A Herpdigest Special Issue- 11/20/13 A Primer on Ranavirus Herpdigest is The Only Free Weekly Electronic Newsletter That Reports on the Latest News on Herpetological Conservation, Husbandry and Science Publisher/Editor- Allen Salzberg _________________________________________________________ HerpDigest is a non-profit organization that is totally dependent on your generosity for its continued existence: Please make a donation on top of your order. _________________________________________________________________ Check with your Human Resources Department-Do they have a Matching Gifts program? _______________________________________________________________ Only 3 TURTLE and 1 SEA TURTLES, 2014 calendars are left -same price as last year, $14.99 each, plus $6.00 S&H $2.00 for every additional calendar. To order, and see photos of the front and back covers as well as a sample photo of an inside page from both calendars go to http://www.herpdigest.org/calendars.html DON’T WAIT UNTIL WE ARE OUT OF STOCK OF ALL CALENDARS.
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Are Back But we are running out of them, 18 different kinds are already sold out. half were herps. Go to the url below to see what is left. Act Now. Don’t wait until the last minute when there is nothing left. Don’t wait until Black Friday or Cyber Tuesday. Great Hanukah & Christmas gifts. For a $25.00 donation we will thank you with 4 magnets of your choice. (For each additional magnet add $6.00.) Please include three alternatives. S&H is included. See order form below. All magnets are 2.5" x 3.5" Mylar UV protected on a flat magnetic back. To see the extensive collection and information on how to order go to http://herpdigest.org/donate.html or The University & College Herp Diplomas For a $25.00 donation we will thank you with a personalized diploma of your choice (add $20 for each additional personalized diploma). S&H is included. Give as gifts to others or yourself and help HerpDigest stay alive. Diplomas available Turtle University (Sea) Turtle University, Frog University, Snake University, Gecko, Salamander State, Bearded Dragon and Chameleon Universities. For information on how to order. Go to http://herpdigest.org/diplomas.html Both great gifts for you, family and friends. Animal or Herp lover. __________________________________________________________________________________________________ Please send all questions or correspondence to the author Dave Lee Dave Lee, torres...@aol.com If you are a turtle, a frog or even a tadpole you need to read this— It’s really scary! A Primer on Ranaviruses. David S. Lee, The Tortoise Reserve Forget vampires and zombies there is a real cold-blooded killer out there, it’s a pathogen named Ranavirus. As the label implies it is not exactly frog friendly, but it also causes illness and death in salamanders, reptiles and fish. This virus is now found worldwide. Transmission is rapid and can result from either direct or indirect contact with infected animals. The virus enters cells of the host and takes over the cell processes for its own replication. Ranaviruses can infect multiple cell types and cell death can occur in as little as 9 hours, quickly leading to loss of organ function. Susceptibility varies with species. In some frogs, for example, mortality can result in just 3 days. Experiments done on infected and uninfected salamanders showed the virus could be transmitted when the salamanders were in contact for as little as 1 second. Exposure to water or soil contaminated with Ranavirus can also result in disease. History and background Ranaviruses are believed to have evolved in fish and only later began to infect amphibians and reptiles (Jancovich et al 2010). It was first reported from amphibians in the 1960’s in a population of northern leopard frogs, Lithobates pipiens (Granoff, et al. 1965), yet the impact of widespread virus related die-offs was not recognized until the 1990’s. Research experiments demonstrate that the virus can be transmitted within and between three of the higher classes of vertebrates. Since the mid-1990’s Ranavirus has been taking a devastating toll on native populations of reptiles and amphibians across the U.S. Especially hard hit are frogs, toads, salamanders and their larvae, as well as turtles. Hundreds of thousands of these animals have died from the lethal virus and the disease continues to spread. The cause of the sudden appearance of a global plague from this pathogen is uncertain, but possibilities include trade in food and ornamental fish, reptiles, amphibians, and/or its emergence from unknown reservoir hosts resulting from changes in the environment. Ranavirus has been documented as being responsible for amphibian die-offs, some of them massive events, in over 20 states. To date over 85 species of turtles and amphibians have been involved with the die-offs where mortality can range from a few individuals to thousands. In some cases where amphibian breeding sites support a number of frogs and/or salamanders the die-offs involve multiple species. Ninety-four percent of the known cases of Ranavirus have been reported since 1998. While to some degree this represents a growing awareness of the problem, this figure strongly suggest that the virus is becoming more common and widespread. Die-offs of amphibians have been reported on private, state and federal lands, including several national parks and wildlife refuges. To date, most of the species involved are relatively common and widespread, but the virus has caused problems for populations of threatened and endangered species. The reasons for the emergence of Ranavirus in wild populations vary from site to site. Often there are stress related elements, man-made or natural, associated with outbreaks. Disturbance of the site and/or pollution are important but not necessarily required factors. The virus exists in aquatic habitats: ponds, lakes, permanent wetlands and vernal amphibian breeding sites. Turtles and breeding adult amphibians moving into recently flooded wetlands are likely carriers of the disease. A common factor in Ranavirus outbreaks is the rapid drying of wetlands. This apparently results because it concentrates turtles and amphibians and accelerates metamorphosis. The immune system of amphibians is suppressed during transformation to the adult stage, increasing the chance of pathogen infections, and disease. Ranaviruses are members of the Iridoviridae, a group of double stranded DNA viruses. There are six recognized species and numerous strains, however in North America viruses related to the Ambystoma tigrinum virus (ATV) and Frog virus 3 (FV3) appear to be the most important to reptiles and amphibians. The Bohle iridovirus (BIV) from Australia is also of concern. Some ranaviruses may be able to infect animals from more than one class (e.g. amphibians, reptiles, and fish). The incubation period is variable- 5 days to several weeks. The virus was identified in skin, intestines and kidneys of African clawed frogs, Xenopus lavis, within 3 hours of introducing them to infected water (Robert et al. 2011). Ambient temperatures, dose of virus exposure, immunosuppression, the host’s developmental stage, and species differences in susceptibility to various Ranavirus strains probably affect infection timing. Ranaviruses replicate at temperatures between 12 and 32 C, because of this, birds and mammals are not suitable hosts (Chinchar 2002). The virus can remain viable in frozen fish for over 2 years (Langdon1989). Three genera of viruses of the family Iridoviridae affect fish. Ranaviruses and Megalocytiviruses are pathogens that have recently appeared. Both types cause severe disease outbreaks, occur globally, and affect a broad spectrum of hosts. The haematopoietic necrosis virus from Australia was the first Ranavirus to cause epizootic mortality in fish. Like other ranaviruses it lacks host specificity. A distinct but closely related virus, European catfish virus, occurs in fish in Europe, while very similar ranaviruses appear in fish and amphibians in Europe, Asia, Australia, North America and South America. These viruses can be distinguished from one another and this could allow policies of the World Organization for Animal Health (OIE) to minimize the spread of these viruses. However, at this time limited information and variations in disease expression create difficulties in sampling strategies, and there remains uncertainty surrounding the taxonomy of some ranaviruses (Whittington, et al. 2010). All types of amphibians including salamanders, newts, frogs and toads are susceptible. Larvae and metamorphic stages are most often associated with massive mortality events. Adult amphibian morbidity and mortality is reported less often, but has been observed in the wild, as well as in captivity. Some species may have covert infections and be able to shed and transmit virus to other susceptible animals without ever exhibiting clinical signs. Likewise, non-lethal infections have been documented and it is likely that these latent infections explain the persistence and emergence of the disease in both wild and captive populations. Ranaviruses found in fish, amphibians, and types of other reptiles may serve as reservoirs for susceptible chelonians. Ranavirus is but one of a number of viral diseases that have been reported in turtles. The two important viral diseases of freshwater and terrestrial chelonians include Herpesvirus disease in tortoises (multiple clinical signs and high mortality may occur) and Iridoviral (Ranavirus) disease. See Origgi (chapter 57, Mader, 2006) for a review of Herpesvirus Disease of tortoises and Jacobson (2007) for a general and comprehensive review of chelonian viral diseases. Recently, outbreaks of Ranavirus have also been documented in lizards (Stohr 2013). Johnston et al. (2008) report affected species that included captive Burmese star tortoises, Geochelone platynota, a free-ranging gopher tortoise, Gopherus polyphemus, free-ranging eastern box turtles Terrapene carolina carolina, and a Florida box turtle Terrepene carolina bauri. They also found evidence for Ranavirus infection in archived material from previously unexplained mass mortality events of eastern box turtles from Georgia in 1991 and Texas in 1998. Ranavirus infections were also found in sympatric species of amphibians at two locations with infected chelonians. The profiles of Ranavirus isolated from a dead Burmese star tortoise and a southern leopard frog, Rana utricularia, found nearby, were similar. These findings support the ideas that certain amphibians and chelonians are infected with a similar virus and that different viruses exist among different chelonians. Amphibians may serve as the major reservoir host for susceptible chelonians. This study also demonstrated that significant Ranavirus infections are likely more widespread in chelonians than previously suspected. Eastern populations of tiger salamanders (Ambystoma tigrinum tigrinum) are listed at some level of conservation concern in almost every state in which they occur, in most they are considered endangered. Most populations are isolated, disjunct, and both their overall numbers and range are declining. Titus and Green (2013) reported Ranavirus in populations of tiger salamanders on Long Island. Thus, the threat of this virus to populations of endangered species is no longer to be considered as just a potential one, it’s real. Death from the virus is not pretty. In amphibians the disease is likened to Ebola or epizootic hemorragice disease due to body swelling and hemorrhaging. Hemorrhagic lesions are characteristic of fish with Ranavirus infections and often in reptiles as well. Tissue necrosis is extensive because the virus commandeers multiple cell types. Recognition Infection does not always cause disease. Long-term non-clinical carriers have been identified. Clinical signs vary depending on the host and a number of other factors. In infected fish the hematopoietic tissue is usually severely affected. General pathological signs include pale gills and liver, friable kidneys and livers, and ecchymosis and petechiation on ventral body surfaces. Fish with Ranavirus often exhibit no external symptoms. Both fresh and saltwater species are affected, and the virus can be spread to animals eating live, dead, or previously frozen infected fish. In amphibians ranaviral outbreaks can result in sudden onset of illness; in a wetland often hundreds or thousands of sick individuals are seen over a 1-5 day period. Overall mortality rates in larvae and juveniles will exceed 90%. A good indication of disease is lethargic animals swimming erratically and weakly, or on their sides. Infected frogs and salamanders typically have subtle to severe hemorrhages on their ventral surface, particularly at the base of the hind limbs, and around the vent. In some cases hemorrhages are present from the chin to the tip of the tail; at other times they may appear in specific sites or as irregular patches. The abdomen may also become enlarged and reddened (red leg-like symptoms) and amphibians may have skin ulceration and/or epithelial proliferation. Mild to severe fluid accumulations can appear under the skin of the abdomen and hind legs. Hemorrhaging also occurs in multiple tissues, especially the liver, kidney, heart tissue, and digestive tract. Red-tinged or clear fluid accumulations may appear in the body cavity. Turtles infected with this virus show overall weakness, swollen eyelids, exhibit discharge from the mouth and nose, and the tongue and palate often shows dull white or thick yellow plaques. At times turtles may have ulcers on the bottom of their feet. Conjunctivitis and subcutaneous edema of the palpebra and neck have also been noted. Mortality is high and other clinical signs can include pharyngeal ulcers, skin sloughing, and marked lethargy (Duncan 2011). White dissection plaques can also be found in the pharynx and esophagus. Infections spread throughout the body affecting many organs, including blood vessels. Other studies show consistent lesions in affected turtles included necrotizing stomatitis and/or esophagitis, fibrinous and necrotizing splenitis, and multicentric fibrinoid vasculitis. Intracytoplasmic inclusion bodies were rarely observed in affected tissues (Johnston et al. 2008). In terrestrial turtles lesions are perhaps more difficult to detect as they are primarily in the oral cavity and associate with internal organs (typically respiratory and gastrointestinal), but can also include eye and nasal discharges. Aquatic turtles exhibit hemorrhages and ulcers, with the ulcerations occurring along respiratory and digestive tracts. Death results from organ dysfunction and secondary infection by other pathogens. Potential Impacts There is no question that Ranavirus outbreaks are now common and the virus is widespread. Allender et al. (2013) examined 606 eastern box turtles from across the southeastern United States and found a 1.3% prevalence of Ranavirus. There was a higher infection rate in juveniles than adults, but the difference was not significant. This seemingly low percentage of infected box turtles is misleading, and the authors of the study suggest that the low detection prevalence is a result of the quick time from exposure to development of the disease and death of the turtles. This would mirror the findings for amphibian Ranavirus mortality with the difference being the sick and dead amphibians are more easily identified due to their seasonal concentrations at breeding sites. In that a number of our endangered and threatened species have restricted distributions and survive in relatively small populations, they are potential targets for extinctions resulting from ranaviruses. So too are numerous peripheral populations of reptiles and amphibians, many of which are state-listed as species of conservation concern. Species endemic to specific springs and spring runs, those confined to narrow elevation zones on isolated mountains, and fish and turtles whose distributions are limited to single drainage systems would seem very vulnerable. In the latter case infected bait-fish released by fisherman could contaminate independent drainage systems with novel ranaviruses. Subterranean species of blind cave-dwelling fishes and salamanders are at risk. Aquatic cave-dwelling animals typically live at very low population levels and entire underground aquatic systems could quickly succumb to the virus. Lee (1969) reported on the occurrence of bullfrogs in pools deep within cave systems, and other types of amphibians commonly inhabit the twilight zones of caves. Both represent potential avenues of Ranavirus transport into subterranean systems where the cool ambient temperatures of cave systems would prove favorable to the virus. The spread of Ranavirus to sites harboring isolated amphibians could result in loss of subpopulations. Narrow range endemics, as well as relict, disjunct and peripheral populations are vulnerable, and could be quickly extirpated. Distant transport of the pathogen by contaminated boots, field equipment, or release of infected animals to biologically significant remote sites is a real concern. This is exacerbated by the fact that because of the different strains of Ranavirus, and their ability to infect a wide spectrum of hosts. Over time the emergence of novel viruses could occur across a broad landscape. Furthermore, roads, pipe lines, and development have fragmented landscapes to the point that if isolated populations of even common and widespread species are extirpated, natural recolonization is unlikely to occur. Nonetheless, development induced isolation does not fully protect sites from exposure to ranavirus as there are a number of anthropomorphic dispersal mechanisms for the virus. People maintaining captive collections of turtles and tortoises outdoors run the risk of locally occurring amphibians infecting them. Native frogs frequently take up residence in outdoor pools set up for aquatic turtles, and even a single infected frog could easily contaminate an entire collection. Additionally, in that Ranavirus can survive in fresh and frozen fish this is another means for the disease to infect facilities maintaining captive turtles. How we are likely aiding and abetting: This virus can remain viable outside a host for 30 days or more. Boots and field equipment that come in contact with water and sediments contaminated with Ranavirus can later spread the pathogen to other areas. This is also likely to occur with the chytrid fungus (Batrachochtrium dendrobatidis) that affects amphibians. The spread of these diseases is also the result of visitation to wetlands for recreational activities. Studies conducted in the Great Smokey Mountains National Park found a higher Ranavirus prevalence in salamanders at sites with high public access. Additionally, livestock and agricultural pesticides in wetland areas stress hosts increasing the likelihood of Ranavirus outbreaks. The release of individual captive animals is an ongoing problem. Virus-infected pets, both commercially purchased and wild-caught captives, can harbor asymptomatic ranaviruses, and the serendipitous broadcasting of the disease to native species is a major concern. While some states have regulations forbidding the release of captive reptiles and amphibians into the wild, they are almost impossible to enforce. Additionally many well-intended people and organizations translocate animals to new localities as natural habitats are lost to development. This is yet another avenue for the unintended dispersal of Ranavirus. Attention needs to be focused on wildlife rehabilitation centers. Often their goal is to help sick animals resolve their health issues so they can eventually be released. Fortunately the staffs of the centers can be trained to identify clinical signs and reptiles and amphibians can be tested for the virus prior to release. Establishment of exotic species may also be a problem. Ranavirus has recently been documented in Anolis lizards in Florida (Stohr 2013). South Florida is probably the exotic Anolis capital of the world; in many areas it is impossible to even find the original native species as they have largely been replaced with introduced species. Florida has the most diverse assemblage of native lizards in eastern North America, a diversity now threatened by ranaviruses. Problems will surface as these lizards, as well as other exotic species, continue to spread and exploit native plant communities. Fish hatcheries and commercial aquaculture practices can rapidly produce new Ranavirus strains. Different studies have shown that ranaviruses collected from hosts raised and maintained in captive facilities, such as bullfrog farms and bait-stores selling minnows, were more virulent than those found in wild populations. The appearance of this virus in Japan is suspected to have originated from captive-raised frogs being released into the wild (Une et al. 2009). The number of fish hatcheries, fish farms, and commercial facilities where people pay to catch farm-raised fish in the United States is phenomenal. In North Carolina alone sales of farm-raised freshwater fish exceeds $16.5 million. On a worldwide basis 47% of the food fish consumed are farm raised. The hatcheries, run both by federal and state agencies, as well as those managed by the private sector, present some major issues. The hatcheries overseen by wildlife agencies maintain their facilities for stocking streams and lakes for fishermen, while the private hatcheries sell the hatchery-produced fish to individuals wishing to stock, or restock private farm ponds. In both cases the fish are dispersed widely, as is the potential for the rapid wholesale spread of Ranavirus infected fish. Nelson (2010) reported Ranavirus from two ponds at Harrison Lake National Fish Hatchery in Charles City County, Virginia. Based on this the author then examined tadpoles from four warm-water fish hatcheries in Virginia to determine if they were infected with Ranavirus. The virus was detected in tadpoles in three of the four warm-water Virginia hatcheries. Temperature and the length of time a pond is filled with water were significant predictors of the proportion of tadpoles that tested positive for Ranavirus. Similar results were found by Nelson over multiple years. Obviously precautions should be taken to ensure that ranaviruses are not spread when fish are transferred from one hatchery to another, or to the wild, but also of concern is the likelihood of the spread of the virus via the native amphibians that use hatcheries and fish farms as breeding sites. Some garden centers that supply plants and other items for backyard outdoor pools also sell tadpoles for stocking garden ponds. While the wholesale suppliers of these tadpoles vary from store to store, clearly the stock does not necessarily come from local sources. No matter the origin of these tadpoles, as well as the fish and aquatic plants offered for sale from the same display containers, they are potential dispersal agents for Ranavirus. In addition biological supply companies supply tadpoles for classroom use so that students can witness metamorphosis, often the young frogs are released after they transform. Maryland has posted a warning about this practice as it relates to the spread of Ranavirus and other diseases on their Natural Resources web site (http://www.dnr.state.md.us/wildlife/Plants_Wildlife/herps/catalogue_frogs.asp ). The release of unwanted ‘minnows’ and salamanders used as fishing bait is yet another avenue for spreading the virus. Lee and Knight (1968) described the commercial sale of native salamanders for fishing bait in the eastern United States in the 1960s. While for the most part the commercial aspects of this are no longer in effect due to current wildlife regulations, the non-commercial practice continues. The appearance of Ranavirus outbreaks in the central United States was attributed to the sale and use of infected tiger salamander larvae (Ambystoma tigrinum) for fishing bait (Ridenhour and Storfer 2008). This salamander is also widely used as bait in the Southwestern states. This, of course, leads to the question as to the extent of infections being spread from large lots of turtles distributed to domestic and foreign retail stores from our Southeastern turtle farms. They annually market over 200,000 hatchling turtles, mostly red-eared sliders, within the United States, and sell 10 million overseas. In addition there are turtle farms where turtles are raised for meat, and others specializing in exotic and other high-end species for sale to the hobbyist. Due to the nature of turtle farming, where large numbers of adult turtles are maintained in overcrowded conditions where breeding stocks constantly are being supplemented with additional wild-caught turtles, not only are outbreaks of ranaviruses likely, it has been demonstrated that novel strains have developed in aquaculture breeding facilities. Furthermore, native amphibians make use of the farm ponds and, in many cases, the turtles are fed heads and other scraps of fish raised on commercial fish farms. This presents yet another way in which the virus can enter turtle farm operations. Not only are the young widely distributed with the chance of retail purchased pet turtles being released when they are no longer wanted, but as in other outdoor aquatic farming operations, storms and floods can result is mass escape of infected breeding stocks. Storm induced spillover of Ranavirus infected water into local creeks and streams could also present a problem. Robert et al. (2007) identified African clawed frogs as a possible vector for Ranavirus. They found that adult frogs typically clear FV3 infections within a few weeks, but viral DNA was still present in their kidneys several months after they were experimentally infected. The virus was also detected in seemingly healthy frogs that were not deliberately infected. In this study the authors hypothesized that “covert FV3 infection” may occur in Xenopus. Based on this, and other aspects of their study, these findings suggest that FV3 can become dormant in resistant species making some species viral reservoirs. The use of African clawed frogs for this research is interesting in that during the 1950s and 60s this species was widely used for pregnancy testing. The species was imported in large numbers and shipped to clinics and hospitals throughout the country. In following decades a smaller species, the African Dwarf clawed frog, Hymenochirus sp., was imported and commonly sold along with aquarium fish in pet stores. The commercial global distribution of African clawed frogs is reportedly responsible for spreading chytrid fungus and accounts for the extinctions of various native frog faunas --i.e., 30 species wiped out in a Panama forest (Lee 2013). Today there are a number of businesses, like Xenopus Express, that supply clawed frogs for medical use, research centers, and the pet trade throughout the country and as well as in international sales. As pointed out earlier (Lee 2012) Ranavirus could become particularly troublesome as a result of turtle races where wild caught, non-native captives, and pet store purchased turtles and tortoises, are all mixed together at the events. Some of these events are even held back to back with frog-jumping contest. While this virus is a serious concern, the potential impact on native turtle populations is but one of a number of important reasons that these turtle derbies should be restructured, if not eliminated altogether. A committee of people working in meetings for months would be hard pressed to come up with a more cost efficient and effective means than turtle derbies to spread a deadly pathogen into our native populations of reptiles and amphibians. Add to this mix the various wholesale farm-bred fish, frogs and turtles imported from Asia and sold live as food items in Asian markets across the United States. These would prove likely vectors for ranaviruses, and possibly a source for establishing new strains of the disease in this country. A decade or so back I purchased several adult frogs from an Asian market for testing and they all were positive for chytrid fungus. This was brought to the attention of our state wildlife agency, but they were unwilling to enforce their injurious wildlife regulations due to possible ethnic backlash. Another issue is the release of fish, turtles and frogs by Buddhists-- a practice resulting from a cultural/religious history going back at least 2,000 years. Because of this certain Buddhists sects release store purchased birds, fish, turtles and other creatures. The belief is that freeing animals back into the wild is a means of achieving blessing, and turtles and tortoises are considered as the most karmaically valuable animals to release. The people are not particularly concerned with the survival of the animal; to receive blessings they simply buy and release them. This same practice occurs not just in Asia, but also in the US and Canada with goldfish and hatchling sliders being the most common subjects for release (see Maclachlan 2011 and Laio and Lee 2012). At other times Buddhists will purchase and release creatures when family members are sick, believing it helps with the healing process. Releases may occur daily until the person is fully recovered. This practice is feasible because of the low cost of a number of commercially available species. Twenty young sliders, for example, can be purchased in China for the US equivalent of $15 (CNY 100). The releases are not limited to hatchlings; adult and sub-adult sliders are often released en mass. And lets not forget all the captive animals held in classrooms, typically individual locally caught creatures, brought in by students and held on display for the remainder of the school year. Usually they are assigned to some student to release prior to the summer recess. Diagnosis, Testing and Treatment for Ranavirus: Polymerase chain reaction (PCR) is the most useful test and is becoming more widely available. Real-time PCR techniques allow detection of smaller amounts of virus, but to identify the group type (ATV or FV3 virus-like) of Ranavirus present, conventional PCR with DNA sequencing is required. Determining the specific species of Ranavirus usually requires cell culture, virus isolation, and molecular characterization. These techniques are not widely available outside of research laboratories. Conventional PCR can provide false-positive results if confirmatory DNA sequencing or Southern blot analysis is not performed. Histopathology is helpful to screen for lesions in sick animals, but lesions tend to be nonspecific unless intracytoplasmic inclusion bodies are seen. Virus isolation, immunohistochemistry, transmission electron microscopy, cell culture, and serology (not widely available or validated for most species) have also been used to identify infected animals (Duncan 2011). For laboratory analysis the best choice for tissue samples are ones collected at necropsy, especially liver, kidney and skin (if lesions are present). Frozen tissues are required for virus isolation and are generally best for molecular analysis as well, however, freezing does not work for histology. For histology, tissues should be submitted fresh or fixed in 70% ethanol or 10% neutral buffered formalin. Ethanol-preserved tissues may be used for some molecular testing. Formalin-fixed tissues may also be used for some molecular testing if the length of time in formalin is minimal (days to weeks). It is possible to perform PCR on paraffin-embedded tissues. Samples can also be collected from clinically ill animals via cloacal or pharyngeal swabs, tissue biopsy (tail clips), or blood samples. Plastic handled, rayon tipped swabs are preferable for collection of PCR samples. If living animals are tested, results should be interpreted with caution, recognizing test limitations- a positive test result is more reliable than a negative result. Test sensitivity for antemortem PCR increases with time post-exposure and development of clinical signs of illness (Duncan 2011). Individual laboratories can provide more information regarding screening . While Ranavirus outbreaks are typically fatal, Allison et al. (2013) developed protocols to treat diseased turtles and prevent the virus from spilling over to other captive animals at the Maryland Zoo. Their work resulted in the survival of 14 of 27 captive eastern box turtles after an outbreak of Ranavirus in the summer of 2011. Their methods included strict quarantine guidelines, modified environments, intensive care-including nutritional support, and extensive multimodal medical treatment by the zoo’s veterinary staff. The surviving turtles all successfully over wintered, far exceeding previous survival rates for box turtles with this virus. Hauserman et al. (2013) used 11 of these turtles to determine if they had developed an immunity response to the virus. Seven turtles were inoculated with a dose of the same strain of the virus and four controls were injected with an equal volume of saline. The turtles were monitored for 9 weeks. Only one of the re-infected and none of the controls died. Except for the turtle that died, the inoculated turtles showed only minor signs of the virus, suggesting that the turtles acquired some level of immunity from their earlier exposure. The single box turtle that died exhibited intracytoplasmic inclusion bodies in the kidney, lungs, pancreas, liver, and vas deferens; vasculitis in the spleen, pancreas, lungs and liver; nephritis; pneumonia; esophagitis; hepatitis; and enteritis. (It appears that the virus gained access to the zoo’s outdoor box turtle exhibit by a visitor adding an additional turtle to the group. When the exposed turtles were brought in for treatment, one additional, previously unmarked, individual was discovered in the group.) Quantitative tests have been developed that are 100% effective in detecting frog virus (FV3) in turtles. FV3 DNA can be identified in whole blood samples, oral swabs and cloacal swabs. Clinical indications of viral infections seen in experimentally infected red-eared sliders include lethargy, conjunctivitis, oral plaques and ulcers, while those in box turtles were fractures and diarrhea. Treatment with anti-viral therapy is reported to have poor success. Red-eared sliders, Trachemys scripta scripta, that were experimentally exposed to the FV3 virus had higher mortality rates when maintained at 22 C than at 28 C, suggesting that Ranavirus is less successful at higher temperatures. Analysis of infected box turtles showed a single oral dose of valcyclovir to have a positive effect, and that it may prove to be useful against the virus (Allender et al. 2013). Precautions and actually doing something about this: Education regarding handling, maintaining, breeding, transporting, and selling farm-raised fish, bait fish, ornamental fish for outdoor ponds, and wild and captive bred pet trade reptiles and amphibians will become increasingly important. Restoration projects and stream and lake stocking that involve release of fish, reptiles and amphibians will need to verify that the released animals are free of the virus. In addition, field biologists, recreationists and the general public will likewise need to be aware of the issues caused by ranaviruses. It is important that wildlife biologists working for government agencies, zoological facilities and wildlife rehabilitation centers understand the threat posed by ranaviruses and take proactive roles in preventing further spread of the disease. Monitoring subsets of wild populations and captive collections of turtles and amphibians would be beneficial to track and control the spread and extent of this virus. Populations of rare and endangered species deserve special attention and commercial imports and shipments of fish, reptiles and amphibians, particularly ones reared en mass on farms and shipped in wholesale quantities for retail sales to the public, need to be regularly checked for the disease. Partly as a result of concerns for Ravavirus outbreaks a number of state agencies have started taking a close look at turtle races. Maryland’s DNR has made it known that turtles and frogs entered in race events can not be released back into the wild once the races are over. Both Pennsylvania and Maryland have started enforcing the illegal entry of state protected species (in PA this includes box turtles) in turtle races. In part, based on Herp Digest’s Internet circulation of concerns about these races (Lee 2012), several race sponsors canceled races all together (e.g., Moss 2013b). At a number of events turtle advocate organizations are screening turtles to help insure that visibly sick and diseased turtles are not entered in the events, or allowed to have contact with other turtles. In the summer of 2013 a number of race sponsors across the country were approached by conservation organizations and asked to alter the way the races are currently conducted or to plan alternate events (e.g., Moss 2013a). The Tortoise Reserve has information prepared by the veterinary community on the various reptile diseases that could be spread by turtle race activities. This is available to individuals or organizations interested in educating the various race sponsors via the Tortoise Reserve. Subsequently the Center for Biological Diversity started contacting sponsors of turtle races explaining their unintended consequences and suggesting that they modify the way races are conducted. Prior to the 4th of July turtle race in BelAir, Maryland, the Susquehannock Wildlife Society posted an online commentary about the problems the annual race was causing native wildlife asking the race sponsors to suspend the event in future years (http://www.daggerpress.com/2013/06/30/susquehannock-wildlife-society-calls-on-public-to-leave-wildlife-in-the-wild-this-fourth-of-july/ ). The majority of the comments posted were quite supportive of the Society’s position, but it is interesting to read the mind-sets of some of the people commenting on this post who strongly believe that such traditional events should not be altered. Preventing the spread of this virus will be taxing for those of us working daily with captive reptiles and amphibians. Disinfection of supplies, equipment, water dishes and caging that come in contact with the animals, or their water, is important. One minute contact with solutions of 3 percent bleach, 0.75 percent Nolvalsan ® (chlorhexidine dicetate), or 1 percent Virkon S ® (potassium peroxymonosulfate) are effective in killing the virus. Nolvalsan ® is least toxic to amphibians. Disposable vinyl gloves should be rinsed, disinfected, or changed when handling different animals. While doing this is often not practical under field conditions, or when dealing with captive collections, minimally it should become standard protocol when changing field sites, or when exchanging specimens and housing between live collections. Particular care needs to be taken in and around habitats such as isolated wetlands that harbor peripheral populations, endemic species, species of state concern, and threatened and endangered species. Protocols need to be developed for people requesting access to these sites, and access will probably best be limited to those with permits and training. The release of captive amphibians and turtles will need to be limited to animals that have been tested for the virus. Stocking streams and lakes from fish hatcheries may no longer be a viable option. Additionally the sales of live fish, salamanders, frogs and turtles commercially raised on farms for bait, stocking, food and pets will need to be addressed, as possible release of these creatures into the wild by well-intended people will be difficult if not impossible to enforce. People overseeing zoos and private collections of amphibians and turtles need to be aware of the problem, particularly when acquiring new stock. Grey and Miller (2013) and Hoverman et al. (2012) point out “natural resource agencies should consider conducting surveillance studies to identify infection hotspots, where ranavirus prevalence exceeds 40 percent.” Once hotspots are located agencies can identify the mechanisms driving them, determine effects on populations, and come up with intervention strategies. Green et al. (2009) provide recommendations regarding sample size to detect the presence of Ranavirus as it relates to approximate host population size, and a 95% confidence level for detection. Large numbers of individuals from any given population will need to be tested to attain meaningful confidence levels. It is interesting to note that the US Department of Agriculture is poised to act quickly to oversee and regulate interstate movement of domestic animals and products that might possibly be infected with diseases where outbreaks can effect livestock, or spread Mediterranean fruit flies. Yet, diseases that seriously impact non-commercial native wildlife, and can be easily spread by our activities, continue to remain unchecked. People interested in, and working with, reptiles and amphibians approach them from different perspectives. Academic researchers investigating wild populations deal with these animals quite differently than those working with them in labs. Amateur herpetologists who enjoy finding reptiles and amphibians in the field often share little in common with those who maintain captive collections of various color morphs and non-native species. Zoos, museums and nature centers are interested in educational displays, while veterinarians and wildlife rehabilitation groups focus on the health of individual animals. In addition, there are commercial collectors, exporters, importers, wholesale distributors, reptile show sponsors and pet shops. Boy Scouts working on merit badges, people rescuing turtles from roads, and retail purchasers are yet other user groups. They, along with the wildlife agencies overseeing the welfare of these animals, all need to be educated as to the plastic nature of ranaviruses if we are to have any hope of keeping this disease from becoming an outright worldwide plague. Due to the broad, and growing, range of host species this pathogen is becoming a major threat to a significant portion of the earth’s vertebrate fauna. With one in three species of amphibians and over 40% of the world’s turtles already at risk of extinction the virus poses a serious additional threat to global biodiversity. Its impact likewise represents a significant problem for aquatic community composition and to the overall functioning of wetland and terrestrial ecosystems. The serious consequences of this virus, excuse the pun, going viral cannot be overstated. We are witnessing a disease that can covertly breach the protective boundaries of state and national parks, wildlife refuges, and any number of private wildlife sanctuaries and similar lands that have been set aside to permanently preserve natural systems. With the potential consequences of expanding Ranavirus outbreaks on our native frogs and toads perhaps a follow up book to Rachel Carlson’s 1962 classic Silent Spring entitled Silent Night is now in order. A more fitting title might be Night of the Living Dead. [For additional information see Manual Diagnostic Test for Aquatic Animals 2012. Infections with ranavirus. Chapter 2.1.2: 71-91, and the literature cited within http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/2.1.02_RANAVIRUS.pdf ] Acknowledgements: I thank Gregory A. Lewbart VMD, College of Veterinary Medicine, N.C. State University for reviewing this manuscript. Literature Cited: Allender, M., M. A. Mitchell, and S. Cox. 2013. Epidemiology and treatment of ranaviral diseases in North American Chelonians (abstract). Box Turtle Conservation Workshop (March 22-23, 2013), NC Zoological Park, Asheboro, NC. Allison, N. W., R. Sim, K. J. Murphy, K. Barrett, and E. Bronson. 2013. Husbandry techniques used during a Ranavirus outbreak in eastern box turtles (Terrapene carolina carolina) at the Maryland Zoo in Baltimore (abstract). Box Turtle Conservation Workshop (March 22-23, 2013), NC Zoological Park, Asheboro, NC. Cinchar, V. D. 2002. Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Archives of Virology 147(3): 147-470. Duncan, A. E. 2011. Ranavirus. American Association of Zoo Veterinarians Infectious Disease Committee Manual. (Jan 2011) Granoff, A., P. E. Came, and K. A. Rafferty. 1965. The isolation and properties of viruses from Rana pipiens: their possible relationships to the renal adenocarcinoma of the leopard frog. Annals of New York Academy of Science 126(1): 273-255. Gray, M. J. and D. L. Miller. 2013. The Rise of Ranavirus. The Wildlife Society 7(1): 51-55. Green, D. E., M. J. Gray and D. L. Miller. 2009. Disease monitoring and biosecurity. 481-506. in C. K. Dodd Jr. (ed.). Amphibian Ecology and Conservation: a handbook of techniques. Techniques in Ecology and Conservation Series. Oxford Univ. Press 529 pp. Hausmann, J. C., A. N. Wack, M. C. Allendar, M. R. Cranfield, K. J. Murphy, K. Barrett, J. L. Romero, J. F. Wellehan, C. Zink, and E. Bronson. 2013. Experimental challenge study of Ranavirus infection in previously infected eastern box turtles (Terrapene carolina carolina) to assess immunity. Box Turtle Conservation Workshop (March 22-23, 2013), NC Zoological Park, Asheboro, NC. Hoverman, J. T., M. J. Gray, D. L. Miller and N. A. Haislip. 2012. Widespread occurrence of ranavirus in pond-breeding amphibian populations. EcoHealth 9(1): 36-48. Jacobson E. R. 2007. Infectious Diseases and Pathology of Reptiles. CRC Press, Boca Raton, FL. 716 pp. Jancovich, J. B., M. Bremont, J. W. Touchman and B. L. Jacobs. 2010. Evidence for multiple recent host species shifts among the Ranaviruses (Family Iridoviridae). Jour. Virology. 84 (6): 2636-2647. Johnson, A. J., A. P. Pessier, J. F. Wellehan, A. Childress, T. M. Norton, N. L. Steadman, D. C. Bloom, W. Belzer, V. R. Titus, R. Wagner, J. W. Brooks, J. Spratt, and E. R. Jacobson. 2008. Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States. Jour. Wildlife Disease 44(4): 851-863. Langdon, J. S. 1989. Experimental transmission and pathogenicty of epizootic haematopoietic necrosis virus (EHNV) in redfin perch, Perica fluviatilis, and 11 other teteosts. Jour. Fish Disease 12: 295-310. Liao, S. K. and D. S. Lee. 2013. Turtles without passports: red-eared sliders come to China. Radiata 22(1): 35-52. Lee, D. S. 1969. Notes on the feeding behavior of cave-dwelling bullfrogs. Herpetologica 25(3): 211-212. Lee, D. S. 2012. Hot tracks, fast turtles- the unforeseen consequences of well-intended turtle derbies. Bull. Chicago Herp. Soc. 47(10): 121-130. Lee, D. S. and E. L. Knight. 1968. The use of salamanders as fishing bait. Bull. Maryland Herpetological Society 4(4): 86-88. Lee, J. J. 2013. African clawed frog spreads deadly amphibian fungus. National Geographic (May 2013) Mader D. R. 2006. Reptile Medicine and Surgery, Second Edition. Elsevier/Saunders Co., Phila. 1262 pp. Maclachlan, M. 2011. Food fight: Controversy a new over live-animal markets. Capitol Weekly, 3 Feb. 2011. Moss, T. 2013a. Danville turtle races won’t have turtles next year. The News Gazette [Illinois] (08/20/2013) Moss, T. 2013b. UI scientist worried about the cause of turtle deaths. The News Gazette [Illinois] (06/04/2013) Nelson, J. 2010. The presence of amphibian Ranavirus in Virginia warm water fish hatchery ponds. VCU Digital Archives. http://hdl.handle.net/10156/2932 Ridenhour, B. J. and A. T. Storter. 2008. Geographically variable selection in Ambystoma tigrinum virus (Iridoviridae) throughout the western USA. Jour of Evolutionary Biology 21(4): 1151-1159. Robert, J. L. Abramowitz, J. Gantress and H. D. Morales. 2007. Xenopus laevis: a possible vector of Ranavirus infection? Jour. Wildlife Disease. 43(4):645-652. Robert, J., E. George, F. De Jesus Andino and G. Chen. 2011. Waterborne infectivity of ranavirus frog virus 3 in Xenopus laevis. Virology 417(2): 410-417. Stohr, A. 2013. Repeated detection of Ranaviruses in Anolis lizards from Florida. 20th Annual ARVA Conference. (14-19 September 2013) Indianapolis, Indiana. Titus, Valorie R. and Timothy M. Green. 2013. Presence of Ranavirus in green frogs and eastern tiger salamanders on Long Island, New York. Herpetological Review. 44 (2): 266-267. Une, Y., A Sakuma, H. Matsueda, K. Nakel and M. Murakami. 2009. Ranavirus outbreak in North American Bullfrogs (Rana catesbeiana), Japan, 2008. Emerging Infectious Disease 15(7): http://wwwnc.cdc.gov/edi/article/15/7/08-1636.htm. Whittington, R. J., J. A. Becker, and M. M. Dennis. 2010. Iridovirus infections in finfish-critical review with emphasis on ranaviruses. Journal Fish Disease 33(2): 95-122. The Tortoise Reserve, P.O. Box 7082, White Lake, North Carolina 27614 (torres...@aol.com) Epilogue: I fear the above may seem to be simply preaching gloom and doom; it is important for all of us to remain upbeat in attempts to protect our native fauna. Since it is unlikely that agencies will be able to respond quickly with proactive solutions there is another option. If we can stick with the current plan and the continued lack of coverage of several major news outlets regarding our total denial of human-induced climate change, this could eliminate the issue of Ranavirus outbreaks in short order. Increased ambient temperatures in temperate climates and throughout the world’s oceans should prevent the spread of ranaviruses. Increased temperatures resulting from global warming will prevent the ability of the virus to replicate itself, many of its host species will become extinct, and the issue will soon be resolved. While temperatures are not likely to increase significantly in boreal regions to prevent the survival and transmission of ranaviruses, there are very few reptiles and amphibians now living in those zones and fewer yet that are confined to them.
