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Prevalence of Ranavirus in Virginia Turtles as Detected by Tail-Clip Sampling Versus Oral-Cloacal Swabbing


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Ranaviruses are emerging infectious diseases that infect amphibians, fish, and reptiles. Several cases of morbidity and mortality in captive and natural populations of reptiles have been attributed to ranaviruses, but research in this taxon has been limited. We used oral-cloacal swabs and tail clips to survey two species, Chrysemys picta picta (Eastern Painted Turtles) and Sternotherus odoratus (Common Musk Turtles), in three water bodies in central Virginia to determine if ranaviruses were present. Prevalence of ranavirus in C. p. picta ranged from 4.8–31.6% at the three sites. Ranavirus was not detected in S. odoratus, but only oral-cloacal swabs were used in this species because of the cornified tail tip. While tail-tip tissues from all three study sites indicated presence of ranavirus in C. p. picta, no oral-cloacal swabs from these same turtles tested positive. We therefore suggest that oral-cloacal swabbing may yield false negatives when ranavirus is present in turtles, and that tissue sampling may be more appropriate for monitoring ranavirus in turtles.
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2013 20(2):325–332
Prevalence of Ranavirus in Virginia Turtles as Detected by
Tail-Clip Sampling versus Oral-Cloacal Swabbing
Rachel M. Goodman1,*, Debra L. Miller2,3, and Yonathan T. Ararso1
Abstract - Ranaviruses are emerging infectious diseases that infect amphibians, sh, and
reptiles. Several cases of morbidity and mortality in captive and natural populations of
reptiles have been attributed to ranaviruses, but research in this taxon has been limited.
We used oral-cloacal swabs and tail clips to survey two species, Chrysemys picta picta
(Eastern Painted Turtles) and Sternotherus odoratus (Common Musk Turtles), in three
water bodies in central Virginia to determine if ranaviruses were present. Prevalence
of ranavirus in C. p. picta ranged from 4.8–31.6% at the three sites. Ranavirus was not
detected in S. odoratus, but only oral-cloacal swabs were used in this species because of
the cornied tail tip. While tail-tip tissues from all three study sites indicated presence of
ranavirus in C. p. picta, no oral-cloacal swabs from these same turtles tested positive. We
therefore suggest that oral-cloacal swabbing may yield false negatives when ranavirus
is present in turtles, and that tissue sampling may be more appropriate for monitoring
ranavirus in turtles.
Biodiversity is declining worldwide, and many biologists believe we are
witnessing the sixth mass extinction in the history of life (Barnosky et al.
2011, Wake and Vredenburg 2008). Nearly half of all amphibian populations
are in decline (IUCN et al. 2008), and reptiles may face similar levels of en-
dangerment (Gibbons et al. 2000, IUCN 2010, Reading et al. 2010). Many
factors have contributed to population declines and extirpations: habitat de-
struction and degradation, pollution, global climate change, introduction of
non-native species, and emerging infectious diseases (Wells 2007, Wilcove
et al. 1998). Globally, two thirds of freshwater turtle and tortoise species are
considered threatened or endangered (IUCN 2010), and infectious diseases
may be a contributing factor (Ernst and Lovich 2009). Emerging infectious
diseases contribute to population declines, and ranaviruses (family Irido-
viridae; genus Ranavirus) are of concern because they infect multiple taxa,
including fish, reptiles, and amphibians (Chinchar 2002). Currently we have
limited research on the susceptibility of this wide range of potential hosts and
the potential for transfer among species.
Ranaviruses are double-stranded DNA viruses that infect reptiles, am-
phibians, and fish and have caused mortality events in each taxon (reviewed
in Chinchar 2002). The importance of ranaviruses in amphibian population
1Biology Department, Box 74, Hampden-Sydney College, Hampden-Sydney, VA 23943.
2Veterinary Diagnostic and Investigational Laboratory, University of Georgia, Tifton, GA
31793. 3Current address - University of Tennessee, Center for Wildlife Health, Knoxville,
TN 37996. *Corresponding author -
Northeastern Naturalist Vol. 20, No. 2
declines has only recently been recognized, although they have caused more
die-offs in North America than the more-studied fungal pathogen Batracho-
chytrium dendrobatidis (Daszak et al. 1999, Duffus 2009, Gray et al. 2009).
Among fish, iridovirus infections have been reported on several continents
and can cause economic damage in commercial freshwater fisheries (Ahne et
al. 1997, Whittington et al. 2010). The importance of ranaviruses for reptilian
population dynamics is unknown, but several cases of morbidity and mortal-
ity in captive and natural populations have been attributed to the pathogen
(De Voe et al. 2004, Hyatt et al. 2002, Marschang et al. 2011). Research thus
far has been limited to description and isolation of viruses from infections in
captive and wild species (Chen et al. 1999, De Voe et al. 2004, Johnson et al.
2008, Marschang et al. 1999, Westhouse et al. 1996), and clinical challenges
of two North American species, Terrapene ornata ornata Agassiz (Ornate Box
Turtle) and Trachemys scripta elegans Weid-Neuwied (Red-eared Slider),
and two Australian species, Emydura krefftii Gray (Krefft's River Turtle)
and Eiseya latisternum Gray (Saw-shelled Turtle) (Ariel 1997, Johnson et al.
2007). Signs of ranavirus infection in turtles reported in these studies include
lethargy, respiratory distress, anorexia, cutaneous erythema, ocular and nasal
discharge, and oral ulceration and plaques. Surveillance of ranavirus in reptile
populations is important to determine whether associated disease threatens
persistence, and whether sub-lethally infected reptiles may serve as reservoirs
for the pathogen that threatens co-occurring species. Also, this work in reptiles
is necessary to gain an understanding of the complete epidemiology, including
interspecific transmission, of ranaviruses. In the current study, we used and
compared oral-cloacal swabbing and tissue sampling for ranavirus surveillance
in two species of turtles, Chrysemys picta picta Schneider (Eastern Painted
Turtles) and Sternotherus odoratus Latreille (Common Musk Turtles), in three
water bodies in Virginia.
Field Site Description
The study was conducted at three sites in Prince Edward County, VA: Briery
Creek Lake in Briery Creek Wildlife Management Area (north end; 37°12.0'N,
78°27.0'W), and two ponds on the campus of Hampden-Sydney College (HSC),
Chalgrove (37°14.5'N, 78°27.8'W) and Tadpole Hole (37°14.7'N, 78°27.2'W).
Chalgrove and Tadpole Hole are both approximately 1 ha and located 0.8 km apart.
Briery Creek Lake is a 342-ha lake managed by the Virginia Department of Game
and Inland Fisheries and is located 4.5 km south of the HSC ponds (Fig. 1).
Turtles were collected during 24 May–1 July 2010. We changed trapping
sites every week, and trapped at each site twice, with 6–10 visits per site.
Traps were set 1–2 m from shore and included four Promar collapsible crab/
fish traps with dual-ring entrance, a Sundeck turtle trap with a bait tower (Item
#840876, Heinsohn’s Country Store,
R.M. Goodman, D.L. Miller, and Y.T. Ararso2013 327
htm), and a floating turtle tunnel (Item#840460, Heinsohn’s Country Store).
Because all turtle traps could capture more than one turtle at a time, there was
a small risk that pathogen transmission could occur among individuals within
the traps.
Upon removal from traps, turtles were weighed, measured for mass and
length, and individually marked using combinations of notches filed into
scutes. We used and compared two methods of sampling for ranavirus, oral-
cloacal swabbing and tail clips, for use in the polymerase chain reaction (PCR).
We swabbed turtles with plastic, sterile, cotton-tipped applicators (Puritan
model 25-806 2PC), first rolling it inside the mouth and then inside the cloaca
for 3–5 seconds each. The distal-most 0.5 cm of the tip of the tail was collected
only from species not possessing cornified tail tips (i.e., C. p. picta) using a
new, sterile scalpel blade for each animal. Both tissue samples and swabs were
stored in 1-ml vials containing 70% ethanol. Turtles were released at the site of
capture immediately after sampling.
A total of 106 turtles, including C. p. picta (n = 63) and S. odoratus (n = 43),
were captured, and all turtles appeared clinically normal. Chrysemys picta picta
Figure 1. Map of three water bodies in central Virginia where turtles were sampled for
ranavirus: Chalgrove, Tadpole Hole, and Briery Creek Lake. The star indicates the area at
Briery Creek Lake where turtle trapping was conducted (across most of shoreline at other
sites). GPS coordinates are given in the Methods section.
Northeastern Naturalist Vol. 20, No. 2
were collected at all sites, whereas S. odoratus were only collected from Briery
and Chalgrove (Table 1). Among the samples collected, only those from species
and sites with sample sizes of approximately 20 were tested. All traps, rubber
boots and waders, and other gear were scrubbed, soaked in a 1% chlorhexidine
diacetate (Fort Dodge Nolvasan Solution) for at least one minute, and rinsed in
water between use at different water bodies.
Genomic DNA was extracted from the tissues or swabs using a commer-
cially available kit (DNeasy Blood and Tissue Kit, Qiagen, Inc., Valencia, CA).
Negative and positive extraction controls were included. Conventional PCR
was performed using the protocol and primer sets (MCP4 and MCP5) found in
Mao et al. (1996, 1997) and targeting an approximately 500-base pair sequence
of the major capsid protein (MCP) gene. The PCR products were resolved via
electrophoresis on a 1.0% agarose gel. Controls for each PCR run included two
negative controls (water and gDNA extracted from a ranavirus-negative tad-
pole) and two positive controls (cultured ranavirus and gDNA extracted from
a ranavirus-positive tadpole). The PCR protocol was repeated once more on all
samples to verify results.
Only oral-cloacal swabs were tested for S. odoratus, and none were positive
for ranavirus (Table 1). While tail tips from all three study sites indicated pres-
ence of ranavirus among C. p. picta, none of the oral-cloacal swabs from these
same turtles tested positive (Table 1). However, two of the eight ranavirus-
positive individuals that tested positive for ranavirus via tissue samples did not
have accompanying oral-cloacal swabs because they were too small for effec-
tive use of technique (i.e., juveniles). Based on tail-tissue sampling, prevalence
of ranavirus in C. p. picta was 4.8% in Briery, 31.6% in Chalgrove, and 17.4%
in Tadpole Hole.
We found evidence of ranavirus infection in C. p. picta in our three study
sites using tail-tissue sampling; however, oral-cloacal swab sampling failed to
detect the pathogen. These ndings suggest that oral-cloacal swabbing may yield
false negatives when ranavirus is present in turtles, and that tissue sampling may
be more appropriate. Gray et al. (2012) conducted a controlled infection study
with Lithobates catesbeianus Shaw (American Bullfrog) tadpoles and found
Table 1. Ranavirus infections in turtles from three water bodies in central Virginia.
Chrysemys picta picta Sternotherus odoratus
Tissues Swabs Swabs
Water body n Ranavirus + n Ranavirus + n Ranavirus +
Briery 21 1 (4.8%) 21 0 (0.0%) 21 0 (0.0%)
Chalgrove 19 6 (31.6%) 8 0 (0.0%) 22 0 (0.0%)
Tadpole Hole 23 4 (17.4%) 21 0 (0.0%) - -
R.M. Goodman, D.L. Miller, and Y.T. Ararso2013 329
false-negative and false-positive rates of 20% and 6% for tail samples, and 22%
and 12% for swabs, respectively, using liver samples as the standard for virus
infection. Those results suggest a similar rate of false negatives for tail and swab
samples in an amphibian, whereas our eld surveillance study suggests a differ-
ence between the methods in a reptile. Further comparisons in additional species
may be warranted.
Necropsy and histology provide the most certain evidence for ranaviral dis-
ease (Miller and Gray 2010); however, lethal sampling is not desirable in the
absence of morbidity or mortality events. Oral-cloacal swabbing is the least
invasive method of sampling, but the current study indicates the sensitivity
of this testing method may be low. While not compared to testing internal or-
gans, tail-tip sampling appears to be more sensitive than oral-cloacal swabbing
and was able to detect ranavirus in C. p. picta using moderate sample sizes of
around twenty individuals. Future research may investigate other potential
areas for superficial tissue sampling on catch-and-release specimens, particu-
larly for species with a cornified or boney tail tip that is used in courtship and
copulation (Ernst and Lovich 2009). In such species, we recommend an ap-
proximately 5-mm diameter skin (epidermal and dermal) biopsy taken from
the mid-dorsal tail.
Compared to common rates for amphibians, prevalence of ranavirus in-
fection in turtles was low in the three water bodies sampled. Research in
amphibians indicates that prevalence can vary widely, depending on the spe-
cies and date of sampling. Using tissue collected from all major organs, Gray
et al. (2007) found ranavirus prevalence of 15–57% in tadpoles in undisturbed
and cattle accessed ponds, depending on the species and sampling period.
Using tail tips and liver samples, Brunner et al. (2004) found prevalence of
46–100% in dispersing metamorph salamanders following an epidemic, but
only 7% prevalence in adults returning to ponds in the following spring. A
recent survey of injured/rehab and free-ranging Terrapene carolina carolina
L. (Eastern Box Turtle) found prevalence of ca. 3% from blood samples col-
lected from injured/sick turtles submitted to rehab centers/medical facilities in
the southeastern US (Allender et al. 2011). This same study was able to detect
ranavirus in swab samples collected from injured/sick T. c. carolina, a species
that spends large amounts of time on dry land, submitted to the medical facility
in Tennessee. Our study differs from Allender et al. (2011) in that we surveyed
a heavily aquatic species and, if it holds true that water is an excellent me-
dium for ranavirus (Chinchar 2002), one would expect greater prevalence in
turtles spending more time in water. However, a recent survey of free-ranging
C. picta and Emydoidea blandingii Holbrook (Blanding’s Turtle) in Illinois
found 0% prevalence for ranavirus using blood samples and oral swabs (Al-
lender et al. 2009). Explanations for the low prevalence and lack of ranavirus
in the two species we studied include possible resistance to infection or ability
to clear infection in these species. Infection rates and ability to clear ranavirus
vary among amphibian species exposed to standardized virus treatments, and
also according to ranavirus isolate (Hoverman et al. 2010, 2011). Thus far, this
Northeastern Naturalist Vol. 20, No. 2
comparative analysis of infection rates has not been investigated in turtles or
any reptile. Given our findings, the marked declines of turtle populations, and
the fact that many turtle species are syntopic with amphibians and fish (poten-
tial hosts of ranaviruses), further investigation, including controlled laboratory
studies, is needed to determine the impact of ranaviruses on turtles.
We thank Hampden-Sydney College, the Biology Department, and the Honors Pro-
gram for providing funding and support for this research. All work in this study was
approved by the Hampden-Sydney College Animal Care and Use Committee and per-
formed under scientic collection permit 38354 from Virginia Department of Game and
Inland Fisheries. We thank Briery Creek Wildlife Management for permitting us to work
on the site, Lisa Whittington for assistance conducting laboratory tests at the University
of Georgia, and Zach Harrelson, Allen Luck, Sam Smith, and Erica Rutherford for as-
sistance in the eld.
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... Ranaviruses have been identified in reptiles and amphibians across the United States, often leading to mass mortality events (Gray et al. 2009;Duffus et al. 2015), but in other cases causing persistent infections in the absence of die-offs (e.g., Johnson et al. 2008;Greer et al. 2009). While ranavirus infections have been documented in the Southeast, Midwest, and numerous areas across the United States (Allender et al. 2011;Gray et al. 2012;Goodman et al. 2013;Duffus et al. 2015), infections have not been detected in Arkansas. ...
... Ranaviruses have been identified in reptiles and amphibians across the United States, often leading to mass mortality events (Gray et al. 2009;Duffus et al. 2015), but in other cases causing persistent infections in the absence of die-offs (e.g., Johnson et al. 2008;Greer et al. 2009). While ranavirus infections have been documented in the Southeast, Midwest, and numerous areas across the United States (Allender et al. 2011;Gray et al. 2012;Goodman et al. 2013;Duffus et al. 2015), infections have not been detected in Arkansas. ...
... We deployed five baited hoop nets (diameter 1 m, mesh 2.5 cm) on 10 October 2015 at a single site in WNWR (Fig. 1) and checked them the following day. Upon capture, we determined the species, sex, mass, and length of each individual, and checked for any clinical signs of ranavirus infection such as ocular discharge, dermatitis, or necrotic oral tissue (Allender et al. 2013). In order to assess ranavirus prevalence and species specificity, we collected a tail clip (~1.0 cm) from each turtle with a sterile razor blade, placed it in a snaptop tube (Fisherbrand®, Cat. ...
Full-text available
Survey for the prevalence of ranaviruses in turtles of the Wapanocca Wildlife Refuge
... A second positive case was also found in 2018, in a wood turtle (Glyptemys insculpta) (Canadian Wildlife Health Cooperative blog 2018). In the USA, the majority of reported cases of ranavirus have involved the eastern box turtle (Terrapene carolina carolina) (e.g., De Voe et al., 2004;Allender et al., 2011;Winzeler et al., 2018); a study on its prevalence in Eastern painted turtles was carried out in Virginia, USA (Goodman, Miller & Ararso, 2013). ...
... Signs can appear similar to those of other infectious agents such as mycoplasma and herpesvirus infections, bacterial infection secondary to trauma, as well as non-infectious issues such as Vitamin A deficiency. Evidence also suggests that reptiles can be asymptomatic carriers of ranviruses (e.g., Goodman, Miller & Ararso, 2013;Goodman, Hargadon & Carter, 2018). ...
... Prevalence of ranavirus in the USA has been found to be variable. PCR on oral-cloacal swabs and tail clips were used to survey two species in three water bodies in Virginia; the Eastern painted turtle, Chrysemys picta picta, and the Common musk turtle, Sternotherus odoratus (Goodman, Miller & Ararso, 2013). They found a prevalence of 4.8-31.6% in painted turtles, and zero in musk turtles. ...
Full-text available
Background Ontario, Canada is home to eight native species of turtles; all eight are federally listed as Species At Risk, due to anthropogenic threats. However, until recently, reports of infectious disease have been lacking. Ranavirus is seen as an emerging threat for ectotherms globally, with mass die-offs most often reported in amphibians. Ranavirus has been detected in Ontario’s amphibian populations, can be transmitted via water, and can be transmitted from amphibians to turtles. However, no studies on the prevalence of this virus in Ontario’s turtles have previously been carried out. With recent reports of two confirmed positive case of ranavirus in turtles in Ontario, a knowledge of the ecology of ranavirus in Ontario’s turtles has become even more important. This study estimates the prevalence of ranavirus in Ontario’s turtles, and investigates the hypothesis that this is a newly emergent disease. Methods Sixty-three samples were tested for ranavirus via PCR. These included a variety of turtle species, across their home range in Southern Ontario. Fifty-two of the samples originated from the liver and kidney of turtles who had succumbed to traumatic injuries after being admitted to the Ontario Turtle Conservation Centre; ten of the samples were taken from cloacal swabs, lesion swabs, or tail clips collected from live turtles showing signs of clinical disease. One of the live turtles was later euthanized for humane reasons and PCR was also carried out on the liver/kidney. Results None of the 63 samples were found to be positive for ranavirus via PCR. The zero prevalence found in this study translates into a population prevalence estimate of less than 5%, with no change in prevalence from 2014–2018. Discussion This is the first report on the prevalence of ranavirus in Ontario’s turtles, and will help build an understanding of the ecology of this virus in Ontario. Ranavirus has historically been underreported in reptiles, but there has been an increase in global reports recently, most likely due to increased awareness. A carrier state is thought to exist in reptiles which makes surveillance in the population via random sampling a viable method of detection of prevalence. The first report of ranavirus in Ontario turtles occurred in 2018. This study suggests a continued low population prevalence for the years 2014–2018, however. Ongoing surveillance is necessary, as well as investigation of the eDNA presence in waterways as compared to the PCR of resident turtles, to further understand the sensitivity of these species to ranavirus infection. The utilization of qPCR would be helpful, to better quantify any positives encountered.
... During 24 May -1 July of 2010, turtles were trapped for a ranavirus surveillance study by Goodman et al. (2013). We trapped at each site twice for one week during this period, using four Promar collapsible crab/fish traps with dual-ring entrance, a Sundeck turtle trap with a bait tower (Item #840876, Heinsohn's Country Store), and a floating turtle tunnel (Item#840460, Heinsohn's Country Store). ...
... 4.5 km from H-SC water bodies; Goodman, pers. obs.,Goodman et al. 2013). Trapping efforts during 2010 yielded several captures of Chrysemys picta picta and Sternotherus odoratus at both Chalgrove and Tadpole, indicating resident populations of both species on campus. ...
... Studies on ranavirus in turtles have included isolation of ranaviruses from natural populations and reports of deaths and declines in captive and wild species [12,18,19]; experiments have examined susceptibility and transmission of ranavirus between species and the importance of exposure dose and rearing temperature to host susceptibility and mortality [18,[20][21][22][23][24]. While ranavirus outbreaks with high mortality may result in disconcerting headlines, limited surveys have also found ranavirus infection occurring at low prevalence in populations without apparent die-offs [25][26][27]. Ranavirus is now well documented on six continents [12], but significant questions remain regarding distribution and factors influencing morbidity and mortality in wild reptile populations. Experimental studies examining the effect of environmental stressors on ranavirus infection are especially lacking in reptiles. ...
... All samples were tested in duplicate using Applied Biosystems™ StepOne Real-time PCR machine with two negative and two positive controls in each run (pure water and DNA extracted from cultured FV3 ranavirus). Samples with Ct values <30 for both runs were considered positive for ranavirus, according to standards established for this machine using known negative and positive controls from water, cultured ranavirus, and ranavirus-infected reptiles [25,26]. If Ct values from two samples for an individual were not both <30 or if one approximated 30, we ran two additional PCR reactions. ...
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Ranaviruses are an important wildlife pathogen of fish, amphibians, and reptiles. Previous studies have shown that susceptibility and severity of infection can vary with age, host species, virus strain, temperature, population density, and presence of environmental stressors. Experiments are limited with respect to interactions between this pathogen and environmental stressors in reptiles. In this study, we exposed hatchling red-eared slider turtles (Trachemys scripta elegans) to herbicide and ranavirus treatments to examine direct effects and interactions on growth, morbidity, and mortality. Turtles were assigned to one of three herbicide treatments or a control group. Turtles were exposed to atrazine, Roundup ProMax®, or Rodeo® via water bath during the first 3 weeks of the experiment. After 1 week, turtles were exposed to either a control (cell culture medium) or ranavirus-infected cell lysate via injection into the pectoral muscles. Necropsies were performed upon death or upon euthanasia after 5 weeks. Tissues were collected for histopathology and detection of ranavirus DNA via quantitative PCR. Only 57.5% of turtles exposed to ranavirus tested positive for ranaviral DNA at the time of death. Turtles exposed to ranavirus died sooner and lost more mass and carapace length, but not plastron length, than did controls. Exposure to environmentally relevant concentrations of herbicides did not impact infection rate, morbidity, or mortality of hatchling turtles due to ranavirus exposure. We also found no direct effects of herbicide or interactions with ranavirus exposure on growth or survival time. Results of this study should be interpreted in the context of the modest ranavirus infection rate achieved, the general lack of growth, and the unplanned presence of an additional pathogen in our study
... Similar to amphibians, susceptibility to ranavirus infections and manifestation of clinical signs in turtles vary depending on developmental stages (Duffus et al. 2015), species, and temperature . A study of Eastern box turtles (Terrapene carolina carolina) in the US reported a prevalence less than 5% in a population without abnormal mortality events (Allender et al. 2013), while in asymptomatic wild Eastern painted turtles (Chrysemys picta picta) the reported prevalence was between 4.8-31.6% in ponds without mortality events (Goodman et al. 2013). While ranavirus cases have been confirmed in North America, the few reports in Canada are potentially from a combination of a lack of surveillance in turtles, as ranavirus surveys are focused on amphibians, and subtle phenotypic manifestations of disease in turtles in general. ...
... Unfortunately, since the virus may be transmitted via the water between ectotherm species (Bandín and Dopazo, 2011;Brenes et al. 2014), the ongoing carrier state does presents opportunities to perpetuate the virus in the water bodies and therefore act as a reservoir for other species. While amphibians have been suggested as potential reservoir hosts for chelonians (Johnson et al. 2008), other studies suggested that reptiles can act as asymptomatic carriers of ranaviruses (Goodman et al. 2013(Goodman et al. , 2018Brenes et al. 2014). Reptiles can act as host and transmit ranavirus to amphibians, and while reptiles do not experience mortality, infected amphibian larvae can experience up to 100% mortality (Brenes et al. 2014). ...
Ranaviruses have been associated with chelonian mortality. In Canada, the first two cases of ranavirus were detected in turtles in 2018 in Ontario, although a subsequent survey of its prevalence failed to detect additional positive cases. To confirm the prevalence of ranavirus in turtles in Ontario, we used a more sensitive method to investigate if lower level persistent infection was present in the population. Here we report results via a combination of qPCR, PCR, Sanger sequencing and genome sequencing from turtles from across Ontario, with no clinical signs of illness. We found 2 positives with high viral load and 5 positives with low viral load. Histopathology found subtle histological changes. DNA sequences identified two types of frog virus 3 (FV3), and genome sequencing identified a ranavirus similar to wild-type FV3. Our results show that the virus has been present in Ontario’s turtles as subclinical infections.
... This could be due either to the types of samples used or the detection method used. Previous studies have shown that more invasive samples, for example, tail clips, may be better for ranavirus detection (Goodman et al., 2013). ...
... 53,54 Virus has also been detected in cloacal swabs and blood of infected animals, 53,55 and there is some indication that more invasive sampling of tissues may lead to higher rates of identification of infected animals. 56 An ELISA has been described for the detection of antibodies against ranaviruses in chelonians, but is not commercially available. 57 ...
Methods for the detection of pathogens associated with respiratory disease in reptiles, including viruses, bacteria, fungi, and parasites, are constantly evolving as is the understanding of the specific roles played by various pathogens in disease processes. Some are known to be primary pathogens with high prevalence in captive reptiles, for example, serpentoviruses in pythons or mycoplasma in tortoises. Others are very commonly found in reptiles with respiratory disease but are most often considered secondary, for example, gram-negative bacteria. Detection methods as well as specific pathogens associated with upper- and lower-respiratory disease are discussed.
Although reptiles have often been overlooked in research, information on viruses of reptiles has been growing steadily in recent decades as has our understanding of the importance of these animals in the ecosystem. As ectotherms, their immune systems are dependent on temperature, among other factors, and interactions between infection and disease are complex and dependent on host, pathogen, and environmental factors. This chapter provides an overview of the viruses described in reptiles so far, as well as insight into some of the diseases caused by viruses in this group of animals. It also discusses the reptile immune system and the host reaction to infection. Influences of the environment on development of disease are in many cases not well understood, and this chapter includes a discussion of some important progress in this field. Studies of the effects of viruses on wild, pet, and farmed reptiles are limited, but indicate that viral disease can strongly affect individual populations in the wild, and that human action and the animal trade likely play a role in disease epidemiology.
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Disease outbreaks among wildlife have surged in recent decades alongside climate change, although it remains unclear how climate change alters disease dynamics across different geographic regions. We amassed a global, spatiotemporal dataset describing parasite prevalence across 7346 wildlife populations and 2021 host-parasite combinations, compiling local weather and climate records at each location. We found that hosts from cool and warm climates experienced increased disease risk at abnormally warm and cool temperatures, respectively, as predicted by the thermal mismatch hypothesis. This effect was greatest in ectothermic hosts and similar in terrestrial and freshwater systems. Projections based on climate change models indicate that ectothermic wildlife hosts from temperate and tropical zones may experience sharp increases and moderate reductions in disease risk, respectively, though the magnitude of these changes depends on parasite identity.
Chelonians are increasingly challenged by anthropogenic threats and disease. This article summarizes recent literature and clinical experiences regarding 4 emerging infectious diseases in turtles and tortoises: ranaviruses, cryptosporidiosis, intranuclear coccodiosis of Testudines, and Emydomyces testavorans.
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Infections with Ranavirus and herpesvirus have contributed to numerous morbidity and mortality reports in chelonians worldwide. To better understand the prevalence of these viruses in healthy and declining populations, a survey for these viruses was performed on two aquatic turtle species in 2007 using polymerase chain reaction assays. Blood and oral swabs were taken from 47 painted turtles, Chrysemys picta, and 58 Blanding's turtles, Emydoidea blandingii. Results demonstrated no positive cases using this method in these populations. The lack of positive Ranavirus test results may indicate that these turtles have never been exposed to virus, have been exposed but have cleared the virus, are not shedding the virus in oral swabs or blood, or that oral swabs are inappropriate samples to assess ranaviral shedding in these species. Similarly, the lack of positive herpesvirus test results may indicate that these turtles have never been exposed to virus, have been exposed but have a latent infection, are not shedding the virus in oral swabs or blood, or that oral swabs are also inappropriate samples to assess herpesviral infection in these species.
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Virulent parasites cannot persist in small host populations unless the parasite also has a reservoir host. We hypothesize that, in hosts with complex life histories, one stage may act as an intraspecific reservoir for another. In amphibians, for example, larvae often occur at high densities, but these densities are ephemeral and fixed in space, whereas metamorphs are long-lived and vagile but may be very sparse. Parasite persistence is unlikely in either stage alone, but transmission between stages could maintain virulent parasites in seasonally fluctuating amphibian populations. We examined this hypothesis with a lethal ranavirus, Ambystoma tigrinumvirus (ATV), that causes recurrent epidemics in larval tiger salamander populations, but which has no reservoir host and degrades quickly in the environment. Although exposure to ATV is generally lethal, larvae and metamorphs maintained sublethal, transmissible infections for .5 mo. Field data corroborate the persistence of ATV between epidemics in sublethally infected metamorphs. Three-quarters of dispersing metamorphs during one epidemic were infected, and apparently healthy metamorphs returning to breed harbored ATV infections. Our results suggest that larval epidemics amplify virus prevalence and sublethally infected metamorphs (re)introduce the virus into uninfected larval populations. Intraspecific res- ervoirs may explain the persistence of parasites in and declines of small, isolated amphibian populations.
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 A virus was isolated from tissues of 2 diseased Hermann’s tortoises (Testudo hermanni) and preliminarily characterized as an iridovirus. This conclusion was based on the presence of inclusion bodies in the cytoplasm of infected cells, sensitivity to chloroform, inhibition of virus replication by 5-iodo-2′-desoxyuridine and the size and icosahedral morphology of viral particles. The virus was able to replicate in several reptilian, avian and mammalian cell lines at 28°C, but not at 37°C. Restriction enzyme analysis showed resistance of the ral DNA to digestion with HpaII due to methylation of the internal cytosine at CCGG sequences. Part of the genomic region encoding the major capsid protein was amplified by PCR and subjected to sequence analysis. Comparative analysis of the obtained nucleotide sequence revealed that the isolate is closely related to frog virus 3, the type species of the genus Ranavirus.
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Ranaviruses have been identified as the etiologic agent in many amphibian die-offs across the globe. Polymerase chain reaction (PCR) is commonly used to detect ranavirus infection in amphibian hosts, but the test results may vary between tissue samples obtained by lethal and non-lethal procedures. Testing liver samples for infection is a common lethal sampling technique to estimate ranavirus prevalence because the pathogen often targets this organ and the liver is easy to identify and collect. However, tail clips or swabs may be more practicable for ranavirus surveillance programs compared with collecting and euthanizing animals, especially for uncommon species. Using PCR results from liver samples for comparison, we defined false-positive test results as occurrences when a non-lethal technique indicated positive but the liver sample was negative. Similarly, we defined false-negative test results as occurrences when a non-lethal technique was negative but the liver sample was positive. Using these decision rules, we estimated false-negative and false-positive rates for tail clips and swabs. Our study was conducted in a controlled facility using American bullfrog Lithobates catesbeianus tadpoles; false-positive and false-negative rates were estimated after different periods of time following exposure to ranavirus. False-negative and false-positive rates were 20 and 6%, respectively, for tail samples, and 22 and 12%, respectively, for swabs. False-negative rates were constant over time, but false-positive rates decreased with post-exposure duration. Our results suggest that non-lethal sampling techniques can be useful for ranavirus surveillance, although the prevalence of infection may be underestimated when compared to results obtained with liver samples.
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Research that identifies the potential host range of generalist pathogens as well as variation in host susceptibility is critical for understanding and predicting the dynamics of infectious diseases within ecological communities. Ranaviruses have been linked to amphibian die-off events worldwide with the greatest number of reported mortality events occurring in the United States. While reports of ranavirus-associated mortality events continue to accumulate, few data exist comparing the relative susceptibility of different species. Using a series of laboratory exposure experiments and comparative phylogenetics, we compared the susceptibilities of 19 amphibian species from two salamander families and five anurans families for two ranavirus isolates: frog virus 3 (FV3) and an FV3-like isolate from an American bullfrog culture facility. We discovered that ranaviruses were capable of infecting 17 of the 19 larval amphibian species tested with mortality ranging from 0 to 100%. Phylogenetic comparative methods demonstrated that species within the anuran family Ranidae were generally more susceptible to ranavirus infection compared to species from the other five families. We also found that susceptibility to infection was associated with species that breed in semi-permanent ponds, develop rapidly as larvae, and have limited range sizes. Collectively, these results suggest that phylogeny, life history characteristics, and habitat associations of amphibians have the potential to impact susceptibility to ranaviruses.
Biologists are nearly unanimous in their belief that humanity is in the process of extirpating a significant portion of the earth's spe­ cies. The ways in which we are doing so reflect the magnitude and scale of human enterprise. Everything from highway construction to cattle ranch­ ing to leaky bait buckets has been implicated in the demise or endan­ germent of particular species. Ac­ cording to Wilson (1992), most of these activities fall into four major categories, which he terms "the mind­ less horsemen of the environmental apocalypse": overexploitation, habi­ tat destruction, the introduction of non-native (alien) species, and the spread of diseases carried by alien species. To these categories may be added a fifth, pollution, although it can also be considered a form of habitat destruction. Surprisingly, there have been reIa­ tively few analyses of the extent to which each of these factors-much less the more specific deeds encomDavid S. Wilcove is a senior ecologist at the Environmental Defense Fund, Wash­ ington, DC 20009. David Rothstein re­ ceived his J.D. in 1997 from Northeastern
Systemic infections of teleost fishes caused by iridoviruses have recently been recognized in Australia, Asia, Europe and the USA. These iridoviruses are different from those of the established genera Lymphocystivirus and Goldfish Virus 1-like Viruses of the family Iridoviridae. The agents exhibit similar physicochemical properties, are antigenically related and prove to be of high virulence to different teleost fishes in aquaculture. The first iridovirus, epizootic haematopoietic necrosis virus, responsible for an epizootic outbreak of haematopoietic necrosis in redfin perch, was reported in Australia. Some years later, similar iridovirus epizootics occurred in sheatfish and catfish in Europe. The Australian and the European isolates proved to be antigenically related and showed properties in common with frog virus 3, the type species of the genus Ranavirus of the Iridoviridae. Further iridovirus isolates from fish, amphibians and reptiles exhibited a close relationship with each other and with frog virus 3. It is important to note that the Australian amphibian iridovirus, Bohle iridovirus, was experimentally transmitted to teleost fish inducing high mortalities. The occurrence of similar viruses in different host species in the aquatic environment and their inter-species transmission emphasize the importance of health control in aquaculture.