascar. In this study, we provide information

about the frequency and distribution of interactions between exotic (dogs and

cats) and native carnivores (Eupleridae) in the Betampona Natural Reserve

(BNR), Madagascar, using non-invasive camera trap surveys. Domestic dogs

(Canis familiaris) were the most frequently detected carnivore species within the

BNR and we found that indirect interactions between exotic and native carnivores

were frequent (n=236). Indirect interactions were more likely to occur near the

research station (Incidence rate ratio=0.91), which may constitute a disease

transmission hotspot for carnivores at BNR. The intervals between capture of

native and exotic carnivores suggest that there is potential for pathogen

transmission between species in BNR. These capture intervals were significantly

shorter near the edge of the reserve (p=0.04). These data could be used to

8

implement biosecurity measures to monitor interactions and prevent disease

transmission between species at the domestic animal and wildlife interface.

Introduction

Exotic mammals constitute a major threat to native wildlife. Among them,

dogs, cats, and foxes have considerable effects on the distribution and

abundance of native predators as well as their prey through predation (Ritchie et

al. 2014), competition (Vanak and Gompper 2009), hybridization (Leonard et al.

2013), and disease transmission (Hughes and Macdonald 2013). These direct

impacts of invasive predators may also be compounded with other ecological

disturbances and further reduce biodiversity through additive and synergistic

effects (Doherty et al. 2015). For example, domestic cats (Felis catus) and red

fox (Vulpes vulpes) are responsible for the death of billions of birds, mammals,

and reptiles every year and at least 14 extinction events, especially in island

ecosystems (Medina et al. 2014).

Madagascar represents one of the world’s top biodiversity hotspots. It

ranks among the richest in terms of biodiversity and is also one of the most

threatened ecosystems in the world (Myers et al. 2000). The Malagasy

carnivores represented by ten endemic species all belong to a single-family

endemic to Madagascar, the Eupleridae, and are arguably one of the least

known and most threatened families of carnivores in the world (Yoder et al. 2003,

Brooke et al. 2014). Additionally, four species of exotic carnivores are known to

occur in Madagascar, the domestic dog (Canis familiaris), the domestic cat (Felis

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catus), the African wildcat (Felis silvestris), and the Indian civet (Viverricula

indica). A felid carnivore, the fitoaty, was recently identified but its affiliation or

differentiation from felids known to occur in Madagascar is awaiting confirmation

(Borgerson 2013, Farris et al. 2015b). Recent research suggests that these

exotic carnivores negatively affect the occupancy and detection probabilities of

several species of lemurs and native Malagasy carnivores even in protected

areas (Farris et al. 2015a, Farris et al. 2016, Farris et al. 2017). In addition, the

Eupleridae are threatened by habitat loss and some are also consumed as

bushmeat in villages neighboring protected areas or killed for retaliation after a

predatory event on livestock (Golden et al. 2015, Kotschwar Logan et al. 2015).

Native carnivores roaming in villages or interacting with domestic animals in the

vicinity of human habitats may also be exposed to pathogens from exotic animal

species.

In fact, the increase in native-exotic carnivore interactions presents

potential for pathogen pollution, the introduction of a parasite into a new

geographic area or a naïve host species (Cunningham et al. 2003). When the

pathogen continually invades and exerts pressure on the native population due to

an artificially increased population of the reservoir host (usually domesticated

animals), the phenomenon is called disease spillover (Power and Mitchell 2004).

Several diseases harbored by dogs and cats are able to infect and spill

over to wild animals’ species. Canine distemper, for example, is a viral disease

caused by a morbillivirus and is considered one of the most important pathogens

of carnivores worldwide (Deem et al. 2000). The canine distemper virus is

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commonly found in domestic dogs but has spilled over to a variety of wild

carnivores including painted dogs, tigers, and lions (Deem et al. 2000, Terio and

Craft 2013). Canine distemper has caused declines in populations and put wild

carnivores at an increased risk of extinction (Roelke-Parker et al. 1996, Gilbert et

al. 2014, Gilbert et al. 2015). Similarly, the canine parvovirus may be transmitted

from domestic animals to a wide variety of hosts including several species of

Felidae, Canidae, Procyonidae, Mustelidae, Ursidae, and Viverridae (Steinel et

al. 2001, Allison et al. 2013). The prevalence of antibodies against parvovirus

and canine distemper in dogs living in rural areas neighboring natural habitats,

including in Madagascar is generally high (in Madagascar the seroprevalence of

dogs to canine parvovirus and canine distemper virus is 67% and 45%

respectively) and these pathogens may be transmitted to the native carnivores

(Belsare and Gompper 2014, Pomerantz et al. 2016).

Disease transmission between species depends in part on the

susceptibility of the hosts, the contact between infected and susceptible animals,

and the length of time the pathogen can survive in the environment. These

pathogens differ by their route of transmission; the canine distemper virus is

usually transmitted between animals through direct contact given it does not

survive for long periods in the external environment (48 hours at 25 C) (Shen and

Gorham 1980). Canine parvovirus, on the other hand, can be transmitted

indirectly through environmental contamination and may remain infective for

several months in fecal matter (Pollock 1982). Assessing the interactions, both

spatially and temporally, between exotic and native carnivores is important for

11

understanding the potential for disease transmission and for developing targeted

mitigation and preventative actions to limit these occurrences.

In this study, our first objective was to provide a survey of the native and

exotic carnivore community of the Betampona Natural Reserve (BNR). Secondly,

we assessed the spatial and temporal patterns of interactions between native

and exotic carnivores within the BNR. Finally, our third objective was to evaluate

the potential for disease transmission between carnivores by determining the

frequency of interactions that occur within a critical time window that relates to a

pathogen-specific survival time in the external environment.

Methods

Study area

The Betampona Natural Reserve is one of the last remnants of lowland

tropical rainforest in Madagascar.It is located approximately 40 km northwest of

Toamasina in Eastern Madagascar (Rendrirendry research station GPS

coordinates: -17.9315, 049.2033) is a 2,228 ha lowland rainforest with a mean

elevation of 270m (range 92-571m). The climate in the region is hot and humid

with annual rainfall amounts of over 2000 mm, average humidity between 81%

and 91%, and the average annual temperature is 24 C (16-32 C). The reserve

consists of dense primary forest and lined by secondary forest at the borders,

characterized by the invasion of exotic guava tree (Psidium cattleianum) or the

Molluca raspberry (Rubus mollucanus) (Ghulam et al. 2011, Ghulam et al. 2014).

12

Betampona is a strict nature reserve managed by the Madagascar National

Parks (MNP) and the Madagascar Fauna and Flora Group (MFG) acts as the

research partner in the reserve. Access of tourists and villagers is not permitted

in the protected area and only a small number of scientists and staff members

from these institutions (less than 30) have access to the forest reserve.

The reserve is isolated from any other forest patch and is surrounded by

agricultural areas and human habitation. Each of the eight villages in the vicinity

of the reserve (less than 1km from the border) is composed of 20-50 households

on average with a total population of 80-200 people per village. The largest

village, Andratambe, is composed by 83 households and is located in the

southeastern corner of the BNR (Andratambe GPS coordinate: -17.9333,

049.2157) (Figure 1). The main activity of villagers in the region is agriculture and

the main crop is rice and to a lesser extent maize, sugar cane, cloves, coffee,

banana and manioc. Most people raise poultry outdoors, pigs and dogs are

common in the region and all are free ranging.

Camera trap survey

We established a camera trap grid composed of 30 passive, infra-red

camera trap stations covering 9.6 km2 (estimated by minimum convex polygon

without any buffer (Mohr 1947, Quantum 2013)). We spaced camera trap

stations 500m (SD=108m) apart on average based on the small home ranges of

Madagascar’s carnivores, excluding the fosa (Cryptoprocta ferox) (Figure 1)

13

(Goodman 2012). We conducted the camera trap survey from June 08th to

October 27th, 2015. Each station consisted of a single camera trap (Model M880

Moultrie, Birmingham, AL, USA) placed on man-made or animal trails and at trail

intersections to maximize captures of carnivores. We set up cameras 10-20 cm

above ground, approximately 1-2m on the side of the trail to capture passing

wildlife (Glen et al. 2013). We set all cameras to take three successive photos

per trigger with a 10 second delay between consecutive triggers. Cameras

automatically stamped date, time and moon phase onto each image. We allowed

cameras to run 24 h day-1 and checked cameras every 5-7 days to change

memory cards and to ensure proper functioning. We did not use any baits or lure.

Geographical Information System data

We recorded localization of each camera trap station using a handheld

GPS device (Garmin GPSMAP 62s; Garmin International Inc., Olathe, KS, USA)

and georeferenced to the Universal Transverse Mercator (UTM) coordinate

system zone 39 South with World Geodetic System Datum of 1984 (WGS84).

We obtained data layers for topography, land use and land cover for the BNR

and vicinity using Ikonos-2 imagery (Ghulam et al. 2014).

Data analysis

14

We identified photographed animals down to species level using

morphological characteristics (Goodman 2012). We treated photos of the same

species taken by the same camera more than 30 minutes apart as an

independent camera trap event following Bitetti et al. (2006). We defined a trap-

night as a 24h period in which the camera trap was functioning properly. For

each species, we calculated their trapping success (number of capture

events/trap nights x 100) as an indicator of relative activity.

Occupancy

To evaluate the proportion of the reserve occupied by each carnivore

species, we conducted single-season occupancy analyses of carnivores in the

reserve. Briefly, the method involves surveying sites (here, a camera trap station)

multiple times (trap-nights) and, within a maximum likelihood-framework, aims to

estimate the proportion of sites that are occupied, knowing the species is not

always detected even when present (MacKenzie et al. 2002, Bailey et al. 2004).

We created capture histories for each of the native and exotic carnivore species

using daily capture events to determine the presence (1) or absence (0) of each

species at each camera station. We analyzed capture histories using the

program PRESENCE (Patuxent Wildlife Research Centre, USGS, Maryland,

USA) (Hines 2006) to provide an estimate of species occurrence while

accounting for variation in detection probability (Bailey et al. 2004).

15

Interspecific interactions data analysis

We assessed the potential for disease transmission between an exotic

carnivore and a native species by quantifying the frequency of direct and indirect

interactions between species. A direct interaction was defined as the

simultaneous presence of an exotic and native carnivore in the same

photograph. An indirect interaction was defined as a visit of one species (native)

to a camera trap station after the visit of another species (exotic). Given our

objectives and the lack of information regarding pathogens disseminated by

native carnivores in Madagascar, we only considered indirect interactions in one

direction, visit of an exotic followed by the visit of a native carnivore.

To evaluate the factors influencing the number of interactions and the

interval between visits of an exotic and a native carnivore, we considered six

environmental variables. These were:

a) forest type, defined as degraded or primary forest based on the land cover and

the presence or absence of invasive plants (guava, Madagascar cardamom

and/or molucca raspberry) in a 250 m buffer around the camera trap station; b)

Distance to edge, as the distance from each camera station to the nearest border

of the reserve; c) Distance to village, as the distance from each camera station to

the nearest village; d) Distance to Andratambe, as the distance from each

camera station to the largest village in the vicinity of the BNR; e) Distance to

Rendrirendry, as the distance from each camera station to the research station;

and f) Elevation, as altitude above sea level for each camera trap station.

16

Elevation was included to reflect the apparent preference of some Eupleridae

species to low elevation (IUCN 2015, Farris et al. 2016).

To avoid including collinear explanatory variables in models, we calculated

the variance inflation factor (VIF), where each variable was set as the explained

variable in a linear regression model and the other variables were included as

explanatory variables. The VIF is calculated as VIF=1/(1-R2). Variables with VIF

higher than 2 were not included in the same model (Zuur et al. 2010).

We calculated the time elapsed between the visit of an exotic carnivore

and the visit of a native carnivore using the date and time stamped on each

picture. We quantified indirect interactions that occurred within specified critical

time windows of 2 days (short interval), and 100 days (long interval). These

critical time windows were determined to account for the estimated survival times

of the canine distemper virus and the canine parvovirus respectively, under local

temperature and humidity conditions (average temperature and humidity: 25 C

and 90% respectively) (Shen and Gorham 1980, Pollock 1982).

As the count data were over-dispersed, we used negative binomial

regression analyses to test whether the number of indirect interactions between

exotic and native carnivores was associated with the sampled environmental

variables listed previously (Gardner et al. 1995). Since the trapping effort was

unequal at each camera trap station, the number of camera trap-nights was used

as an offset variable. We ran negative binomial regression models using the

nbinom function from the package MASS in R (Ripley et al. 2015).

17

We computed and ranked models using the Akaike’s Information Criterion

adjusted for small sample size (AICc) and report the incidence rate ratio for each

significant variable in the most supported models (ΔAICc<2). We conducted a

Pearson chi-square goodness of fit test to assess the fit of the final model.

We used the number of short-interval indirect interactions (maximum of

two days between the visit of an exotic and a native carnivore at a camera trap)

to obtain a generalized density map of their distribution within the BNR. For this,

we used the function heatmap in Quantum GIS (qGIS) (Quantum 2013), setting

the Kernel density estimation function to Epanechnikov and a radius of 500m.

We further analyzed the relationship between the time interval separating

the visit of an exotic and a native carnivore with selected environmental variables

using linear regression models. Since the interval between visits was not

normally distributed, they were log-transformed and used as the dependent

variable in a series of regression models. The explanatory variables presented

above as well as the number of interaction events per trap-night recorded at a

station were used as explanatory variables. Beginning with an initially full model

containing all the explanatory variables, a backward stepwise approach was

used with p>0.05 as the rejection criterion and the least important variables

(based on p values) being removed first (Burnham and Anderson 2002).

We carried out statistical data analyses using R (Team 2014) and

produced maps using qGIS (Quantum 2013).

18

Results

The camera trapping survey produced a total of 2,556 images of

carnivores from eight species over a total trapping effort of 2,598 trap-nights

(median: 105 nights per station, range: 25-141 nights per station) (Figure 1).

We identified eight species of carnivores from the camera traps including

five natives and three exotic carnivores (Table 1). The domestic dogs were the

most frequently detected carnivore in the BNR with 240 camera capture events

followed by the fosa (Cryptoprocta ferox; 130 capture events), the ring-tailed

vontsira (Galidia elegans; 75 capture events), the broad-striped vontsira

(Galidictis fasciata; 44 capture events), and the brown-tailed vontsira (Salanoia

concolor; 43 capture events). Felid species (Felis sp.), falanouc (Eupleres

goudotii) and Indian civets (Viverricula indica) were infrequently detected at

camera trap stations with 20, 4 and 1 events respectively.

The occupancy of the fosa was the highest followed closely by domestic

dog while the ring-tailed vontsira had the lowest occupancy of any carnivore

(Table 1). The occupancy for the felid species, the falanouc and the Indian civet

could not be calculated due to low detection rates so their naïve occupancy is

reported. Results of the camera trapping survey are presented in Table 1.

Carnivore interactions and critical time windows

We recorded no direct interactions between native and exotic carnivore

species at the camera trap stations. We recorded indirect interactions between

exotic and native carnivores on 21 of the 30 cameras deployed and documented

19

236 indirect interactions (exotic carnivore visit followed by the visit of a native

carnivore). The number of indirect interactions at each camera station varied

from 0.00 to 0.52 events per trap-night.

Preliminary analyses suggested that distance to the edge and distance to

the closest village are collinear (VIF=2.38). Similarly, distance to the largest

village (Andratambe) and distance to the research station (Rendrirendry) are also

collinear (VIF=8.33). Therefore, both of these variable pairings were not included

in the same model to explain the number of indirect interactions and the interval

between visits.

The best supported negative binomial regression model shows that the

number of indirect interactions is positively associated with the distance to the

research station Rendrirendry (Beta=-0.10 SE=0.02) (Table 2). For every 100m

further from the Rendrirendry research station, the rate of interactions would be

expected to decrease by 9%, while holding all other variables constant (Table 3,

Figure 2).

The interval of time between the visit of an exotic carnivore followed by the

visit of a native carnivore at a camera station ranged from 1 hour to 92 days

(median 6 days). All recorded indirect interactions at camera traps occurred

within 100 days, which corresponds to the estimated survival time of the canine

parvovirus in the environment (Figure 3). Fifty-one (22%) indirect interactions

were qualified as short interval and occurred within two days, which corresponds

to the estimated survival time of the canine distemper virus in the environment

20

(Figure 3). Areas near the research station had the highest density of short

interval indirect interactions (Figure 4).

The final model showed that a larger distance from the edge of the

reserve is associated with longer time intervals between the visit of an exotic and

a native carnivore (p=0.04) (Table 4, Figure 5). The adjusted R-squared value for

this model was 0.16 (Table 4, Figure 5).

Discussion

With this study, we aimed to explore the spatio-temporal interactions

between domestic and native carnivores in the Betampona Natural Reserve and

to evaluate the potential for disease transmission between species. Our findings

highlight the potential for pathogen pollution from exotic to native carnivores at

BNR.

First, we showed that activities of domestic dogs are abundant and widely

distributed throughout the reserve. In fact, dogs were the most detected

carnivore species and were detected on 83% of the camera traps in Betampona.

The occupancy of dogs in Betampona (91%) was similar to the occupancy of

dogs in the fragmented forest portion of the Ranomafana national park (87%)

(Gerber et al. 2012), but is higher than what was found even in the most

degraded habitat of the Makira National Park (61%) (Farris et al. 2015c). The

widespread distribution of dogs in the BNR is of concern because they are

expected to affect native carnivores and particularly the small and medium sized

species (Vanak and Gompper 2010, Vanak et al. 2013, Ritchie et al. 2013).

21

The presence of free-ranging dogs in natural habitats may have multiple

detrimental effects on wild animals (Young et al. 2011). On the one hand, the

increased activities of dogs and other exotic carnivores in natural habitats can

reduce the occupancy and detectability of sympatric wild animals or affect their

behavior (Vanak and Gompper 2009, 2010, Silva-Rodríguez and Sieving 2012,

Zapata-Ríos and Branch 2016, Farris et al 2017). On the other hand, one

carnivore species may be attracted to areas where another species occur, which

could facilitate the transmission of diseases from one species to another (Belsare

et al. 2014, Cruz et al. 2015).

No direct interactions were detected during our study. This is consistent

with previous studies showing that direct interactions between domestic and wild

species tend to be rare compared to indirect interactions (Kukielka et al. 2013,

Sepúlveda et al. 2014, Payne et al. 2015). This could denote an asymmetrical

interaction between the carnivores and the behavioral avoidance of the

subordinate (here native carnivore) of areas occupied by the dominant (exotic

carnivore) species. It would be important to uncover patterns consistent with

asymmetrical interactions and fear between exotic and native carnivores in

Betampona but this is beyond the scope of this study.Here, we aimed to uncover

interactions relevant to the transmission of diseases that can occur either through

direct contact or merely through shared habitat use. For this study, given the lack

of information regarding pathogens disseminated by native carnivores in

Madagascar and given the concern of pathogen spillover from domestic animals

22

to endangered wildlife, we only considered indirect interactions in one direction,

visit of an exotic followed by the visit of a native carnivore.

Our results show frequent indirect interactions between exotic and native

carnivores, especially near the Rendrirendry research station. This may indicate

that this area is preferred by the exotic carnivores as well as the native

carnivores due to prey abundance or other habitat characteristics. It is possible

that dogs, rats and other peridomestic species are attracted to Rendrirendry

because of the constant presence of researchers and the associated garbage

accumulation. Since rats constitute part of the diet of most Eupleridae (Dollar et

al. 2007, Goodman 2012), it may also favor increased Eupleridae activities in this

area. Elsewhere, the occurrence of different species of carnivores was positively

associated with garbage availability. In the trans-Himalayan mountains for

example, red fox occurrence and density of domestic dogs was positively

associated with garbage availability (Ghoshal et al. 2016).

Another possibility for explaining the increased number of interactions in

this area is that Rendrirendry constitutes the main entrance point to the reserve.

It marks the beginning of the main trail that leads into the core of the protected

area so dogs and other carnivores that are entering or exiting the reserve may

favor going through Rendrirendry through this path. Research on carnivore

behavior and detection probabilities show that they tend to prefer traveling along

trail systems which would consequently increase the opportunities for

interactions near the research station (Harmsen et al. 2010, Cusack et al. 2015).

23

A second area, approximately 2km north of the research station seem to

have a high density of short interval interactions between exotic and native

carnivore (Figure 4). The reason for the increased interactions in this area is

unclear but may also reflect habitat preference or the preferred used of trails. In

fact, this area constitutes the intersection between the main trail and two smaller

secondary trails. The increased density of interactions in this area may be due to

the high number of carnivores using this intersection. The effect of intersecting

trails on the number of interaction between species requires further research.

Similarly, studies of habitat preference, diet, and behavior of native carnivores in

the BNR are needed to tease apart the contribution of these factors in the

patterns of interactions we uncovered.

Frequent interactions between animals of the same or different species

may lead to an increase in pathogen transmission (Altizer et al. 2003, Böhm et al.

2009, Ogada et al. 2012). For example, a study suggests that river otters (Lontra

provocax) have acquired canine distemper virus from exotic American minks

(Neovison vison) and domestic dogs as a result of interspecies interactions,

especially near latrines (Sepúlveda et al. 2014). Similarly, frequent interactions

between ungulate species near water points were associated with the potential

transmission of Mycobacterium bovis (Kukielka et al. 2013, Barasona et al. 2014,

Cowie et al. 2015). These areas of increased risks of transmission or disease

prevalence are referred to as transmission foci or disease transmission

“hotspots” (Paull et al. 2012).

24

We also showed that a large proportion of indirect interactions occur within

critical time windows that would allow the transmission of environmentally

transmitted pathogens. We used canine distemper virus and canine parvovirus

as examples of pathogens that may be transmitted indirectly and with varying

abilities to survive in the environment. One the one hand, canine parvovirus

exemplifies a pathogen that is mainly transmitted indirectly. It is acquired by a

susceptible individual through indirect contact and may survive for long periods of

time in the environment (Pollock 1982). Canine distemper on the other hand, is

mainly transmitted by direct contact but it may survive in the environment for

short periods of time (Shen and Gorham 1986). These pathogens are known to

occur in rural areas of Madagascar and a large proportion of domestic carnivores

in Western Madagascar show evidence of prior exposure to either or both canine

parvovirus and canine distemper virus (Pomerantz et al. 2016). The prevalence

of these pathogens in carnivores from the BNR ecosystem is unknown and

warrants further investigation. It is important to note however, that the

susceptibility of Eupleridae to these pathogens is unknown and that indirect

interactions between infective and susceptible individuals does not necessarily

result in pathogen transmission (Courtenay et al. 2001). Our results only indicate

that there is a potential for pathogen transfer between species in BNR and

interactions occur mainly near the research station.

In addition, our inability to individually identify the exotic and native

carnivores that frequent the reserve and interact within the protected area raises

the possibility that we are repeatedly capturing only a small number of individuals

25

at the camera trap stations. This could have implications for the risks of disease

transmission, as the incidence rate of infections in a population is variable and

may be influenced by the proportion of individuals with large home ranges

(Belsare and Gompper 2015). Nevertheless, our findings highlight the fact that

some exotic animals are highly active throughout the reserve and may spread

pathogens in the areas where they occur. Identifying the specific individuals that

are involved in these interactions within the BNR and assessing the patterns of

pathogen shedding in these animals would be important to assess the risks of

disease transmission from exotic to native carnivores.

We also show that the intervals between the visit of an exotic and a native

carnivore tend to be shorter near the edge of the reserve than in the interior.

However, as the low adjusted r-squared value indicates (adjusted r

squared=0.16), this result should be interpreted with caution. One or more

variables that have not been measured during the course of this study could be

important for explaining the variation in the interval between the visit of an exotic

and an endemic carnivore. For example, the distance to water bodies was linked

to increased interactions between domestic and wildlife species elsewhere

(Kukielka et al 2013). Here, the fact that there is a statistically significant

association between the distance to the edge and the interval between the visits

of carnivores suggests that the distance to the edge could have implications for

disease transmission. The process leading to changes in community structure

and abundances at the periphery of natural habitats compared to the interior is

known as “edge effects” (Murcia 1995). On the one hand, habitat edges and

26

fragmentation are associated with higher prevalence of diseases and density of

parasites (Glass et al. 1995, Suzán et al. 2008, Perrott and Armstrong 2011). On

the other hand, edges may be detrimental to the pathogens by increasing solar

radiation, decreasing moisture near the margins and ultimately decreasing

environmental survival of parasites (Murcia 1995, Hulbert and Boag 2001).

Whether edge habitats are indeed associated with higher parasite densities,

longer parasite survival in the environment, or an increase in disease exposure in

animals inhabiting the BNR is unclear and warrants further investigation.

Understanding the ecological relationship between habitat alterations,

animal interactions and disease transmission is important for improving our

overall comprehension of the links between environmental change and health.

Our study showed that exotic and native carnivores interact indirectly within the

Betampona Natural Reserve ecosystem and that these interactions are not

uniformly distributed across the landscape. In fact, the areas neighboring the

research station and the edges of the reserve may constitute foci of pathogen

transmission between carnivore species. This may help develop strategies for

biodiversity conservation by reducing contact rates between species at the

domestic animal-wildlife interface in the BNR.

Table 1: Number of capture events, trapping success and naïve occupancy of native and exotic carnivore species caught

on camera in the Betampona Natural Reserve. Photographic sampling of carnivores occurred at 30 camera stations from

June 5th to October 27th 2015 at the Betampona Natural Reserve, Madagascar.

English name Species

Type of

carnivore

Capture

events*

Trapping

success**

Occupancy

(SE)

Fosa Cryptoprocta ferox Native 130 5.00 0.95 (0.08)

Ring-tailed vontsira Galidia elegans Native 75 2.89 0.62 (0.12)

Broad-striped vontsira Galidictis fasciata Native 44 1.69 0.80 (0.09)

Brown-tailed vontsira Salanoia concolor Native 43 1.66 0.68 (0.13)

Falanouc Eupleres goudotii Native 4 0.15 0.03***

Domestic dog Canis familiaris Exotic 240 9.24 0.91 (0.07)

Cat Felis sp. Exotic 20 0.77 0.13***

Indian civet Viverricula indica Exotic 1 0.04 0.03***

Exotic carnivore species are indicated in bold. *: Capture events: number of independent photographic capture of

carnivores at camera stations in the Betampona Natural Reserve, Madagascar. **Trapping success: number of capture

events per trap-night x100. *** Indicates naïve occupancy: proportion of cameras where a given species was detected.

29

Table 2: Comparison of the ten highest-ranking models built to explain the number of indirect interactions between native

and exotic carnivore species in the Betampona Natural Reserve.

Candidate model ΔAICc ωi

Distance to Rendrirendry 0 0.75

Distance to Rendrirendry + type of forest 3.8 0.11

Distance to Andratambe 4.2 0.09

Distance to Rendrirendry + type of forest +elevation 7.02 0.02

Distance to closest village 7.9 0.02

Distance to Rendrirendry + type of forest + elevation + distance to edge 8.29 0.01

Distance to edge 10.83 0.00

Elevation 11.38 0.00

Type of forest 14.9 0.00

Intercept only 16.41 0.00

30

ΔAICc: Difference between each model Akaike Information Criterion value (AIC) and the one of the lowest AIC; ωi: Akaike

weight of the model.

Table 3: Estimates and Incidence rate ratios (IRR) of the variable associated with the number of indirect interactions

within the Betampona Natural Reserve.

Estimate

(SE) IRR IRR 95% CI

Z value

Pr(>|z|)

(Intercept) -2.34 (0.24) 0.10 (0.06-0.16) -4.41 0.00

Distance Rendrirendry -0.10 (0.02) 0.91 (0.89-0.93) -5.33 0.00***

*** p<0.001

31

Table 4: Linear regression evaluating the effects of environmental variables on the interval between visits of carnivores at

a camera station within the Betampona Reserve.

Estimate (SE) t value Pr(>|t|)

(Intercept) 3.47 (0.65) 5.31 0.00

Distance to edge 0.08 (0.03) 2.24 0.04*

Events per trap-night 0.66 (1.55) 0.42 0.68

*p<0.05

33

Figure 1 Map of the Betampona Natural Reserve (BNR) and placement of camera trap stations in different types of forest.

Locations of villages in the vicinity of the BNR are indicated by triangles. Numbers at each camera trap station indicate the

number of trap-nights the camera was active.

34

35

Figure 2: Negative binomial regression model showing the relationship between the distance from Rendrirendry (research

station) and the number of indirect interactions per trap-night. Indirect interaction is defined as the visit of an exotic

carnivore followed by the visit of a native carnivore at a camera trap station Incidence rate ratio = 0.91 (95% confidence

interval: 0.89–0.93); P <0.001.

37

Figure 3: Number of indirect interactions between exotic and native carnivores according to the number of days

separating visits of an exotic and native carnivore. The vertical colored lines indicate the critical time windows

relative to the estimated survival of the canine distemper virus (2 days; red), Sarcoptes scabiei, (7 days; green) and

the canine parvovirus (100 days; blue) in the environment.

39

Figure 4: Heat map of the density of short interval indirect interactions within the Betampona Natural Reserve. Short

interval indirect interactions are defined as a maximum of two days separating the visit of an exotic and a native carnivore

at a camera trap station.

41

Figure 5: Association between the time interval between camera capture events of exotic and native carnivores at camera

trap station and distance from the edge of the reserve Estimate: 0.08 (SE = 0.03); P value = 0.04

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Evolution 1: 3-1

Chapter 2: Prevalence of antibodies to selected viruses and parasites

in introduced and endemic carnivores in western Madagascar

Published in Journal of wildlife diseases as: “ Pomerantz, J., Rasambainarivo, F.

T., Dollar, L., Rahajanirina, L. P., Andrianaivoarivelo, R., Parker, P., & Dubovi, E.

(2016). Prevalence of antibodies to selected viruses and parasites in introduced

and endemic carnivores in western Madagascar. Journal of wildlife

diseases, 52(3), 544-552.”

ABSTRACT

Introduced animals are impacting endemic populations through predation,

competition, and disease transmission. Populations of endemic carnivores in

Madagascar are declining and pathogens transmitted from introduced animal

species may further endanger these unique species. Here, we assess the

exposure of introduced and endemic carnivore species to common viral and

parasitic pathogens in two national parks of Madagascar (Kirindy Mitea National

Park and Ankarafantsika National Park) and their neighboring villages. We also

identified variables associated with the presence of antibodies to these

pathogens in fosa (Cryptoprocta ferox). We show that introduced and endemic

species are exposed to canine parvovirus, canine herpesvirus, feline calicivirus

and Toxoplasma gondii. Dogs (Canis familiaris) and cats (Felis catus) may

56

constitute sources of infection for these pathogens. Prevalence of antibodies to

Toxoplasma in captured fosa was over 93% and adults were more likely to be

exposed than immature individuals. This study is the first to report the prevalence

of antibodies to common viral and parasitic pathogens in introduced and endemic

carnivores in Madagascar and provides a basis upon which to evaluate and

manage the risks of pathogen transmission across species.

Key words: Carnivore, conservation medicine, Cryptoprocta ferox, Fosa,

serosurvey, Toxoplasma.

INTRODUCTION

Domestic dogs (Canis familiaris) and cats (Felis catus) have been

introduced virtually on every landmass, reaching populations of 700 and 500

million respectively (Pollinger et al. 2010; Hughes and Macdonald 2013; Serpell

2000; Vigne et al. 2004; Driscoll et al. 2007). These domestic animals are

impacting wildlife populations on continental landmasses and have contributed to

multiple wildlife extinction on islands (Courchamp et al. 2003; Medina et al.

2011). Introduced animals can exclude endemic species by predation or by

reducing available resources (Vanak and Gompper 2009 2010). Additionally,

domestic animals may transmit pathogens to endemic wildlife through direct

contact or environmental contamination. This pathogen “spillover” is a particular

threat to endangered species and may lead to local extinction (Woodroffe 1999;

Daszak et al. 2000). For example, rabies and canine distemper, spilling over from

dogs, have decimated populations of African wild dogs, Ethiopian wolves and

57

African lions (Gascoyne et al. 1993; Laurenson et al. 1998). Pathogens can also

be an important factor determining the outcomes of trophic or competitive

interactions through “apparent competition” (Holt 1977).

Madagascar harbors four introduced species of carnivores, the domestic

dog, the domestic cat, the African wildcat (Felis silvestris) and the Indian civet

(Viverricula indica) and 10 endemic carnivore species belonging to the family

Eupleridae (Yoder et al. 2003). Of the Euplerid species, all but the Malagasy ring-

tailed mongoose (Galidia elegans) are listed in one of the threatened categories

on the IUCN red list (IUCN 2014). Malagasy carnivores appear sensitive to

habitat degradation and are further endangered by bushmeat hunting (Golden

2009; Gerber et al. 2012). Furthermore, research on Eupleridae has shown a

negative correlation between detection of endemic carnivores or primates and

introduced animal activity at camera sites (Gerber et al. 2012; Farris et al. 2014).

As in other ecosystems, the reason for the observed decline is likely a

combination of factors including resource competition, predation, and disease

(Vanak and Gompper 2009; Knobel et al. 2013; Belsare and Gompper 2014).

The fosa (Cryptoprocta ferox) is the largest extant native carnivore in

Madagascar. It is listed as endangered on the IUCN red list and threatened by

habitat loss and hunting (IUCN 2014). Fosa are solitary, cathemeral carnivores

ranging throughout Madagascar. They are found at low densities mainly in

forested areas but are also known to visit villages and prey on poultry (Hawkins

and Racey 2005; Kotschwar Logan et al. 2015). These raids into villages may

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facilitate the interactions with domestic animals and the transmission of

pathogens across species.

Despite the importance of the fosa as the top predator in the ecosystem

and the growing awareness about the risks of pathogen transmission between

domestic and wild animals, little information is available about the pathogens

affecting introduced and endemic carnivores in Madagascar. With this study, our

objectives were to: (1) assess the exposure of introduced and endemic

carnivores to selected viral and parasitic pathogens at two sites in Madagascar

and (2) identify variables associated with the presence of antibodies for these

diseases in fosa.

MATERIALS AND METHODS

Study sites

This study was conducted in 2 protected areas in Madagascar: the

Ankarafantsika National Park (ANP) and Kirindy Mitea National Park (KMNP) as

well as villages located around these protected areas. The ANP covers 65,520

ha of dry deciduous forest in northwestern Madagascar. Animals were trapped in

the Ampijoroa area at 16°15’S, 46°48’E. Within the national park altitude varies

between 80-370 m above sea level. Total rainfall is between 100 and 1500 mm

per year, 95% of this falling between November and April. The mean annual

temperature is 26 C (11.4 C - 39.3 C).

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The KMNP covers 152,000 ha of dry deciduous forest in western

Madagascar. Animals were trapped around the research station at 20°47’17” S,

44°10’08” E. Altitude in this area varies between 50–100 m. Mean annual rainfall

is approximately 700 mm. KMNP exhibits a dry season from April to early

November and a hotter, rainy season during the remainder of the year.The

temperature at the site varies from 7– 40 C, with an annual mean temperature of

24 C.

Sample collection

Samples from introduced and endemic carnivores were collected as part

of an ongoing ecological study of carnivores in 2000, 2001, 2003-2006, 2008,

2012 and 2013 in ANP and in 2005-2006 in KMNP. Samples were collected

mainly during the dry season (April-November) at both sites.

Free ranging carnivores were trapped in cage traps (Tomahawk Live Trap,

Tomahawk, Wisconsin USA) and anesthetized by intramuscular injection of

tiletamine-zolazepam hydrochloride (Telazol Ò, Fort Dodge Animal Health, Fort

Dodge, Iowa, USA) dosed at approximately 10 mg/kg or a combination of

ketamine (Ketaset Ò, Fort Dodge Animal Health) dosed at approximately 4mg/kg

plus medetomidine (Domitor Ò, Pfizer, New York, New York, USA) dosed at

approximately 0.1mg/kg. The injection was administered to the trapped animal

via short blowpipe and dart (Pneu-Dart, Inc., Williamsport, Pennsylvania, USA or

Telinject dart, Telinject USA Inc., Agua Dulce, California, USA).

60

Once anesthetized, each animal underwent an external physical

examination and a passive radio-frequency identification (RFID) tag was inserted

subcutaneously between the shoulder blades for future identification. The sex

and age category of the animal were recorded. Animals were classified as

immature or adult based on body size, dentition and allometric measurements.

Blood (not exceeding 1% of the animal body weight) was collected by

venipuncture of the lateral saphenous vein and immediately placed in serum

separator tubes (Corvac Sherwood Medical, Saint Louis, Missouri 63103, USA).

Animals were left to recover in the trap and released at the capture site.

Domestic dogs and cats from villages located around the National Parks

were also sampled. Dogs and cats in this region consist of owned and unowned

animals that are all free-ranging. None of the dogs or cats had a previous history

of vaccination. Samples from owned dogs and cats were collected with the

owners’ consent. Animals were classified as immature or adult based on body

size, dentition, allometric measurements and behavior. Only dogs and cats ≥5

mo old were sampled to avoid detection of maternal antibodies (Greene 1998).

Dogs and cats were manually restrained or anesthetized with tiletamine-

zolazepam (as described previously for fosa) and blood (not exceeding 1% of

body weight) was collected from the cephalic, medial/lateral saphenous, or

jugular vein and placed in serum separator tubes.

Laboratory analysis

Blood was allowed to clot before being centrifuged for 15 min at 400 x g.

Serum was pipetted and placed into cryotubes (cryovials, Nalgene Company,

61

Rochester, New York) and immediately frozen in liquid nitrogen for storage and

transport. Serum samples were submitted to the New York State Animal Health

Diagnostic Laboratory (Ithaca, New York, USA) for serological analysis.

Depending on volume of serum available from each animal, serologic analyses

were conducted to detect antibodies to a combination of the following pathogens:

canine adenovirus (CAV), canine coronavirus, canine distemper virus (CDV),

canine herpesvirus (CHV) and feline calicivirus (FCV) by serum neutralization

(SN) assays, canine parvovirus (CPV-2) by hemagglutination inhibition test (HI),

feline herpes virus (FHV), feline coronavirus, feline immunodeficiency virus (FIV),

feline leukemia virus (FeLV) and Toxoplasma gondii by enzyme linked

immunosorbent assay (ELISA). Table 1 presents the viruses and parasites for

which exposure of introduced and endemic carnivores was tested along with their

mode of transmission and the cut-off values for each serological test. Levels of

antibody titers, indicative of prior exposure in euplerids are unknown; therefore,

we used a conservative approach and considered low positive titers as “suspect”.

Statistical analysis

Prevalence of antibodies were estimated for each pathogens and exact

binomial methods were used to calculate 95% confidence intervals (CI). When

animals were captured and sampled more than once (4 individuals), only data

from the first capture event were included in statistical analyses.

As host biology, habitat and behavior may influence exposure to

pathogens, associations between demographic and temporal variables and

antibody status of selected diseases were evaluated in fosa using logistic

62

regressions. We also assessed the association between presence of antibodies

to multiple pathogens as a marker of potential coinfection. Univariable logistic

regression models were constructed to investigate, separately, the association

between putative risk factors and the presence of antibodies to the pathogens.

The following variables were evaluated: age category (Juvenile, Subadult, Adult),

sex, location (ANP, KMNP), year of sampling, and presence of antibodies to

another virus or parasite. Only variables that showed an association (P<0.2)

were included in a multivariable model. Multivariable logistic regression models

were built employing a backward elimination approach (P>0.05 as rejection

criteria). The fit of the logistic regression model was assessed using Hosmer-

Lemeshow goodness of fit test (Hosmer Lemeshow 2000). From the final

models, the odds ratios (OR) and 95% CI were calculated for each predictor

variable to estimate the magnitude of its association with pathogen exposure.

Statistical analyses were conducted using the base package of R version 3.0.3

(R-Core Team 2014).

RESULTS

Animals sampled

A total of 240 animals from 6 species, namely domestic dog, domestic cat,

African wildcat, fosa, narrow-striped mongoose (Mungotictis decemlineata) and

Indian civets, were evaluated and sampled (Table 2). All animals appeared

healthy upon physical examination. Domestic cats and Indian civets were

sampled only in ANP and narrow striped mongooses were only trapped in

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KMNP. Not every animal was tested for antibodies to all pathogens due to limited

serum availability.

Antibody prevalences

Antibodies to Toxoplasma gondii were detected in all species but the

Indian civet and the overall prevalence was 67%. Dogs were also positive for

antibodies to CAV (14%), CDV (45%), CPV (67%) and CHV (20%).A large

proportion of fosa (42/45) presented antibodies to Toxoplasma, of which 33 had

high titers (>1024) suggestive of a recent or active infection. In addition, fosa

were seropositive to FCV (23%), CHV (9%) and CAV (4%) with maximum titers

detected 640, 60 and 48 respectively. Fosa also had low titers (suspected

positive) to CDV and CPV. One narrow striped mongoose was positive to canine

parvovirus.

No animal had detectable antibody to feline herpes virus or feline

coronavirus. The proportions of animals with positive or suspected positive for

antibody to each pathogen are presented in Table 3.

Risk factors for exposure to viral and parasitic diseases

Presence of antibodies to another pathogen as well as temporal,

environmental and demographic variables were considered as putative risk

factors for the presence of antibodies (high positive titers) to CAV, CHV, FCV

and Toxoplasma in fosa. Using univariable models, presence of antibodies to

Toxoplasma was associated with age (P=0.03) and presence of antibodies to

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FCV (P=0.17). Presence of antibodies to CHV was associated with the exposure

to FCV (P=0.07) and location (P= 0.18). No variables were associated with the

presence of antibodies to CAV.

Using multivariable logistic regression models, only age was retained as a

risk factor for the exposure to Toxoplasma (P=0.03). Adults were significantly

more likely to be exposed to Toxoplasma than immature individuals (OR=17.5).

Prevalence of antibodies to Toxoplasma in immature and adult fosa are

presented in Table 4. Prevalence of antibodies to Toxoplasma in both study sites

are presented in Table 5.

No variables were significantly associated with exposure to FCV and CHV.

DISCUSSION

This study evaluated the prevalence of antibodies to eight pathogens of

introduced and endemic carnivores living in or around two protected areas of

Madagascar. We show that these animals are exposed to a large array of

introduced pathogens. Regardless of species, age category, sampling location

and year, these carnivores presented antibodies to Toxoplasma (68%), canine

parvovirus (34%), canine distemper virus (22%), feline calicivirus (21%), canine

herpesvirus (13%) and canine adenovirus (8%). These pathogens are potential

threats to the conservation of endemic carnivores and may contribute to wild

carnivore population declines. Dogs and cats are often considered the primary

reservoir of these pathogens and a source of infection for wild animals. As such,

monitoring domestic dogs’ and cats’ exposure to these viruses and parasites is

65

an important first step to evaluate and manage the risks of disease transmission

across species (Cleaveland et al. 2001; Slater 2001; Belsare and Gompper

2014). Secondly, identifying risk factors in fosa can guide allocation of limited

resources for effective management and the conservation of this endangered

species.

Antibodies to Toxoplasma were detected in 68% of the sera including 93%

of fosa. Toxoplasma gondii is a zoonotic, globally distributed protozoan parasite

capable of infecting a wide range of animals including mammals and birds

(Tenter et al. 2000; Vitaliano et al. 2014). The only recognized definitive hosts of

Toxoplasma are domestic and wild felids, which shed the parasite in their feces.

Humans and animals are then infected through ingestion of infective forms of the

parasite from contaminated environment or through eating an infected prey item.

Alternatively, vertical transmission of the parasite can also occur (Miller et al.

2008; Calero-Bernal et al. 2013). Madagascar has no endemic felid and the

presence of Toxoplasma in free ranging fosa indicates parasite spillover from an

introduced cat species, either Felis catus or Felis silvestris. In a captive fosa, T.

gondii caused encephalomyelitis resulting in ataxia, muscular atrophy and

eventually the death of the animal (Corpa et al. 2013). However, the high

prevalence of antibodies to Toxoplasma detected in free ranging fosa at both

sites of this study suggests that Toxoplasma infection may not be universally

lethal in fosa.

Here, the presence of antibodies to Toxoplasma of fosa was significantly

associated with age. Adults (OR=17.5; P=0.03) were more likely to have

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antibodies to Toxoplasma than immature individuals (juveniles and subadults).

This finding is consistent with other studies that have investigated the effect of

age on the presence of antibodies to Toxoplasma in various wild animal species

(Åkerstedt et al. 2010; Garcıa-Bocanegra et al. 2010). This suggests horizontal

transmission of the parasite, which may occur through ingestion of an

intermediate host. The diet of free ranging fosa consists of a wide range of

animal species including the black rat (Rattus rattus) (Dollar et al. 2007), a

potential intermediate host of the parasite. Similar prevalence of antibodies in

fosa at both sites may result from a comparable environmental exposition due to

similar cat populations in the villages (Dollar, pers. Obs).

A large proportion of dogs (69%) were exposed to canine parvovirus.

Canine parvovirus is transmitted indirectly from environmental contamination and

via the fecal oral route. The virus is also highly resistant and can survive for

months in the environment. These characteristics may facilitate the persistence

of the virus in the environment and its transmission to wild carnivore population

(Fiorello 2004; Woodroffe and Donnelly 2011). Cross species transmission of

CPV-2 is well documented and antibodies to CPV-2 were found in several

species of Felidae, Canidae, Procyonidae, Mustelidae, Ursidae, and Viverridae.

Infection of free ranging wildlife was attributed to increasing population of

domestic dogs and habitat overlap between species. The high prevalence of this

pathogen in dogs from Madagascar could facilitate disease spillover from

domestic to endemic carnivores.

67

In this study, one giant striped mongoose was positive for antibody to

CPV-2 and 9% of fosa had low positive titers 1:10-1:20 anti-CPV-2. This shows

that euplerids may be exposed to a parvovirus but no clinical signs were detected

in the captured endemic carnivores. Pathogenic effects of CPV-2 in euplerids are

unknown but in other wild carnivore species, CPV may cause nonsuppurative

myocarditis in pups (Hayes et al. 1979; McCandlish et al. 1981) or

gastrointestinal signs in animals of all ages (Appel 1987). The absence of high

positive titers in fosa may indicate that most of the infected fosa do not survive

the infection by CPV-2, an inadequate diagnostic method or the lack of exposure

to fecal matters from infected dogs.

Similarly, canine distemper is arguably one of the most important viral

diseases affecting both domestic and wild carnivores worldwide (Deem et al.

2000; Timm et al. 2009; Terio and Craft 2013). It has caused morbidity and

mortalities in a wide range of captive and free ranging species including Canidae,

Felidae, Mustelidae, Ailuridae and Ursidae (Deem et al. 2000; Cottrell et al. 2013;

Seimon et al. 2013) and domestic dogs were identified or suspected as the origin

of infection. In fact, studies suggest that tiger populations are 25 times more

likely to go extinct if affected by canine distemper and the most significant factors

influencing infection were virus prevalence in the reservoir population (dogs) and

its contact rate with wildlife (Gilbert et al. 2014).

Here, we show that fosa only had low antibody titers (1:10) to CDV while

45% of the dogs tested had been exposed to CDV. The low titers detected in

fosa may be due to the persistence of maternally derived antibodies, an early

68

stage in seroconversion, waning titers from exposure to virus, cross reactivity, or

toxicity of the sample for the test cells. On the other hand, the lack of positive

titers (>1:16) to CDV in fosa may indicate: 1) an absence of exposure of fosa to

this directly transmitted virus; 2) a low survival rate of infected fosa; or 3) an

inadequate diagnostic method for canine distemper in this endemic carnivore.

The prevalence of antibodies to FCV in fosa from this study is similar to

the antibody prevalence in wildcats from Scotland, Saudi Arabia, and within the

range of seroprevalence found in hyenas from Tanzania (Daniels et al. 1999;

Ostrowski et al. 2003; Harrison et al. 2004). Feline Calicivirus is a RNA virus from

the family of Caliciviridae causing oral ulcers and upper respiratory tract disease

in affected cats but clinical signs in other taxonomic groups are unknown. This

virus has a short survival time in the environment and is transmitted through

direct contact via saliva and nasal secretion (Radford et al. 2007). It is not known

whether fosa become infected through contact with cats or if the virus may be

transmitted directly between fosa. No fosa exhibited signs consistent with

infection with FCV and further studies are required to monitor the exposure of

fosa to this virus and evaluate its ability to spread within the fosa population.

In conclusion, this study is the first to report the prevalence of antibodies

to common viral and parasitic pathogens in introduced and endemic carnivores in

Madagascar. The use of serological techniques in a cross sectional manner limits

us to discussing exposure to pathogens rather than current infection status.

However, these data provide a basis for monitoring the exposure of carnivores to

these pathogens and for assessing the risks of spillover among carnivores in this

69

biodiverse area. Although we cannot rule out the possibility of false positives and

cross-reaction, our results indicate that free-ranging fosa may be exposed to

several common pathogens of dogs and cats. Further studies using

metagenomic approaches for example, are needed to identify the specific strains

of viruses and parasites that are infecting domestic and endemic carnivores in

Madagascar (Bodewes et al., 2014). Similarly, long-term health monitoring that

may be based on “passive collection” coupled with population survey are needed

to evaluate the potential impact of diseases on wild fosa.

Acknowledgments:

We thank the Madagascar National Parks and the Autorité scientifique

committee (CAFF/CORE) for permission to conduct this research, the

Madagascar Institute for Conservation of Tropical Ecosystem for logistical

assistance in Madagascar, the Pittsburgh Zoo Conservation Fund and

Earthwatch Institute for their support of this research and the many Earthwatch

Institute volunteers who assisted in the field.

70

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Table 1: Selected pathogens tested in endemic and introduced carnivores of western Madagascar between 2002 and

2013, their methods of transmission, serological diagnostic method and positive cut-off values.

Pathogen Method of transmission Serological

diagnostic

Method1

Cutoff value

Suspected Positive

Canine adenovirus Exposure to body fluids; in utero SN 1:8 >1:16

Canine distemper virus Exposure to aerosolized body fluids SN 1:8 >1:16

Canine herpesvirus Exposure to body fluids; in utero SN 1:8 >1:16

Canine parvovirus Exposure to feces HAI 1:10 >1:20

Feline calicivirus Inhalation of aerosolized oronasal

and ocular secretions

SN 1:8 >1:16

79

Feline herpesvirus Inhalation of aerosolized oronasal

and ocular secretions

SN 1:8 >1:16

Feline coronavirus Exposure to feces, body fluids; in

utero

SN 1:8 >1:16

Feline immunodeficiency

virus

Bite wounds ELISA Positive/Negative

Toxoplasma gondii Ingestion of infected tissues or fecal

oocysts; in utero

ELISA 1:64

1: SN: Serum Neutralization, HAI: hemagglutination inhibition, ELISA: Enzyme Linked Immunosorbent Assay

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Table 2: Number of endemic and introduced carnivores captured by site and species in western Madagascar between

2002 and 2013.

Common Name

Species ANP1

KMNP

1

Domestic dog Canis familiaris 58 2

Fosa Cryptoprocta ferox 22 32

African wildcat Felis silvestris 27 6

Domestic cat Felis catus 80 0

Narrow-striped

mongoose

Mungotictis

decimlineata 0 6

Indian civet Viverricula indica 7 0

1ANP: Ankarafantsika National Park, KMNP: Kirindy Mitea National Park.

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Table 3: Proportion of samples with positive or suspected titers to selected

pathogens in different species of domestic and endemic carnivores in Western

Madagascar.

CAV2 CDV2 CHV2

Species1 n

Suspe

ct

(95%C

I)3

Positive

(95%CI)3 n

Suspect

(95%CI)3

Positive

(95%CI)3 n

Suspect

(95%CI)

3

Positive

(95%CI)

3

C.

familiaris

5

1 0

0.14

(0.06-

0.27)

4

9

0.14

(0.06-

0.28)

0.45

(0.31-

0.6)

5

0

0.06

(0.02-

0.18)

0.2 (0.1-

0.34)

Cr. ferox

4

4

0.02

(0-

0.12)

0.04

(0.01-

0.15)

4

3

0.07

(0.02-

0.2) 0

4

3

0.16

(0.07-

0.3)

0.09

(0.03-

0.23)

F.

silvestris 2 0 0 2 0 0 2 0 0

F. catus - - - - - - - - -

M.

decimlin

eata 5 0 0 5 0 0 6 0 0

V. indica 1 0 0 1 0 0 - - -

1

0

3

0.01

(0-

0.06)

0.08

(0.04-

0.16)

9

8

0.1

(0.05-

0.18)

0.22

(0.14-

0.32)

1

0

4

0.1

(0.05-

0.17)

0.13

(0.08-

0.22)

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CPV2 FCV2

Toxoplasm

a2

Species1 n

Suspect

(95%CI)3

Positive

(95%CI)3 n

Suspect

(95%CI)3

Positive

(95%CI)3 n

Positive

(95%CI)3

C.

familiaris

4

9 0

0.67

(0.52-

0.8) 2 0 0

1

6

0.88 (0.6-

0.98)

Cr. ferox

4

4

0.09

(0.03-

0.21) 0

4

4

0.21

(0.11-

0.36)

0.23

(0.13-

0.38)

4

2

0.93 (0.79-

0.98)

F.

silvestris 2 0 0

2

0

0.15

(0.04-

0.39)

0.35

(0.16-

0.59) 8

0.38 (0.1-

0.74)

F. catus - - -

6

1

0.21

(0.12-

0.34)

0.11

(0.05-

0.23)

1

8

0.22 (0.07-

0.48)

M.

decimlin

eata 5

0.6

(0.17-

0.93)

0.2

(0.01-

0.7) 5 0

0.4 (0.07-

0.83) 6

0.17 (0.01-

0.64)

V. indica 1 0 0 0 - - 1 0

1

0

1

0.07

(0.03-

0.13)

0.34

(0.25-

0.44)

1

3

1

0.19

(0.13-

0.27)

0.21

(0.14-

0.29)

9

1

0.67 (0.56-

0.76)

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1 C. familiaris: Canis familiaris, Cr ferox: Cryptoprocta ferox, F. silvestris: Felis

silvestris, F. catus: Felis catus, M. decimlineata: Mungotictis decimlineata, V.

indica: Viverricula indica. 2 CAV: Canine adenovirus, CDV: Canine distemper

virus, CHV: Canine herpesvirus, CPV: Canine parvovirus, FCV: Feline calicivirus,

Toxoplasma: Toxoplasma gondii. 3 95% CI: 95% Confidence interval

Table 4: Prevalence of antibodies to Toxoplasma and odds ratio in different age

categories of fosa.

Age

category n

Prevalence (95%

CI)1 OR (95% CI)1

Immature 6 0.67 (0.25-0.94) 1.00

Adult 38 0.97 (0.84-0.99) 17.5 (1.4 - 432.6)

Total 42 0.93 (0.79-0.98)

1 95% CI: 95% Confidence interval

Table 5: Prevalence of antibodies to Toxoplasma of fosa in two National Parks of

Madagascar.

Location n Prevalence (95% CI)1

ANP1 10 0.9 (0.54-0.99)

KMNP1 32 0.94 (0.78-0.99)

Total 42 0.93 (0.79-0.98)

1ANP: Ankarafantsika National Park, KMNP: Kirindy Mitea National Park

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Chapter 3: Patterns of exposure of carnivores to selected pathogens

in the Betampona Natural Reserve landscape, Madagascar.

Accepted for publication in Journal of wildlife diseases as: Rasambainarivo, F.,

Andriamihajarivo, M. N., Dubovi, E., & Parker, P. G. (2018). Patterns of Exposure

of Carnivores to Selected Pathogens in the Betampona Natural Reserve

Landscape, Madagascar. Journal of wildlife diseases.

ABSTRACT

Carnivores of Madagascar are at increased risk of extinction due to

anthropogenic loss of habitat, hunting, and interactions with introduced

carnivores.Interactions between introduced and native animals also present the

potential for introduction of pathogens into new geographic areas or host

species. Here, we provide serologic data regarding pathogen exposure of

domestic and native carnivores from a protected area in eastern Madagascar,

the Betampona Natural Reserve. For the Eupleridae, we found limited evidence

of exposure to viruses from domestic animals but greater prevalence for

Toxoplasma gondii (39%) and Leptospira spp. (40%). We also evaluated the

associations between the presence of antibodies to selected pathogens and

demographic and spatial variables. We showed that individual characteristics

such as sex and species are associated with exposure to Toxoplasma gondii, but

not Leptospira spp. or CPV. Finally, we investigated the spatial structure of

pathogen exposure in Betampona and found no evidence of spatial structuring,

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indicating the absence of hotspots and agent-free refugia for Toxoplasma gondii,

Leptospira spp. and CPV in the protected area. Our results may be useful for

assessing and monitoring disease risk and for formulating control strategies to

minimize the negative impact of exotic species on the endemic carnivores of

Madagascar.

Key words: Canine parvovirus, Eupleridae, introduced species, Leptospira spp.,

Madagascar, Toxoplasma gondii.

Introduction

Madagascar hosts 7 species of native carnivores from a single family, the

Eupleridae, that is arguably the least studied and most endangered carnivore

family (Yoder et al. 2003, Brooke et al 2014). In recent times, domestic dogs and

cats were introduced on Madagascar where they negatively impact native

carnivores through predation, competition or disease transmission (Farris et al.

2017).

Here, we provide data regarding pathogen exposure of carnivores living in

or near a 2,228 ha protected lowland rainforest in eastern Madagascar,

Betampona Natural Reserve (BNR) [Rendrirendry research station GPS

coordinates: -17.9315, 049.2033] (Figure 1). Because dogs and cats are

considered reservoirs of several pathogens of importance to wildlife, we assess

the prevalence of antibodies against selected pathogens in domestic carnivores

and sympatric Eupleridae.For the Eupleridae, we also investigate the existence

of variables associated with pathogen exposure. Because of species and sex-

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specific behavioral differences, we hypothesize that individual characteristics

influence exposure of Eupleridae to introduced animals’ pathogens. In western

Madagascar, adult fosa are more likely than subadults to be exposed to

pathogens (Pomerantz et al., 2016). Therefore, we expect similar findings in

Eupleridae species of BNR. Spatial variation in the frequency of interactions

between animals could also have implications for the transmission of pathogens

between species. Within BNR, interspecific interactions tend to occur near the

research station (Rasambainarivo et al., 2017), and we predicted pathogen

exposure to follow a similar trend and cluster in the same areas.

Materials and methods

Dogs and cats inhabiting the villages around the protected area were

sampled with the owners’ consent and vaccination histories of animals were

inquired through interviews. Endemic carnivores were captured and sampled

throughout the protected area following methods presented in Pomerantz et al.

(2016).

Serum samples were submitted to the New York State Animal Health

Diagnostic Laboratory (Ithaca, New York, USA) to detect antibodies against

seven pathogens including canine distemper virus, canine parvovirus, canine

adenovirus, canine herpesvirus, feline calicivirus, five serovars of Leptospira spp.

(hereafter Leptospira), and Toxoplasma gondii (hereafter Toxoplasma). For

Leptospira, the five serovars tested are usually associated with domestic animals

or introduced species in the region and may indicate cases of pathogen pollution

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89

(Desvars et al. 2014). We considered animals with titers higher than 1:100 for

one or more serovars as seropositive.

The prevalence of antibodies against each pathogen was estimated and

exact binomial methods were used to calculate 95% confidence intervals (CI). In

Eupleridae, the association between independent variables and the presence of

antibodies were evaluated using multiple logistic regressions employing a

stepwise backward elimination approach. The following variables were

evaluated: species, age category, sex, presence of antibodies to another

pathogen, and distances from the capture site to the: (1) edge of the forest, (2)

closest village, and (3) Rendrirendry research station. From the final models, the

odds ratios (OR) were calculated for each predictor variable.

The spatial distribution and clustering of exposure to pathogens was

assessed using two methods. First, we used Mantel tests to evaluate whether

pathogen exposure status was related to capture distance between hosts

(Gilbertson et al. 2016). Secondly, we assessed the presence of spatial clusters

of seropositive euplerids by using the Kulldorf spatial scan test (Kulldorff and

Nagarwalla 1995).

For spatial and non-spatial statistical analysis, we used 0.05 as an

indicator of statistical significance. We conducted the statistical analysis in R

(Team 2014) and used the package “ade4” to perform the Mantel tests. Cluster

detection was performed using the software satscan™ v.9.4.4(www.satscan.org).

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Results

We collected samples from 121 individuals from six species including both

species of domestic carnivores inhabiting five villages located near the protected

area and four Eupleridae species from BNR (Table 1). None of the domestic

animals were previously vaccinated.

Dogs had antibodies to canine distemper, canine herpesvirus, canine

parvovirus, feline calicivirus, Toxoplasma and Leptospira. In domestic dogs, the

serovar Grippotyphosa was the most commonly detected leptospiral serovar.

Eupleridae had antibodies to Leptospira, canine parvovirus and Toxoplasma

(Table 2). None of the six euplerids (two ring tailed vontsira, three broad striped

vontsira and one fosa) recaptured between 2015 and 2016 seroconverted for

Leptospira, Toxoplasma or canine parvovirus. Antibodies to Leptospira were

detected in 17/42 individuals (40%) from four species.

The presence of antibodies to Leptospira in Eupleridae was not

associated with any host characteristics or variables. Using multiple logistic

regression models, species and sex were retained as risk factors for the

exposure to Toxoplasma in Eupleridae (Table 3). There was no evidence of

spatial autocorrelation in exposure to Toxoplasma or Leptospira in BNR.

Discussion

This study evaluated the prevalence of antibodies to pathogens of

carnivores living in or near a protected area on Madagascar. First, we show that

domestic carnivores are exposed to pathogens including canine parvovirus,

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canine distemper, Leptospira and Toxoplasma that may spill over to endemic

carnivore species in Madagascar.

In BNR, two ring-tailed vontsira (8%) had antibodies against CPV;

however, none showed clinical signs consistent with parvovirosis at the time of

capture. This low prevalence of CPV in Eupleridae of BNR (3% overall; 95% CI:

1%-12%) is surprising given (1) that CPV-2 often crosses the species barrier

(Steinel et al. 2001); (2) the endemic status of the disease in dogs from

Madagascar; and (3) the frequent interactions between animals in BNR

(Rasambainarivo et al., 2017). It is possible that most Eupleridae exposed to

CPV do not survive infection or are unable to mount an immune response

following the exposure to CPV. It is also possible that the diagnostic method

used here is unable to detect antibodies to CPV in most Eupleridae.

Antibodies to Leptospira were found in (40%) of Eupleridae. The

serological tests suggest that euplerids in BNR are exposed to Leptospira and

particularly the serovar Icterrohemorraghiae. In Madagascar, this variant of

Leptospira is mainly spread by Rattus rattus while native Malagasy mammals

harbor unique species of Leptospira (Dietrich et al. 2014,). Since endemic

carnivorans prey upon both the introduced and endemic rodents, the Eupleridae

may be exposed to several species of Leptospira. It would be important to

determine whether the native species of Leptospira also occur in euplerids and

whether they may constitute a reservoir for this potentially zoonotic pathogen.

Our results show that exposure to Toxoplasma is prevalent among native

carnivores in BNR. Since no felid species are native to Madagascar, the

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presence of Toxoplasma antibodies in Eupleridae indicates a spillover of the

pathogen from cats to euplerids. This parasite was associated with neurological

disease and death in a fosa, and may affect wild euplerid populations (Corpa et

al. 2013).

At this site, 85% of fosa were previously exposed to Toxoplasma, a

prevalence similar to that found in western Madagascar (Pomerantz et al. 2016).

Our results also indicate differences between Eupleridae species in their

exposure to Toxoplasma, which could be due to differences in ranging patterns

or in the diet of the carnivores. Research suggests that the fosa and ring-tailed

vontsira are more flexible regarding habitat degradation and more likely to occur

closer to villages compared to the broad-striped vontsira (Kotschwar Logan et al.

2015). The fosa and ring-tailed vontsira may be more likely to acquire this

parasite in these human-dominated habitats. Regarding diet, all three species

feed on a wide variety of vertebrate and invertebrate species. The fosa and ring-

tailed vontsira tend to prey mainly upon mammals and other vertebrates, while

the broad- striped vontsira preys on species with body masses below 10g, mainly

invertebrates (Hawkins and Racey 2008, Andriatsimietry et al. 2009). This could

influence their exposure to trophically transmitted pathogens.

We found that the prevalence of antibodies to Toxoplasma was higher in

male Eupleridae when adjusting for species. This could be due to sex-specific

behavioral differences. Male fosa and broad-striped vontsira have larger home

ranges than females (Marquard et al. 2011, Lührs et al. 2013). In other

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ecosystems, animals with larger home ranges were associated with higher

prevalence of antibodies to Toxoplasma (Fredebaugh et al. 2011).

Contrary to our expectation, age was not significantly associated with

exposure to Toxoplasma. The absence of statistical association could reflect

biological processes such as the potential vertical transmission of the parasite in

these species or may result from the small sample size. Further research is

required.

Finally, our results indicate that, at this study site, there was no evidence

of spatial autocorrelation or significant clusters of pathogen exposure in

Eupleridae. This suggests that Toxoplasma and Leptospira have spread

relatively evenly in BNR and that the probability of exposure to these pathogens

is similar throughout the protected area. Our inability to detect spatial clustering

of pathogen exposure may also be due to the diagnostic tests employed here.

Further studies examining the spatial distribution of Toxoplasma oocysts and

Leptospira bacteria contamination in prey and the environment would be valuable

to identify areas of greater infection probability.

In conclusion, this study revealed that the Eupleridae of Betampona are

exposed to pathogens commonly carried by domestic animals. We also showed

that individual characteristics are associated with exposure to the felid parasite

Toxoplasma. Continued monitoring of pathogen exposure in endemic carnivores

is needed to determine the long-term effect of these pathogens on population

persistence.

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giant-striped mongoose Galidictis grandidieri Wozencraft 1986 in the sub-

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We would like to thank the Madagascar National Parks (MNP) and the

Autorité scientifique committee of Madagascar (CAFF/CORE) for permission to

conduct this research, the Madagascar Institute for Conservation of Tropical

Ecosystem (MICET) and the Madagascar Fauna and Flora Group for logistical

assistance in Madagascar, Natacha Rasolozaka, Hertz Andrianalizah, Victorice,

Flavien and Emilie Ribault for field assistance. We are grateful to Ingrid Porton

and two anonymous reviewers who provided valuable comments and

suggestions on an earlier version of the manuscript. This project was funded in

part by the Saint Louis Zoo Field Research for Conservation Program, The

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Whitney Harris World Ecology Center, The Madagascar Fauna and Flora Group

and the Rufford Small Grant Foundation.

Table 1: Number of endemic and domestic carnivores sampled by sex and age category in Betampona Madagascar

between 2014 and 2016.

Sex Age category

Common name Species n Females Males Adult Subadult

Domestic dog Canis familiaris 48 19 29 36 12

Domestic cat Felis catus 9 4 5 7 2

Ring-tailed vontsira Galidia elegans 26 16 10 21 5

Broad striped vontsira Galidictis fasciata 28 15 13 25 3

Brown tailed vontsira Salanoia concolor 3 2 1 3 0

Fosa Cryptoprocta ferox 7 2 5 5 2

Table 2: Proportion of samples with positive titers to selected pathogens in different species of domestic and endemic

carnivores in Betampona, Madagascar.

CAV2 CDV2 CHV2

Species1 n Positive

(95%CI)3 n Positive (95%CI)3 n

Positive

(95%CI)3

C. familiaris 48 0.04 (0.01-0.15) 48 0.13 (0.05-0.26) 47 0.02 (0.00-0.13)

F. catus - - - - - -

G. elegans 25 0.00 (0.00-0.17) 25 0.00 (0.00-0.17) 20 0.00 (0.00-0.20)

G. fasciata 26 0.00 (0.00-0.16) 27 0.00 (0.00-0.15) 17 0.18 (0.05-0.44)

S. concolor 3 0.00 (0.00-0.69) 3 0.00 (0.00-0.69) 3 0.00 (0.00-0.69)

Cr. ferox 7 0.00 (0.00-0.43) 7 0.00 (0.00-0.43) 7 0.00 (0.00-0.43)

109 0.01 (0.00-0.06) 110 0.05 (0.02-0.12) 94 0.04 (0.01-0.11)

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CPV2 FCV2 Toxoplasma Leptospira

Species1 n Positive

(95%CI)3 n

Positive

(95%CI)3 n

Positive

(95%CI)3 n

Positive

(95%CI)3

C. familiaris 48 0.56 (0.41-0.70) - - 5 0.80 (0.30-0.99) 47 0.19 (0.10-0.34)

F. catus - - 7 0.43 (0.12-0.80) 6 0.83 (0.36-0.99) 2 0.50 (0.10-0.94)

G. elegans 25 0.08 (0.01-0.28) 25 0.00 (0.00-0.17) 25 0.48 (0.28-0.68) 17 0.47 (0.24-0.71)

G. fasciata 27 0.00 (0.00-0.15) 27 0.00 (0.00-0.15) 27 0.19 (0.07-0.39) 16 0.38 (0.16-0.64)

S. concolor 3 0.00 (0.00-0.69) 3 0.00 (0.00-0.69) 3 0.00 (0.00-0.69) 2 0.50 (0.10-0.94)

Cr. ferox 7 0.00 (0.00-0.43) 7 0.00 (0.00-0.43) 7 0.86 (0.42-0.99) 7 0.43 (0.12-0.80)

Total 110 0.26 (0.19-0.60) 69 0.04 (0.01-0.13) 73 0.44 (0.32-0.56) 91 0.31 (0.22-0.41)

1 C. familiaris: Canis familiaris, Cr ferox: Cryptoprocta ferox, F. catus: Felis catus, G. elegans: Galidia elegans, G. fasciata: Galidictis

fasciata 2 CAV: Canine adenovirus, CDV: Canine distemper virus, CHV: Canine herpesvirus, CPV: Canine parvovirus, FCV: Feline

calicivirus, Toxoplasma: Toxoplasma gondii. Leptospira: Leptospira spp. (serovars: canicola, pomona icterohemorhagiae, hardjo,

grippotyphosa)3 95% CI: 95% Confidence interval.

Table 3: Significant variables (multivariate logistic regression) associated with exposure to Toxoplasma gondii in endemic

carnivores from Betampona, Madagascar.

Variable

P-

value

OR 95% CI

Sex 0.03

Female 1.00

Male 4.13 (1.16-14.78)

Species1 0.003

G. fasciata 1.00

Cr. ferox 24.55 (2.19-275.47)

G. elegans 5.07 (1.31-19.58)

1Cr. ferox: Cryptoprocta ferox, G. elegans: Galidia elegans, G. fasciata: Galidictis fasciata. Due to the small sample size (n=3)

and lack of variation (all negative), S. concolor were not included in logistic regression analyses.

Figure 1: Capture locations of three species of Eupleridae and their status regarding the presence of antibodies against

Toxoplasma gondii within the Betampona Natural Reserve, Madagascar between 2015 and 2016.

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Chapter 4: Sharing is not caring, microbial strain-sharing network among

carnivorans in Betampona, Madagascar.

Fidisoa Rasambainarivo1,2,4 and Patricia G. Parker1,3

1University of Missouri Saint Louis, One University Blvd, Saint Louis MO, 63121,

USA; 2 Madagascar Fauna and Flora Group, BP 442 Toamasina, Madagascar;

3Saint Louis Zoo One Government Drive Saint Louis, MO, 63110, USA

4corresponding author: [email protected]

Abstract

Interactions between domestic animals and wildlife are increasing in the vicinity

of natural habitats and may facilitate the emergence of diseases affecting both

the human and animal populations. Identifying species or groups of individuals

that play a disproportionately central role in the transmission of pathogens within

and between populations of animals could help inform disease mitigation and

conservation strategies. Here, we use network analysis techniques and microbial

genetic tools to uncover the patterns of microbial sharing among sympatric

species of endemic and introduced carnivorans in and around a protected area of

Madagascar. We show that despite a tendency of animals to share microbial

strains with their conspecifics, interspecific microbial exchanges were frequent.

Among the carnivorans inhabiting Betampona, fosa (Cryptoprocta ferox) and

broad-striped vontsira (Galidictis fasciata) shared microbial strains with

significantly fewer animals than dogs. Despite their large home range and

propensity to use both natural and human dominated habitats, fosa were not

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more likely to constitute a bridge between the domestic and native carnivorans.

Taken together, our results suggest that the introduction of a pathogenic agent

that is environmentally transmitted in this community could affect all carnivorans

and underline the importance of animal health management and disease

surveillance.

Introduction

As a result of the increase in human population size near biodiverse

habitats, domestic animals are encroaching into natural areas and interacting

with wild animals. These interactions may facilitate the emergence of wildlife-

borne infectious diseases affecting both humans and livestock or the spillover of

pathogens from domestic to wild animals (Daszak et al. 2000; Landaeta-Aqueveque

et al. 2014; Johnson et al. 2015; Cunningham et al. 2017). For example, outbreaks of

rabies and canine distemper viruses from domestic dogs have led to dramatic

declines in the population size of endangered wild dogs and lions of the

Serengeti Natural Park, while the emergence of Nipah virus transmitted from

bats caused morbidity and mortality in both humans and animals (Roelke-

Parker et al. 1996; Plowright et al. 2015). These disease outbreaks have

prompted numerous research programs and conservation actions to study the

dynamics of disease transmission and to control the spread of the pathogens

(Prager et al. 2012; Belsare and Gompper 2015) .

Often times, however, these studies use classical mathematical models

that assume a homogeneous mixing of individuals and constant transmission

rates to estimate the basic reproductive number of the pathogen and the

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proportion of individuals to vaccinate (Anderson and May 1991). In reality,

heterogeneities exist and individuals vary in their contact rates and therefore

their potential for acquiring and transmitting a pathogen. Understanding and

taking into account the differences in contact rates between individuals may be

useful to develop efficient disease control strategies and improve our ability to

predict the spread of pathogens in a community (Lloyd-Smith et al. 2005;

Rushmore et al. 2013, 2014, 2017). For instance, contact network analysis was

recently used to demonstrate the particular role of male European badgers in

the transmission of Mycobacterium bovis among conspecifics (Silk et al. 2018).

In addition to studying contact and interactions between individuals to

estimate the potential for pathogen transmission, researchers have used the

genetic fingerprinting patterns of microbes to effectively track the transmission

pathways of bacteria among individuals. The combined use of network analysis

and genotyping methodologies further improves our knowledge of disease

dynamics within and between species and provides policy makers with precise

and actionable recommendations to limit the spread of pathogens (Pesapane et

al. 2013; Blyton et al. 2014; VanderWaal et al. 2014).

For mammals, Escherichia coli constitutes an ideal surrogate to model the

transmission of a pathogen transmitted fecal-orally. Specifically, commensal E.

coli is a facultative, anaerobic and mostly non-pathogenic bacterium that is

highly prevalent in the gastrointestinal tracts of mammals and with a genetic

diversity that is sufficient to capture inter-individual variation (Sears et al. 1950;

Gordon and Cowling 2003; Tenaillon et al. 2010). Thus, if individuals have

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genotypically similar E. coli, they are likely to have either transmitted the strain

of microbe between one another via fecal-oral contact or acquired it through a

common environmental source (Vanderwaal et al. 2013; Blyton et al. 2014;

Chiyo et al. 2014; VanderWaal et al. 2014; Springer et al. 2016).

Repetitive extragenic palindromic-PCR (rep-PCR) is a genotypic method

that uses oligonucleotide primers complementary to amplify the repetitive

sequences dispersed throughout the genome of E. coli (Versalovic et al. 1994).

Rep-PCR produces amplicon patterns that are specific for an individual E. coli

strain with high discriminatory power between different subtypes and is used to

identify strain sharing between individuals (Rademaker and De Bruijn 1997;

Goldberg et al. 2008; Rwego et al. 2008; Blyton et al. 2014; VanderWaal et al.

2014; Naziri et al. 2015). BOX-PCR is one of the many rep-PCR techniques and

is one of the commonest methods used to identify sources of fecal

contamination (Dombek et al. 2000; Cesaris et al. 2007; Mohapatra et al. 2007;

Somarelli et al. 2007; Ma et al. 2011).

In this study, we constructed and assessed the characteristics of a

microbial sharing network among sympatric endemic and introduced

carnivorans in and around a protected area of Madagascar, the Betampona

Natural Reserve. The carnivoran community of Betampona is comprised of five

species of carnivorans that belong to a single endemic carnivoran family, the

Eupleridae, and three species of introduced species, domestic dogs, domestic

cats and Indian civet (Rasambainarivo et al. 2017). Similar to other parts of the

island, recent research in Betampona indicates that the probability of occupancy

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and the distribution of Eupleridae are negatively affected by the presence of

introduced dogs and cats (Farris et al. 2015, 2017; Rasambainarivo et al. 2017).

This decline in population size may be due to predation and competition, but

also disease transmission. In fact, previous research indicates that the

endangered carnivorans of Betampona may acquire a variety of pathogens

such as canine parvovirus, Leptospira interrogans and Toxoplasma gondii from

these introduced species (Pomerantz et al. 2016; Rasambainarivo et al. 2018).

Identifying species or individuals that are central to the transmission of

microbes within the carnivoran community of Betampona may help in disease

management and to design effective mitigation and monitoring strategies. Our

first objective was to uncover the microbial strain-sharing network of an

environmentally transmitted microbe between carnivorans in the Betampona

Natural Reserve landscape. Second, we wanted to identify species or groups of

individuals that may play a central role in the transmission of microbes between

carnivorans. Because fosa (Cryptoprocta ferox) have the largest home ranges

among the carnivorans of Betampona and it is likely to span the protected area

and human –dominated habitats (Lührs and Kappeler 2013; Lührs et al. 2013),

we predicted that fosa would act as a bridge between different groups and

species of carnivorans inhabiting the larger BNR landscape. Third, we aimed to

identify characteristics that would influence the probability of sharing a microbial

subtype. We wanted to assess whether individuals of specific species, age

category or sex would be linked to more individuals on the microbial strain-

sharing network than others and whether animals of the same age category,

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sex or species were more likely to share microbial strains. Because of

behavioral differences among species and the higher likelihood of intraspecific

social interactions, we hypothesized that dogs would share microbial subtypes

with other individuals compared to other species and, individuals of a species

would tend to share microbial subtypes with individuals of their own species

rather than with individuals of other species. Similarly, because of widespread

male bias in the exposure of pathogens, we predicted that, regardless of

species, males would be more likely to play a central role in the microbial

sharing network of the carnivorans of Betampona (Perkins et al. 2008; Guerra-

Silveira and Abad-Franch 2013; McDonald et al. 2014).

Materials and methods

Study site

Betampona Natural Reserve is located approximately 40 km northwest of

Toamasina in Eastern Madagascar (Rendrirendry research station GPS

coordinates: -17.9315, 049.2033). BNR is a 2,228 ha lowland rainforest with a

mean elevation of 270m (range 92-571m), which consists of dense primary

forest and lined by secondary forest at the borders, characterized by the

invasion of exotic guava tree (Psidium cattleianum) or the Molluca Raspberry

(Rubus mollucanus) (Ghulam 2014; Ghulam et al. 2014). Betampona is a strict

nature reserve managed by the Madagascar National Parks, and the

Madagascar Fauna and Flora Group acts as the research partner in the

reserve. Access of tourists and villagers is not permitted and only a small

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number of scientists and staff members from these institutions (fewer than 30)

have access to the protected area.

The reserve is isolated from any other forest patch and is surrounded by

agricultural areas and human habitation. Each of the eight villages in the vicinity

of the reserve (less than 1km from the border) is composed of 20-50

households on average with a total population of 80-200 people per village. The

main activity of villagers in the region is agriculture and the main crop is rice and

to a lesser extent maize, sugar cane, cloves, coffee, banana and manioc. Most

people raise poultry outdoors, pigs and dogs are common in the region and all

are free ranging.

Sample collection

Fecal samples of carnivorans were collected when the animals were

captured as part of an ongoing health evaluation program (Rasambainarivo et

al. 2018), insuring correct identification of the individual and avoiding sample

duplication. All animal handling procedures were conducted according to IACUC

guidelines (University of Missouri St Louis and Saint Louis Zoo) and under

permits issued by the Madagascar National Parks. Because of the potential for

frequent temporal turnover of E. coli strains in an individual, we only considered

carnivorans sampled within a eight week period (November-December 2016)

(Blyton et al. 2013). Fecal samples were collected from naturally voided feces

immediately after defecation or directly from the rectum using a sterile swab.

Samples were stored in sterile tubes that were frozen within 8h of collection until

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further processing. Fecal samples were then homogenized and streaked onto

MacConkey Agar plates. After incubation for 24 hours at 37 C, a maximum of 12

putative E. coli colonies from each plate were randomly selected and grown in

tryptic soy broth for 24 hours at 37 C. DNA was extracted from cultured cells

using Macherey Nagel kits following the manufacturer’s instructions and genetic

subtypes were determined using BOX-PCR and gel electrophoresis.

Similarity of isolates was determined through pairwise comparison of the

densitometric curves generated for each isolate. Isolates were considered to be

matching subtypes if their densitometric curves were >90% similar (Pearson’s

correlation coefficient). Analysis of gel electrophoresis images were performed

using GelJ (Heras et al. 2015)

Data analysis

A microbial strain-sharing network was constructed where each sampled

carnivoran was represented as a node and two nodes were linked by an edge if

they shared at least one E. coli subtype. Edges were undirected and

unweighted meaning that the direction of microbial strain-sharing network

between the two individuals is unknown and two nodes were linked regardless

of the number of strains shared between the individuals. For each individual, we

calculated the betweenness centrality measure, which is loosely defined as the

number of shortest paths (geodesics) between every other pair of individuals

that pass through the focal individual. Betweenness is assumed to indicate an

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individual’s propensity to act as a bridge and transmit a microbe between

groups of otherwise unconnected individuals.

Because of the inherently relational type of observation in a network

analysis and the violation of assumption of independence, generalized linear

mixed models cannot be used to test hypotheses (James et al. 2009; Silk et al.

2017).

To identify the prominence and influence of key attributes associated with

sharing at least one E. coli subtype, we built a series of exponential random

graph models (ERGM). ERGMs model the probability of each edge or link in the

network as a function of network structure and the characteristics of the

individuals within the network. Using ERGM, the unit of analysis is a dyad of

carnivorans and the dependent variable is whether or not the two individuals

share a microbial subtype. Therefore, ERGMs are suited to address questions

related to the number of interactions that an individual has (degree) and the

homophily (are within-sex/species interactions more likely to occur than

between-sex/species ones) (Robins et al. 2007; Robins 2013; Silk and Fisher

2017). Here, we used ERGMs to determine whether individuals of certain

species, age or sex tend to have more links and whether belonging to the same

species, age or sex as well as the geographical distance separating two

individuals influenced the probability that they would share at least one

microbial strain. Model selection was based on the smallest AIC (Akaike

Information Criterion) value. Model convergence was checked by visual

inspection of Monte Carlo Markov Chains for parameter estimates.

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Using the microbial sharing network developed here, we simulated the

introduction and spread of a hypothetical pathogen that has low, intermediate or

high transmissibility and epidemic potential. The transmissibility of a pathogen is

often characterized through the basic reproductive number R0, which is defined

as the average number of secondary infections produced when one infected

individual is introduced into a host population where everyone is susceptible

(Anderson and May 1992). For the simulations we varied the rate at which

individuals get infected (infection rate: b) and the rate at which individuals

recover from the infection (recovery rate: g) such that R0=b/g varied between

0.125 and 5, to reflect the transmissibility estimates of an environmentally

transmitted pathogen such as Cryptosporidium sp. (Guerrant, 1997; Breban et

al., 2007; Thien et al., 2010). For each combination of transmission and

recovery rate, simulations were run 1000 times and each time, a randomly

selected individual was infected.

All statistical analyses were performed using R (Team 2014). The

microbial strain-sharing network was constructed using the ‘igraph’ package and

ERGMs were built using the ‘ergm’ package in R (Csardi and Nepusz 2006;

Hunter et al. 2008).

Results

During the sampling period, we sampled 79 individual carnivorans from 6

species. Table 1 shows the number of carnivorans captured and sampled from

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each species, sex and age category. From each fecal sample, an average of 10

(range 4-12) E. coli isolates were cultured and genotyped using BOX-PCR

methods and a total of 196 unique E. coli DNA fingerprints were identified. On

average, each carnivoran harbored 2 different E.coli subtypes (range : 1-6).

The network of E.coli subtype sharing between the carnivorans of

Betampona was drawn where each individual animal was represented as a

node and two individuals were linked if they shared at least one E. coli subtype

(Figure 1).

Network measures

The density of the network was 0.18, meaning that out of all links possible

among the individuals in the network, 18% were realized. The mean degree in

the microbial sharing network is 14. In other words, on average, each

carnivoran in the BNR landscape shared at least one E.coli subtype with 14

other individual carnivorans (range 0-41). The degree distribution of the

microbial sharing network is presented in Figure 2 and shows a skewed

distribution of degree where a limited number of individuals are sharing

microbial subtypes with a large number of carnivorans in the landscape.

Visual assessments of the effect of species identity on betweenness measures

suggest that there were no differences among species in the betweenness

centrality measures (Figure 2).

Exponential Random Graph Models

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The best exponential random graph model indicates that species and sex

were significantly associated with the probability that an individual shares a

microbial subtype with another carnivoran. This model also includes a term

related to the probability of sharing microbial subtypes with a conspecific. The

parameter estimates of the most supported ERGMs are presented in Table 2.

Compared to domestic dogs, fosa and broad-striped vontsira (Galidictis

fasciata) were less likely to share microbial subtypes with another carnivoran

(Odds Ratio=0.50 and Odds Ratio=0.75 respectively). Males were more likely to

share a microbial subtype with another carnivoran than were females. The

significant and positive coefficient for species-match indicates that animals are

more likely to share at least one E. coli subtype with individuals of their own

species than individuals from other species (Odds Ratio=1.7, p<0.01). The

Goodness of fit test of the model and visual exploration of Markov chain Monte

Carlo indicate a good fit of the model.

Disease simulation

Graphical representation of the SIR (Susceptible- Infected- Recovered)

model simulations are presented in Figure 4. These simulations estimate the

number of infected individuals following the introduction of a pathogen (with

varying transmissibility and recovery rates) in this community.

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Discussion

We explored the characteristics of a microbial sharing network among 79

sympatric carnivorans in the Betampona Natural Reserve landscape,

Madagascar to evaluate the potential for pathogen spread in the community.

We show that the microbial sharing network of carnivorans is highly connected

and only a very limited number of individuals (n=2) do not have at least one E.

coli subtype in common with another carnivoran. These results suggest that,

should a fecal-orally transmitted pathogen capable of infecting and being spread

by all carnivoran species be introduced within the BNR landscape, it could affect

most, if not all, of the carnivorans (Figure 3). In addition, the network structure

presents a heterogeneous degree distribution which is often associated with

disease outbreaks of short duration (Sah et al. 2017). This is further highlighted

through the SIR model simulations (Figure 3), which indicate that even medium

to highly transmissible pathogens would quickly spread in the community and

lead to large outbreaks of short durations. Identifying the individuals that

constitute a bridge between groups or are central to the network would be

important to design cost-effective disease mitigation strategies.

There were no statistically significant differences in the betweenness

centrality measures of carnivorans, meaning that no species are more likely

than others to act as a bridge between groups of unconnected individuals. This

is contrary to our prediction and may result from the frequent and widespread

activity of dogs in the Betampona Natural Reserve and the ability of other

Eupleridae such as the ring-tailed vontsira to use human dominated habitats

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(Logan et al. 2014; Rasambainarivo et al. 2017). Both of these behavioral

patterns may facilitate frequent indirect and interspecific interactions and

potentially the transmission of pathogens among the different species

(Rasambainarivo et al. 2017).

The results of the ERGM show differences among sex and species in the

number of interactions with other individuals. Males have more connections

than females and may be more likely to acquire and transmit a pathogen to a

larger number of individuals compared to females. Male-biased differences in

exposure and susceptibility to diseases are common in both humans and

animals (Guerra-Silveira and Abad-Franch 2013; Mcdonald et al. 2014), which

could result from physiological differences, an increase in contact rates or

differences in ranging patterns (Ferrari et al. 2004; Mougeot et al. 2005; Grear

et al. 2009). For instance, analyses of badger social networks indicate an effect

of sex in between-group interactions but also in the probability of infection with

bovine tuberculosis (Weber et al. 2013; Silk et al. 2018). In the carnivoran

species evaluated here, home range sizes differ between the sexes

(Andriatsimietry et al. 2009; Marquard et al. 2011; Lührs and Kappeler 2013;

Lührs et al. 2013). In both endemic and introduced carnivorans, males tend to

have a significantly larger home range and consequently may be in contact with

fecal matter from more individuals but also disperse fecal-borne bacteria on a

wider geographical scale than females (Lührs et al. 2013; Hall et al. 2016;

Hudson et al. 2017).

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Dogs have significantly more connections than fosa and broad-striped

vontsira. This may be due to behavioral differences related to the sociality of the

carnivoran species. Fosa are considered a solitary species with infrequent

male-male associations and the broad-striped vontsira is a nocturnal species

with individuals that live in pairs or small family groups on the interior of

protected areas (Lührs and Kappeler 2013; Lührs et al. 2013; Schneider and

Kappeler 2013). By contrast, the sociality of domestic dogs is highly variable

and they can live at diverse levels of social organization, from singly in

households to transient groups and packs when free roaming (Serpell, 1995). In

both of the latter cases, free roaming dogs tend to have a high direct and

indirect contact rate with other dogs. A study estimated that, within a 24-hour

period, a free roaming dog could have up to 147 direct contacts with more than

40 other dogs (Bombara et al. 2017). In other species, social interactions were

an important predictor of microbial sharing among individuals and influenced the

number of animals with which an individual shares microbial subtypes

(Vanderwaal et al. 2013; Blyton et al. 2014; Springer et al. 2016).

Our results also indicate that there is homophily among species in the

microbial sharing network. Individuals of the same species were almost twice as

likely to share a microbial subtype than individuals of different species (Odds

Ratio=1.7). This result mirrors findings of microbial subtype sharing in ungulates

of Kenya and could reflect similarities in the space use, habitat preference and

physiological similarities of conspecifics (VanderWaal et al. 2014). Also, the

species homophily in microbial sharing among sympatric carnivorans could be

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119

explained by intraspecific behavioral patterns. Scent marking with feces is an

important form of intraspecific communication of a large variety of animal

species and is used to mark territory boundaries, select and defend potential

mates, signal status or note the availability of resources (Zala 2004; Jordan et

al. 2010). Therefore, it is reasonable to think that conspecifics are more likely to

investigate fecal deposits than individuals of other species and thus acquire the

same microbial subtypes. However, the use of scent marking for interspecific

communication was demonstrated in certain species and may lead to

interspecific disease transmission (Allen et al. 2017). The frequency of

interspecific fecal scent-mark signaling and its potential implication for pathogen

transmission in Eupleridae and other mammalian species requires further

research.

Here we used E. coli as a marker of microbial transmission between

carnivorans in a protected area of Madagascar. Although our results clearly

indicate widespread sharing of microbes and other potential pathogens between

animals, the derived strain-sharing network should be utilized with caution to

model the transmission of other environmentally transmitted pathogens and to

manage the potential disease transmission between individuals. First, E. coli is

a commensal and mostly non-pathogenic bacterium, while it is generally

acknowledged that behavioral changes caused by a diseased state may modify

contact patterns between individuals and therefore transmission patterns of

diseases (Lopes et al. 2016). It is possible that the introduction of a pathogenic

agent in the ecosystem would affect the behavior, mating and ranging patterns

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120

of animals and therefore the spread of the disease. Secondly, the network

drawn here is static, as it represents only the interactions between carnivorans

inhabiting the landscape during a single time period. Because social interactions

and disease transmission between animals have an inherent time component,

studying the network of interactions over time may provide us with more precise

estimates and robust conclusions on the risks and rates of pathogen

transmission (Springer et al. 2016a; Farine 2017, 2018). In several species of

Eupleridae, animal contact and ranging patterns are highly seasonal due to the

mating behavior of the species and the availability of resources (Hawkins and

Racey 2009; Schneider and Kappeler 2013; Schneider et al. 2016). Therefore,

analysis of microbial sharing network among carnivorans inhabiting the BNR

over multiple seasons would be important to study the temporal dynamics of

disease spread in this community and inform conservation strategies.

In conclusion, this study highlights the potential for disease transmission

between introduced and native carnivorans of Madagascar. By using network

analysis and microbial genetics, we show an extensive exchange of microbes

among all species of carnivorans in the landscape. While individuals are more

likely to share a microbe with conspecifics, interspecific microbial exchanges

occur very frequently. This suggests that the introduction of a pathogenic agent

that is environmentally transmitted in this community could affect all carnivorans

and our results underline the importance of animal health management and

disease surveillance.

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121

Acknowledgments

We would like to thank the Madagascar National Parks (MNP) and the Autorité

scientifique committee of Madagascar (CAFF/CORE) for permission to conduct

this research, the Madagascar Institute for Conservation of Tropical Ecosystem

(MICET) and the Madagascar Fauna and Flora Group for logistical assistance in

Madagascar, Natacha Rasolozaka, Mamy Navalona Andriamihajarivo, Hertz

Andrianalizah, Victorice, Flavien and Emilie Ribault for field assistance. This

project was funded in part by the Saint Louis Zoo Field Research for

Conservation Program, The Whitney Harris World Ecology Center, The

Madagascar Fauna and Flora Group and the Rufford Small Grant Foundation.

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dog

ring−tailed vontsira

broad striped vontsira

fosa

Male

Female

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Figure 1: E.coli strain sharing network among sympatric introduced and

endemic carnivorans of Betampona, Madagascar. Shapes represent carnivoran

species, colors represent the sex of the individual and lines represent similarity

(>90%) in DNA fingerprints of at least one E.coli.

Figure 2: Distribution of the number of individual with whom a carnivoran shares a microbial subtype in Betampona,

Madagascar.

Distribution of the number of connexion (degree)

Degree

Num

ber o

f ind

ivid

uals

0 10 20 30 40 50

05

1015

20

137

137

Figure 3: Boxplot of the betweenness centrality measures of carnivorans of Betampona according to species.

Canis familiaris Cryptoprocta ferox Felis catus Galidia elegans Galidictis fasciata

0

50

100

150

200

250

Species

Betw

eenn

ess

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Figure 4: Simulation of disease spread in the carnivoran community using a SIR (Susceptible-Infected-Recovered) model.

Simulations were run 1000 times while varying R0=b/g where b represents the transmission rate of the pathogen and g the

recovery rate. The median number of infected individuals over time is represented (bold solid line) as well as the 10th and

90th quantiles (dashed lines).

0 1 2 3 4 5 6

020

4060

80

Time

NI(t)

R0=0.125

0 1 2 3 4 5 6

020

4060

80Time

NI(t)

R0=0.25

0 1 2 3 4 5 6

020

4060

80

Time

NI(t)

R0=0.5

0 1 2 3 4 5 6

020

4060

80

Time

NI(t)

R0=1

0 1 2 3 4 5 6

020

4060

80

Time

NI(t)

R0=2

0 1 2 3 4 5

020

4060

80

Time

NI(t)

R0=5

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Table 1: Number of carnivorans sampled in the Betampona Natural Reserve landscape and from which E. coli could be

cultured.

Female Male Adult Subadult Total

Dog (Canis familiaris) 15 18 27 6 33

Cat (Felis catus) 2 1 2 1 3

Fosa (Cryptoprocta ferox) 3 4 5 2 7

Ring-tailed vontsira (Galidia elegans) 7 5 11 1 12

Broad-striped vontsira (Galidictis fasciata) 12 12 21 3 24

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Table 2: Result of ERGM model predicting microbial sharing among carnivorans in the Betampona Natural Reserve

landscape.

Estimate Std. Error p-value

Sex (Male) 0.18 0.07 <0.01

Species (Cryptoprocta ferox) -0.70 0.16 <0.01

Species (Felis catus) 0.14 0.18 0.44

Species (Galidia elegans) -0.09 0.10 0.39

Species (Galidictis fasciata) -0.28 0.08 <0.01

Same species 0.54 0.11 <0.01

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Interactions and Pathogen Transmission Between Carnivores