Investigation of laboratory methods for characterization of aquatic viruses in fish infected experimentally with infectious salmon anemia virus.

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon (Salmo salar). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation.

ABORTION AND NEONATAL MORTALITY DUE TO TOXOPLASMA GONDII IN BIGHORN SHEEP (OVIS CANADENSIS).

Low lamb recruitment can be an obstacle to bighorn sheep (Ovis canadensis) conservation and restoration. Causes of abortion and neonate loss in bighorn sheep, which may affect recruitment, are poorly understood. Toxoplasma gondii is a major cause of abortion and stillbirth in domestic small ruminants worldwide, but no reports exist documenting abortion or neonatal death in bighorn sheep attributable to toxoplasmosis. Between March 2019 and May 2021, eight fetal and neonatal bighorn lamb cadavers from four western US states (Idaho, Montana, Nebraska, and Washington) were submitted to the Washington Animal Disease Diagnostic Laboratory for postmortem examination, histologic examination, and ancillary testing to determine the cause of abortion or neonatal death. Necrotizing encephalitis characteristic of toxoplasmosis was identified histologically in six of eight cases, and T. gondii infection was confirmed by PCR in five cases with characteristic lesions. Other lesions attributable to toxoplasmosis were pneumonia (3/5 cases) and myocarditis (2/5 cases). Protozoal cysts were identified histologically within brain, lung, heart, skeletal muscle, adipose tissue, or a combination of samples in all five sheep with PCR-confirmed T. gondii infections. Seroprevalence of T. gondii ranged from 40-81% of adult females sampled in the Washington population in October and November 2018-2021, confirming high rates of exposure before detection of Toxoplasma abortions in this study. Of 1,149 bighorn sheep postmortem samples submitted to Washington Animal Disease Diagnostic Laboratory between January 2000 and May 2021, 21 of which were from fetuses or neonates, a single case of chronic toxoplasmosis was diagnosed in one adult ewe. Recent identification of Toxoplasma abortions in bighorn sheep suggests that toxoplasmosis is an underappreciated cause of reproductive loss. Abortions and neonatal mortalities should be investigated through postmortem and histologic examination, particularly in herds that are chronically small, demographically stagnant, or exhibit reproductive rates lower than expected.

Reproductive tract neoplasia in adult female Asian elephants (Elephas maximus).

Recent reports have highlighted a lower-than-expected prevalence of neoplasia in elephants and suggested mechanisms for cancer resistance. But despite infrequent reports in the literature, uterine neoplasia is common in managed Asian elephants (Elephas maximus). This study is an archival review of reproductive tract neoplasia in 80 adult female Asian elephant mortalities in managed care facilities in the United States from 1988 to 2019. Neoplasms occurred in 64/80 (80%) of cases. Most were in the uterus (63/64; 98%) with only a single case of ovarian neoplasia. Myometrial leiomyomas were present in 57/63 (90%) cases with uterine neoplasia. Uterine adenocarcinoma was present in 8/63 (13%) cases. Remaining cases included endometrial adenoma (2), focal carcinoma in situ in endometrial polyps (1), anaplastic carcinoma (1), endometrial hemangioma (1), primitive neuroectodermal tumor (PNET; 1), and angiosarcoma (1). One case with uterine adenocarcinoma had a separate pelvic mass histologically characterized as an anaplastic sarcoma. Distant metastases were documented in 5/8 (63%) cases of uterine adenocarcinoma, and in the uterine anaplastic carcinoma, PNET, and angiosarcoma. Four uterine adenocarcinomas and one carcinoma in situ were examined immunohistochemically for pan-cytokeratin, vimentin, and estrogen receptor. In all, neoplastic cells were pan-cytokeratin positive and vimentin negative, and in 2 cases were immunoreactive for estrogen receptor. Results show that female reproductive tract neoplasia, particularly of the uterus, is common in Asian elephants and is not limited to leiomyomas. Importantly, uterine neoplasms have the potential to impact fecundity and may represent obstacles to conservation in managed care.

Radiographic and ultrasonographic appearance of pneumonia in a frog.

A 12-year-old female African Clawed Frog (Xenopus laevis) displayed lethargy and anorexia for 1 week. Radiographs detected a moderately reduced volume of aerated lung with lobulated margins with the left lung predominantly affected. Transcelomic ultrasound revealed marked consolidation of the left lung. Postmortem examination confirmed suppurative and histiocytic pneumonia, worse in the left lung, caused by Mycobacterium chelonae. Given that amphibian pulmonary consolidation and celomic effusion can have similar radiographic findings, ultrasound may be helpful to differentiate.

Lifetime cancer prevalence and life history traits in mammals.

BACKGROUND: Cancer is a common diagnosis in many mammalian species, yet they vary in their vulnerability to cancer. The factors driving this variation are unknown, but life history theory offers potential explanations to why cancer defense mechanisms are not equal across species. METHODOLOGY: Here we report the prevalence of neoplasia and malignancy in 37 mammalian species, representing 11 mammalian orders, using 42 years of well curated necropsy data from the San Diego Zoo and San Diego Zoo Safari Park. We collected data on life history components of these species and tested for associations between life history traits and both neoplasia and malignancy, while controlling for phylogenetic history. RESULTS: These results support Peto's paradox, in that we find no association between lifespan and/or body mass and the prevalence of neoplasia or malignancy. However, a positive relationship exists between litter size and prevalence of malignancy (P = 0.005, Adj. R2 = 0.212), suggesting that a species' life history strategy may influence cancer vulnerabilities. Lastly, we tested for the relationship between placental invasiveness and malignancy. We find no evidence for an association between placental depth and malignancy prevalence (P = 0.618, Adj. R2 = 0.068). CONCLUSIONS: Life history theory offers a powerful framework to understand variation in cancer defenses across the tree of life. These findings provide insight into the relationship between life history traits and cancer vulnerabilities, which suggest a trade-off between reproduction and cancer defenses. LAY SUMMARY: Why are some mammals more vulnerable to cancer than others? We test whether life history trade-offs may explain this variation in cancer risk. Bigger, longer-lived animals do not develop more cancer compared to smaller, shorter-lived animals. However, we find a positive association between litter size and cancer prevalence in mammals.

Ranavirus infection dynamics and shedding in American bullfrogs: consequences for spread and detection in trade.

American bullfrogs Lithobates catesbeianus are thought to be important in the global spread of ranaviruses-often lethal viruses of cold-blooded vertebrates-because they are commonly farmed, dominate international trade, and may be 'carriers' of ranavirus infections. However, whether American bullfrogs are easily infected and maintain long-lasting ranavirus infections, or are refractory to or rapidly clear infections, remains unknown. We tracked the dynamics of ranavirus in American bullfrogs through time and with temperature in multiple types of samples and also screened shipments from commercial suppliers to determine whether we could detect subclinical infections. Collectively, we found that tadpoles and juveniles were commonly infected at moderate doses, and while some died, others controlled and appeared to clear their infections. Some individuals, however, harbored subclinical infections for up to 49 d, suggesting that American bullfrogs may be important carriers. Indeed, tadpoles and metamorphosed frogs from 2 of 5 commercial suppliers harbored subclinicial infections. Juveniles at warmer temperatures had less intense but still persistent infections. Because diagnostic performance was strongly related to infection intensity, non-lethal samples (i.e. tail or toe clips, swabs, and environmental DNA) had only a moderate chance of detecting subclinical infections. Even internal tissues may fail to detect subclinical infections. However, viral shedding was correlated with the intensity of infection, so while subclinically infected tadpoles shed virus for 35-49 d, the low levels might lead to little transmission. We suggest that a quantitative focus on virus dynamics within hosts can provide a more nuanced view of ranavirus infections and the risk presented by American bullfrogs in trade.

Phylogenetic investigation of skin sloughing rates in frogs: relationships with skin characteristics and disease-driven declines.

Amphibian skin is highly variable in structure and function across anurans, and plays an important role in physiological homeostasis and immune defence. For example, skin sloughing has been shown to reduce pathogen loads on the skin, such as the lethal fungus Batrachochytrium dendrobatidis ( Bd), but interspecific variation in sloughing frequency is largely unknown. Using phylogenetic linear mixed models, we assessed the relationship between skin turnover rate, skin morphology, ecological traits and overall evidence of Bd-driven declines. We examined skin sloughing rates in 21 frog species from three continents, as well as structural skin characteristics measured from preserved specimens. We found that sloughing rate varies significantly with phylogenetic group, but was not associated with evidence of Bd-driven declines, or other skin characteristics examined. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position. While not related to available evidence of declines, understanding variation in sloughing rate may help explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus).

FATAL RANAVIRUS INFECTION IN A GROUP OF ZOO-HOUSED MELLER'S CHAMELEONS (TRIOCEROS MELLERI).

A group of five juvenile Meller's chameleons (Trioceros melleri) experienced 100% mortality over a period of 1 mo due to ranavirus infection. The index case was found dead without premonitory signs. The three subsequent cases presented with nonspecific clinical signs (lethargy, decreased appetite, ocular discharge) and were ultimately euthanatized. The final case died after initially presenting with skin lesions. Postmortem examination revealed thin body condition in all five animals and mild coelomic effusion and petechiae affecting the tongue and kidneys of one animal. Microscopically, all animals had multifocal necrosis of the spleen, liver, and kidney; four of five animals had necrosis of the nasal cavity; and two of five had necrosis of adrenal tissue, bone marrow, and skin. Numerous basophilic intracytoplasmic inclusions were present in the liver of all animals and nasal mucosa of three of the five animals. Consensus polymerase chain reaction for herpesvirus and adenovirus were negative, whereas ranavirus quantitative polymerase chain reaction was positive. Virus isolation followed by whole genome sequencing and Bayesian phylogenetic analysis classified the isolates as a strain of frog virus 3 (FV3) most closely related to an FV3 isolate responsible for a previous outbreak in the zoo's eastern box turtle (Terrapene carolina carolina) group. This case series documents the first known occurrence of ranavirus-associated disease in chameleons and demonstrates the potential for interspecies transmission between chelonian and squamate reptiles.

The relationship between spindly leg syndrome incidence and water composition, overfeeding, and diet in newly metamorphosed harlequin frogs (Atelopus spp.).

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

Factors influencing detection and co-detection of Ranavirus and Batrachochytrium dendrobatidis in Midwestern North American anuran populations.

Amphibian populations are in decline worldwide as they face a barrage of challenges, including infectious diseases caused by ranaviruses and the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). Here we describe seasonal dynamics of Bd and ranavirus detection in free-ranging post-metamorphic wood frogs Lithobates sylvaticus, boreal chorus frogs Pseudacris maculata/triseriata, and gray treefrogs Hyla versicolor/chrysoscelis, sampled over a 3 season gradient in Minnesota (USA) wetlands. We detected Bd in 36% (n = 259) of individuals sampled in 3 wetlands in 2014, and 33% (n = 255) of individuals sampled in 8 wetlands in 2015. We also detected ranavirus in 60% and 18% of individuals sampled in 2014 and 2015, respectively. Ranavirus and Bd were detected concurrently in 26% and 2% of animals sampled in 2014 and 2015, respectively. We report clinical signs and associated infection status of sampled frogs; of the clinical signs observed, skin discoloration was significantly associated with ranavirus infection. Using generalized estimating equations, we found that species, season, wetland, and a species × season interaction term were significant predictors of Bd detection, whereas test year approached significance as a predictor of ranavirus detection. The odds of detecting both pathogens concurrently was significantly influenced by species, season, a species × season interaction term, year, and environmental ammonia. We propose an amphibian health monitoring scheme that couples population size surveys with seasonal molecular surveys of pathogen presence. This information is crucial to monitoring the health of remaining strongholds of healthy amphibian populations, as they face an uncertain future of further anthropogenic change.

Rapid extirpation of a North American frog coincides with an increase in fungal pathogen prevalence: Historical analysis and implications for reintroduction.

As extinctions continue across the globe, conservation biologists are turning to species reintroduction programs as one optimistic tool for addressing the biodiversity crisis. For repatriation to become a viable strategy, fundamental prerequisites include determining the causes of declines and assessing whether the causes persist in the environment. Invasive species-especially pathogens-are an increasingly significant factor contributing to biodiversity loss. We hypothesized that Batrachochytrium dendrobatidis (Bd), the causative agent of the deadly amphibian disease chytridiomycosis, was important in the rapid (<10 years) localized extirpation of a North American frog (Rana boylii) and that Bd remains widespread among extant amphibians in the region of extirpation. We used an interdisciplinary approach, combining interviews with herpetological experts, analysis of archived field notes and museum specimen collections, and field sampling of the extant amphibian assemblage to examine (1) historical relative abundance of R. boylii; (2) potential causes of R. boylii declines; and (3) historical and contemporary prevalence of Bd. We found that R. boylii were relatively abundant prior to their rapid extirpation, and an increase in Bd prevalence coincided with R. boylii declines during a time of rapid change in the region, wherein backcountry recreation, urban development, and the amphibian pet trade were all on the rise. In addition, extreme flooding during the winter of 1969 coincided with localized extirpations in R. boylii populations observed by interview respondents. We conclude that Bd likely played an important role in the rapid extirpation of R. boylii from southern California and that multiple natural and anthropogenic factors may have worked in concert to make this possible in a relatively short period of time. This study emphasizes the importance of recognizing historical ecological contexts in making future management and reintroduction decisions.

MEDICAL MANAGEMENT OF RECURRENT EOSINOPHILIC GRANULOMA IN TWO BLACK RHINOCEROS (DICEROS BICORNIS).

Recurrent eosinophilic granuloma (EG) in two captive eastern black rhinoceros ( Diceros bicornis michaeli) was effectively managed with glucocorticoids and antihistamines. The first case was a female and the second case was a male. The animals were housed at separate institutions and initially presented with hemorrhagic oral lesions. Multifocal lesions occurred in the second case. Multiple biopsies were taken from each animal, all of which were consistent with EG. Each animal was anesthetized multiple times for surgical treatment but experienced frequent recurrence. Due to lack of response to therapy and the risks and adverse events associated with repeated anesthesia, medical treatment was initiated in both cases using a tapering dose of oral dexamethasone. The lesions dramatically improved, but would recur frequently after treatment. Hydroxyzine, an oral antihistamine, greatly reduced the incidence and severity of the lesions. Medical management with glucocorticoids and antihistamines minimized stressful anesthetic events in both cases and contributed to the successful management of this recurrent disease. The exact pathogenesis of EG in black rhinoceros remains unknown but response to antihistamines suggests an allergic etiology.

NUPTIAL PAD AMPUTATION IN AN AMERICAN BULLFROG (LITHOBATES CATESBEIANUS) WITH SQUAMOUS CELL CARCINOMA.

A 7-yr-old male captive American bullfrog (Lithobates catesbeianus) presented with a 2-wk history of an enlarged, ulcerated nuptial pad on the mediopalmar surface of the first digit of the left carpus. A 3-mm wedge biopsy of the mass was not diagnostic and differentials included an epidermal inclusion cyst or squamous cell carcinoma. No fungal or acid-fast organisms were cultured or noted on impression smear. Wide surgical resection of the mass and associated first digit were performed. Histopathology confirmed squamous cell carcinoma associated with the dermal nuptial gland with neoplastic cells extending close to deep surgical margins. Two months after surgery, no recurrence was noted. Although experimental tumor studies in amphibians are well documented, clinical reports of cutaneous neoplasia management in captive amphibians are scarce. Squamous cell carcinoma should be considered as a differential diagnosis when male anurans present with nuptial gland enlargement.

Rapid Response to Evaluate the Presence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Wild Amphibian Populations in Madagascar.

We performed a rapid response investigation to evaluate the presence and distribution of amphibian pathogens in Madagascar following our identification of amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd) and ranavirus in commercially exported amphibians. This targeted risk-based field surveillance program was conducted from February to April 2014 encompassing 12 regions and 47 survey sites. We simultaneously collected amphibian and environmental samples to increase survey sensitivity and performed sampling both in wilderness areas and commercial amphibian trade facilities. Bd was not detected in any of 508 amphibian skin swabs or 68 water filter samples, suggesting pathogen prevalence was below 0.8%, with 95% confidence during our visit. Ranavirus was detected in 5 of 97 amphibians, including one adult Mantidactylus cowanii and three unidentified larvae from Ranomafana National Park, and one adult Mantidactylus mocquardi from Ankaratra. Ranavirus was also detected in water samples collected from two commercial amphibian export facilities. We also provide the first report of an amphibian mass-mortality event observed in wild amphibians in Madagascar. Although neither Bd nor ranavirus appeared widespread in Madagascar during this investigation, additional health surveys are required to disentangle potential seasonal variations in pathogen abundance and detectability from actual changes in pathogen distribution and rates of spread. Accordingly, our results should be conservatively interpreted until a comparable survey effort during winter months has been performed. It is imperative that biosecurity practices be immediately adopted to limit the unintentional increased spread of disease through the movement of contaminated equipment or direct disposal of contaminated material from wildlife trade facilities. The presence of potentially introduced strains of ranaviruses suggests that Madagascar's reptile species might also be threatened by disease. Standardized population monitoring of key amphibian and reptile species should be established with urgency to enable early detection of potential impacts of disease emergence in this global biodiversity hotspot.

Early 1900 s detection of Batrachochytrium dendrobatidis in Korean amphibians.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is a major conservation concern because of its role in decimating amphibian populations worldwide. We used quantitative PCR to screen 244 museum specimens from the Korean Peninsula, collected between 1911 and 2004, for the presence of Bd to gain insight into its history in Asia. Three specimens of Rugosa emeljanovi (previously Rana or Glandirana rugosa), collected in 1911 from Wonsan, North Korea, tested positive for Bd. Histology of these positive specimens revealed mild hyperkeratosis - a non-specific host response commonly found in Bd-infected frogs - but no Bd zoospores or zoosporangia. Our results indicate that Bd was present in Korea more than 100 years ago, consistent with hypotheses suggesting that Korean amphibians may be infected by endemic Asian Bd strains.

Leaping forward in amphibian health and nutrition.

The Epidemiology Working Group, a subgroup of the participants of the Disney's Animal Kingdom Workshop on "Ex situ Amphibian Medicine and Nutrition," identified a critical need to design and implement approaches that will facilitate the assessment and evaluation of factors impacting amphibian health. In this manuscript, we describe and summarize the outcomes of this workshop with regards (a) the identified gaps in knowledge, (b) identified priorities for closing these gaps, and (c) compile a list of actions to address these priorities. Four general areas of improvement were identified in relation to how measurements are currently being taken to evaluate ex situ amphibian health: nutrition, infectious diseases, husbandry, and integrated biology including genetics and endocrinology. The proposed actions that will be taken in order to address the identified gaps include: (1) identify and quantify major health issues affecting ex situ amphibian populations, (2) identify and coordinate laboratories to conduct analyses using standardized and validated protocols to measure nutritional, infectious diseases, genetic, and hormonal parameters, (3) determine in situ baseline distribution of parameters related to amphibian health, and (4) establish an inter-disciplinary research approach to target specific hypotheses related to amphibian health such as the effects of population genetics (e.g., relatedness, inbreeding) on disease susceptibility, or how environmental parameters are related to chronic stress and hormone production. We think is important to address current gaps in knowledge regarding amphibian health in order to increase the probability to succeed in addressing the issues faced by in situ and ex situ amphibians populations. We are confident that the recommendations provided in this manuscript will facilitate to address these challenges and could have a positive impact in both the health of in situ and ex situ amphibian populations, worldwide.

Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.