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Interactions and Pathogen Transmission BetweenCarnivores in MadagascarFidisoa [email protected]
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Recommended CitationRasambainarivo, Fidisoa, "Interactions and Pathogen Transmission Between Carnivores in Madagascar" (2018). Dissertations. 742.https://irl.umsl.edu/dissertation/742
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.
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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
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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
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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
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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
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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).
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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)
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(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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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
58
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).
59
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
63
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
64
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
66
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
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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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84
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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(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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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
120
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
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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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140
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