Alisa L. Newton

Densovirus associated with sea-star wasting disease and mass mortality

Significance Sea stars inhabiting the Northeast Pacific Coast have recently experienced an extensive outbreak of wasting disease, leading to their degradation and disappearance from many coastal areas. In this paper, we present evidence that the cause of the disease is transmissible from disease-affected animals to apparently healthy individuals, that the disease-causing agent is a virus-sized microorganism, and that the best candidate viral taxon, the sea star-associated densovirus (SSaDV), is in greater abundance in diseased than in healthy sea stars. Populations of at least 20 asteroid species on the Northeast Pacific Coast have recently experienced an extensive outbreak of sea-star (asteroid) wasting disease (SSWD). The disease leads to behavioral changes, lesions, loss of turgor, limb autotomy, and death characterized by rapid degradation (“melting”). Here, we present evidence from experimental challenge studies and field observations that link the mass mortalities to a densovirus (Parvoviridae). Virus-sized material (i.e., <0.2 μm) from symptomatic tissues that was inoculated into asymptomatic asteroids consistently resulted in SSWD signs whereas animals receiving heat-killed (i.e., control) virus-sized inoculum remained asymptomatic. Viral metagenomic investigations revealed the sea star-associated densovirus (SSaDV) as the most likely candidate virus associated with tissues from symptomatic asteroids. Quantification of SSaDV during transmission trials indicated that progression of SSWD paralleled increased SSaDV load. In field surveys, SSaDV loads were more abundant in symptomatic than in asymptomatic asteroids. SSaDV could be detected in plankton, sediments and in nonasteroid echinoderms, providing a possible mechanism for viral spread. SSaDV was detected in museum specimens of asteroids from 1942, suggesting that it has been present on the North American Pacific Coast for at least 72 y. SSaDV is therefore the most promising candidate disease agent responsible for asteroid mass mortality.

Wildlife cancer: a conservation perspective

Until recently, cancer in wildlife was not considered to be a conservation concern. However, with the identification of Tasmanian devil facial tumour disease, sea turtle fibropapillomatosis and sea lion genital carcinoma, it has become apparent that neoplasia can be highly prevalent and have considerable effects on some species. It is also clear that anthropogenic activities contribute to the development of neoplasia in wildlife species, such as beluga whales and bottom-dwelling fish, making them sensitive sentinels of disturbed environments.

Canine Distemper Virus: an Emerging Disease in Wild Endangered Amur Tigers (Panthera tigris altaica)

ABSTRACT Fewer than 500 Amur tigers (Panthera tigris altaica) remain in the wild. Due to low numbers and their solitary and reclusive nature, tiger sightings across their range in the Russian Far East and China are rare; sightings of sick tigers are rarer still. Serious neurologic disease observed in several wild tigers since 2001 suggested disease emergence in this endangered species. To investigate this possibility, histology, immunohistochemistry (IHC), in situ hybridization (ISH), and reverse transcription-PCR (RT-PCR) were performed on tissues from 5 affected tigers that died or were destroyed in 2001, 2004, or 2010. Our results reveal canine distemper virus (CDV) infection as the cause of neurologic disease in two tigers and definitively establish infection in a third. Nonsuppurative encephalitis with demyelination, eosinophilic nuclear viral inclusions, and positive immunolabeling for CDV by IHC and ISH were present in the two tigers with available brain tissue. CDV phosphoprotein (P) and hemagglutinin (H) gene products were obtained from brains of these two tigers by RT-PCR, and a short fragment of CDV P gene sequence was detected in lymph node tissue of a third tiger. Phylogenetically, Amur tiger CDV groups with an Arctic-like strain in Baikal seals (Phoca siberica). Our results, which include mapping the location of positive tigers and recognition of a cluster of cases in 2010, coupled with a lack of reported CDV antibodies in Amur tigers prior to 2000 suggest wide geographic distribution of CDV across the tiger range and recent emergence of CDV as a significant infectious disease threat to endangered Amur tigers in the Russian Far East. IMPORTANCE Recognition of disease emergence in wildlife is a rare occurrence. Here, for the first time, we identify and characterize a canine distemper virus (CDV), the second most common cause of infectious disease death in domestic dogs and a viral disease of global importance in common and endangered carnivores, as the etiology of neurologic disease and fatal encephalitis in wild, endangered Amur tigers. We establish that in 2010 CDV directly or indirectly killed ~1% of Amur tigers. Location of positive cases over an expansive geographic area suggests that CDV is widely distributed across the tiger range. Interspecies interactions are increasing as human populations grow and expand into wildlife habitats. Identifying animal reservoirs for CDV and identifying the CDV strains that are transmissible to and among wildlife species, including Amur tigers and sympatric critically endangered Amur leopards (Panthera pardus orientalis), is essential for guiding conservation and mitigation efforts. Recognition of disease emergence in wildlife is a rare occurrence. Here, for the first time, we identify and characterize a canine distemper virus (CDV), the second most common cause of infectious disease death in domestic dogs and a viral disease of global importance in common and endangered carnivores, as the etiology of neurologic disease and fatal encephalitis in wild, endangered Amur tigers. We establish that in 2010 CDV directly or indirectly killed ~1% of Amur tigers. Location of positive cases over an expansive geographic area suggests that CDV is widely distributed across the tiger range. Interspecies interactions are increasing as human populations grow and expand into wildlife habitats. Identifying animal reservoirs for CDV and identifying the CDV strains that are transmissible to and among wildlife species, including Amur tigers and sympatric critically endangered Amur leopards (Panthera pardus orientalis), is essential for guiding conservation and mitigation efforts.

EFFICACY OF TREATMENT AND LONG-TERM FOLLOW-UP OF BATRACHOCHYTRIUM DENDROBATIDIS PCR-POSITIVE ANURANS FOLLOWING ITRACONAZOLE BATH TREATMENT

Abstract:  All anuran specimens in the Wildlife Conservation Societys collections testing positive for Batrachochytrium dendrobatidis (Bd) were treated with itraconazole and then studied after treatment to assess the long-term effects of itraconazole and the drugs effectiveness in eliminating Bd carriers. Twenty-four individuals and eight colonies of 11 different species (75 total specimens) tested positive for Bd via polymerase chain reaction (PCR) on multicollection survey. All positive individuals and colonies were treated with a 0.01% itraconazole bath solution and retested for Bd via one of two PCR methodologies within 14 days of treatment completion, and all were negative for Bd. A total of 64 animals received secondary follow-up PCR testing at the time of death, 6–8 mo, or 12–15 mo post-treatment. Fourteen animals (14/64, 21.9%) were PCR positive for Bd on second follow-up. The highest percentage positive at second recheck were green-and-black poison dart frogs (Dendrobates auratus; 5/5 specimens, 100%), followed by red-eyed tree frogs (Agalychnis callidryas; 4/11, 36.4%), grey tree frogs (Hyla versicolor; 1/3, 33.3%), and green tree frogs (Hyla cinera; 3/11, 27.3%). Re-testing by PCR performed on 26/28 individuals that died during the study indicated 11/26 (42.3%) were positive (all via DNA extracted from formalin-fixed paraffin-embedded skin sections). However, there was no histologic evidence of chytridiomycosis in any of 27/28 individuals. The small number of deceased animals and effects of postmortem autolysis limited the ability to determine statistical trends in the pathology data, but none of the necropsied specimens showed evidence of itraconazole toxicity. Problems with itraconazole may be species dependent, and this report expands the list of species that can tolerate treatment. Although itraconazole is effective for clearance of most individuals infected with Bd, results of the study suggest that repeat itraconazole treatment and follow-up diagnostics may be required to ensure that subclinical infections are eliminated in amphibian collections.

A Mycoplasma Species of Emydidae Turtles in the Northeastern USA

Abstract Mycoplasma infections can cause significant morbidity and mortality in captive and wild chelonians. As part of a health assessment of endangered bog turtles (Glyptemys muhlenbergii) in the northeastern US, choanal and cloacal swabs from these and other sympatric species, including spotted turtles (Clemmys guttata), eastern box turtles (Terrapene carolina carolina), wood turtles (Glyptemys insculpta), and common snapping turtles (Chelydra serpentina) from 10 sampling sites in the states (US) of Delaware, New Jersey, and Pennsylvania, were tested by PCR for Mycoplasma. Of 108 turtles tested, 63 (58.3%) were PCR positive for Mycoplasma including 58 of 83 bog turtles (70%), three of three (100%) eastern box turtles, and two of 11 (18%) spotted turtles; all snapping turtles (n = 7) and wood turtles (n = 4) were negative. Sequence analysis of portions of the 16S–23S intergenic spacer region and the 16S ribosomal RNA gene revealed a single, unclassified species of Mycoplasma that has been previously reported in eastern box turtles, ornate box turtles (Terrapene ornata ornata), western pond turtles (Emys marmorata), and red-eared sliders (Trachemys scripta elegans). We document a high incidence of Mycoplasma, in the absence of clinical disease, in wild emydid turtles. These findings, along with wide distribution of the identified Mycoplasma sp. across a broad geographic region, suggest this bacterium is likely a commensal inhabitant of bog turtles, and possibly other species of emydid turtles, in the northeastern US.

Three Novel Herpesviruses of Endangered Clemmys and Glyptemys Turtles

The rich diversity of the world’s reptiles is at risk due to significant population declines of broad taxonomic and geographic scope. Significant factors attributed to these declines include habitat loss, pollution, unsustainable collection and infectious disease. To investigate the presence and significance of a potential pathogen on populations of critically endangered bog turtles (Glyptemys muhlenbergii) as well sympatric endangered wood (G. insculpta) and endangered spotted (Clemmys guttata) turtles in the northeastern United States, choanal and cloacal swabs collected from 230 turtles from 19 sites in 5 states were screened for herpesvirus by polymerase chain reaction. We found a high incidence of herpesvirus infection in bog turtles (51.5%; 105/204) and smaller numbers of positive wood (5) and spotted (1) turtles. Sequence and phylogenetic analysis revealed three previously uncharacterized alphaherpesviruses. Glyptemys herpesvirus 1 was the predominant herpesvirus detected and was found exclusively in bog turtles in all states sampled. Glyptemys herpesvirus 2 was found only in wood turtles. Emydid herpesvirus 2 was found in a small number of bog turtles and a single spotted turtle from one state. Based on these findings, Glyptemys herpesvirus 1 appears to be a common infection in the study population, whereas Glyptemys herpesvirus 2 and Emydid herpesvirus 2 were not as frequently detected. Emydid herpesvirus 2 was the only virus detected in more than one species. Herpesviruses are most often associated with subclinical or mild infections in their natural hosts, and no sampled turtles showed overt signs of disease at sampling. However, infection of host-adapted viruses in closely related species can result in significant disease. The pathogenic potential of these viruses, particularly Emydid herpesvirus 2, in sympatric chelonians warrants additional study in order to better understand the relationship of these viruses with their endangered hosts.

Emydid herpesvirus 1 infection in northern map turtles (Graptemys geographica) and painted turtles (Chrysemys picta)

A captive, juvenile, female northern map turtle (Graptemys geographica) was found dead following a brief period of weakness and nasal discharge. Postmortem examination identified pneumonia with necrosis and numerous epithelial, intranuclear viral inclusion bodies, consistent with herpesviral pneumonia. Similar intranuclear inclusions were also associated with foci of hepatocellular and splenic necrosis. Polymerase chain reaction (PCR) screening of fresh, frozen liver for the herpesviral DNA-dependent DNA polymerase gene yielded an amplicon with 99.2% similarity to recently described emydid herpesvirus 1 (EmyHV-1). Molecular screening of turtles housed in enclosures that shared a common circulation system with the affected map turtle identified 4 asymptomatic, EmyHV-1 PCR-positive painted turtles (Chrysemys picta) and 1 asymptomatic northern map turtle. Herpesvirus transmission between painted and map turtles has been previously suggested, and our report provides the molecular characterization of a herpesvirus in asymptomatic painted turtles that can cause fatal herpesvirus-associated disease in northern map turtles.

A Retrospective Study of Mortality in Varanid Lizards (Reptilia:Squamata:Varanidae) at the Bronx Zoo: Implications for Husbandry and Reproductive Management in Zoos

Varanid lizards have been maintained in zoological parks for more than a century, yet few studies to date have attempted to pinpoint significant health issues affecting their management or areas of captive husbandry that are in need of improvement. In an effort to identify and better understand some of the husbandry-related challenges and health issues specifically affecting varanids in zoos, this study examined mortality in 16 species maintained at the Bronx Zoo between 1968 and 2009. Out of 108 records reviewed, complete necropsy reports were available for 85 individuals. Infection-related processes including bacterial (15.3%), protozoal (12.9%), nematode (9.4%), and fungal (3.5%) infections accounted for the greatest number of deaths (47.1%). Noninfectious diseases including female reproductive disorders (7.1%), neoplasia (7.1%), gout (10.8%), and hemipenal prolapse (1.3%) accounted for 29.4% of deaths. Multiple disease agents were responsible for 5.9% of deaths, and a cause for death could not be determined for 17.7% of individuals. Reproductive complications accounted for 11.5% of female deaths, but were identified in 23.1% of females. Although not necessarily the cause for death, gout was present in 18.8% of individuals. Differences in mortality between species, genders, and origin (captive-bred vs. wild-caught) were also evaluated. The results of this study corroborate earlier findings that identify bacterial infections, neoplasia, female reproductive disorders, gout, and endoparasitism as major sources of mortality in captive varanids. In light of these results, we discuss potential etiologies and offer recommendations for improving captive management practices in zoos.

MANAGEMENT OF OSTEOMYELITIS CAUSED BY SALMONELLA ENTERICA SUBSP. HOUTENAE IN A TAYLOR'S CANTIL (AGKISTRODON BILINEATUS TAYLORI) USING AMIKACIN DELIVERED VIA OSMOTIC PUMP

Abstract An adult female Taylors cantil (Agkistrodon bilineatus taylori) presented with marked spinal and mandibular osteomyelitis that cultured positive for Salmonella enterica subsp. houtenae, serovar IV 43:z4,z32:-. Progression of osteomyelitis was arrested by treatment using amikacin (0.026 mg/kg per hour) delivered via subcutaneous osmotic pump for 10 mo, replacing the pump every 4 wk. No adverse effects on renal function were appreciated throughout the course of therapy. Amikacin therapy was discontinued after improvement with treatment, but 5 mo later, bony lesions worsened, and an additional abscess formed at the previous pump site. The animals condition declined and euthanasia was elected. Postmortem examination confirmed marked osteomyelitis with Salmonella infection of same serovar as the initial biopsy. This report highlights the pathogenicity of the S. enterica subsp. houtenae serovar and the ability to deliver effective amikacin dosage via osmotic pump to arrest osteomyelitis due to salmonellosis in a venomous snake.

Avian Hemosporidian Parasite Lineages in Four Species of Free-ranging Migratory Waterbirds from Mongolia, 2008

Abstract Avian hemosporidian parasites have been detected in Asia, but little information is known about the hemosporidian parasite lineages that circulate in waterbirds that migrate along the East Asian and Central Asian migratory flyways to breed in Mongolia. To gather baseline data on hemosporidian parasite presence in Mongolian waterbirds, 151 blood-spot samples (81 hatch year [HY] and 70 after hatch year [AHY]) from Bar-headed Goose (Anser indicus), Ruddy Shelduck (Tadorna ferruginea), Great Cormorant (Phalacrocorax carbo), and Mongolian Gull (Larus mongolicus) were screened for three genera of apicomplexan parasites, Plasmodium, Haemoproteus, and Leucocytozoon, using nested PCR. Of these, 17 samples (11%, 95% confidence interval: 7.1–17.4%), representing all four species, were positive. We identified 10 species (six Plasmodium, one Haemoproteus, and three Leucocytozoon) through mitochondrial DNA sequencing of the cytochrome b gene and BLAST analysis. One lineage shared 100% nucleotide identity to a hemosporidian parasite lineage that has been previously identified as Plasmodium relictum (SGS1). Six lineages were found in AHY birds and five in HY birds, the latter confirming that infection with some of the identified hemosporidian parasites occurred on the breeding grounds. Our data provide important baseline information on hemosporidian parasite lineages found in AHY waterbirds that breed and migrate through Mongolia as well as in HY offspring.