Serpentoviruses Exhibit Diverse Organization and ORF Composition with Evidence of Recombination.

Serpentoviruses are a subfamily of positive sense RNA viruses in the order Nidovirales, family Tobaniviridae, associated with respiratory disease in multiple clades of reptiles. While the broadest viral diversity is reported from captive pythons, other reptiles, including colubrid snakes, turtles, and lizards of captive and free-ranging origin are also known hosts. To better define serpentoviral diversity, eleven novel serpentovirus genomes were sequenced with an Illumina MiSeq and, when necessary, completed with other Sanger sequencing methods. The novel serpentoviral genomes, along with 57 other previously published serpentovirus genomes, were analyzed alongside four outgroup genomes. Genomic analyses included identifying unique genome templates for each serpentovirus clade, as well as analysis of coded protein composition, potential protein function, protein glycosylation sites, differences in phylogenetic history between open-reading frames, and recombination. Serpentoviral genomes contained diverse protein compositions. In addition to the fundamental structural spike, matrix, and nucleoprotein proteins required for virion formation, serpentovirus genomes also included 20 previously uncharacterized proteins. The uncharacterized proteins were homologous to a number of previously characterized proteins, including enzymes, transcription factors, scaffolding, viral resistance, and apoptosis-related proteins. Evidence for recombination was detected in multiple instances in genomes from both captive and free-ranging snakes. These results show serpentovirus as a diverse clade of viruses with genomes that code for a wide diversity of proteins potentially enhanced by recombination events.

One Health Approach to Globalizing, Accelerating, and Focusing Amphibian and Reptile Disease Research-Reflections and Opinions from the First Global Amphibian and Reptile Disease Conference.

The world's reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity.

A novel herpesvirus from a wild-caught Madagascar spider tortoise shows evidence of host-viral coevolution with a duplication event in Durocryptodira.

Herpesviruses can be significant reptile pathogens. Herpesviral infection in a wild-caught, male spider tortoise (Pyxis arachnoides) under human care was detected during a routine wellness examination prior to transition between zoologic organizations. The tortoise had no clinical signs of illness. Oral swabs obtained during a physical examination as part of pre-shipment risk mitigation for infectious disease were submitted for consensus herpesvirus PCR assay and sequencing. Based on comparative sequence analysis, the novel herpesvirus identified is a member of the subfamily Alphaherpesvirinae. Studies of herpesviral phylogeny in chelonian species support branching patterns of turtle herpesviruses that closely mirror those of their hosts. The symmetry of these patterns is suggestive of close codivergence of turtle herpesviruses with their host species. The distribution of these viruses in both tortoises and emydids suggests a phylogenetic duplication event in the herpesviruses after host divergence of the Pleurodira and basal to the divergence of Americhelydia. Herpesviral infections have been documented to cause higher morbidity when introduced to aberrant host species, and significant consideration must be given to the presence of herpesviruses in the management of tortoise collections, particularly collections that include various species of testudines.

In Vitro Characterization and Antiviral Susceptibility of Ophidian Serpentoviruses.

Ophidian serpentoviruses, positive-sense RNA viruses in the order Nidovirales, are important infectious agents of both captive and free-ranging reptiles. Although the clinical significance of these viruses can be variable, some serpentoviruses are pathogenic and potentially fatal in captive snakes. While serpentoviral diversity and disease potential are well documented, little is known about the fundamental properties of these viruses, including their potential host ranges, kinetics of growth, environmental stability, and susceptibility to common disinfectants and viricides. To address this, three serpentoviruses were isolated in culture from three unique PCR-positive python species: Ball python (Python regius), green tree python (Morelia viridis), and Stimson's python (Antaresia stimsoni). A median tissue culture infectious dose (TCID50) was established to characterize viral stability, growth, and susceptibility. All isolates showed an environmental stability of 10-12 days at room temperature (20 °C). While all three viruses produced variable peak titers on three different cell lines when incubated at 32 °C, none of the viruses detectably replicated at 35 °C. All viruses demonstrated a wide susceptibility to sanitizers, with 10% bleach, 2% chlorhexidine, and 70% ethanol inactivating the virus in one minute and 7% peroxide and a quaternary ammonium solution within three minutes. Of seven tested antiviral agents, remdesivir, ribavirin, and NITD-008, showed potent antiviral activity against the three viruses. Finally, the three isolates successfully infected 32 unique tissue culture cell lines representing different diverse reptile taxa and select mammals and birds as detected by epifluorescent immunostaining. This study represents the first characterization of in vitro properties of growth, stability, host range, and inactivation for a serpentovirus. The reported results provide the basis for procedures to mitigate the spread of serpentoviruses in captive snake colonies as well as identify potential non-pharmacologic and pharmacologic treatment options for ophidian serpentoviral infections.

Characterization of expression and prognostic implications of GD2 and GD3 synthase in canine histiocytic sarcoma.

GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope®) in the detection of GD2S and GD3S in canine histiocytic sarcoma (HS) in vitro and optimize this technique in canine formalin-fixed paraffin-embedded (FFPE) tissues. A secondary objective is to evaluate the prognostic significance of GD2S and GD3S on survival. Quantitative RT-PCR compared GD2S and GD3S mRNA expression between three HS cell lines followed by RNAscope® in fixed cell pellets from the DH82 cell line and FFPE tissues. Variables prognostic for survival were determined with Cox proportional hazard model. RNAscope® was validated for detection of GD2S and GD3S and optimized in FFPE tissues. GD2S and GD3S mRNA expression was variable between cell lines. GD2S and GD3S mRNA expression was detected and measured in all tumor tissues; there was no association with prognosis. GD2S and GD3S are expressed in canine HS and successfully detected using the high throughput technique of RNAscope® in FFPE samples. This study provides the foundation for future prospective research of GD2S and GD3S utilizing RNAscope®.

Dermatomycosis Caused by Paranannizziopsis australasiensis in Nonnative Panther Chameleons (Furcifer pardalis) Captured in Central Florida, USA.

Emergent fungal pathogens in herpetofauna are a concern in both wild and captive populations. We diagnosed dermatomycosis by Paranannizziopsis australasiensis in two panther chameleons (Furcifer pardalis) and suspected it in eight others captured from an established free-living nonnative population in Florida, USA. Chameleons developed skin lesions following recent exposure to cold weather conditions while housed in captivity, approximately 10 mo after capture and 12 wk after being placed in outdoor enclosures. Affected animals were treated with oral voriconazole and terbinafine until most cases resolved; however, medications were ultimately discontinued. Paranannizziopsis australasiensis has not previously been described in chameleons, nor in animals originating from a free-ranging population in the USA. Although the source of P. australasiensis infection is uncertain, we discuss several scenarios related to the pet trade and unique situation of chameleon "ranching" present in the USA.

Multisystemic Enterococcosis in Brown Anoles (Anolis sagrei) from Florida, USA.

Beginning in July 2019, numerous free-ranging brown anoles (Anolis sagrei), an invasive lizard species in Florida, USA, were reported with large, soft, subcutaneous masses and disfiguring facial swellings. Postmortem evaluations of six affected animals, including cytology, histology, and electron microscopy, identified the presence of myriad chain-forming coccoid bacteria surrounded by a prominent clear capsule and abundant lightly basophilic matrix material with minimal associated granulomatous inflammation and effacement of normal tissue. Standard PCR and sequencing of the lesions revealed 100% nucleotide identity to Enterococcus lacertideformus. This bacterium was first observed in 2014 as the cause of a severe, multisystemic infection in several species of lizards (geckos and skinks) on Christmas Island, an Australian external territory in the Indian Ocean. Previously, analysis of E. lacertideformus had been hindered by an inability to grow the bacterium in standard culture conditions. We successfully cultured the organism on primary anole kidney cells. Given the growing recognition of host species diversity and geographic distribution noted for this organism, there is potential concern for spread to native North American lizards, especially the green anole (Anolis carolinensis), whose population numbers have apparently decreased due to introduced brown anoles.

Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma.

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-ß), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-ß, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-ß and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-ß H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-ß dots/cell. In summary, RNAscope® is a valid technique to detect TGF-ß and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-ß and PD-L1. Increased TGF-ß and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-ß, PD-L1, and Tregs effect on prognosis.

Immune priming prior to pathogen exposure sheds light on the relationship between host, microbiome and pathogen in disease.

Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.

The invasive pentastome Raillietiella orientalis in a banded water snake from the pet trade.

Raillietiella orientalis is established in Florida and rapidly spreading both geographically and in known host species. A banded water snake (Nerodia fasciata), purchased in Michigan at a regional reptile show, expectorated a pentastome whose morphology and DNA sequence indicated that it was R. orientalis. This event indicates that, through the pet trade, R. orientalis has been spread 1,500 km from its previously known distribution limit. Fecal sample analyses indicated that the snake was shedding large numbers of embryonated eggs for at least several months. The diversity of reptile species that are both known hosts of R. orientalis in Florida and are commonly sold in the pet trade indicates that this invasive pentastome may become a widespread health concern for pet owners and veterinarians.

Piscichuvirus-Associated Severe Meningoencephalomyelitis in Aquatic Turtles, United States, 2009-2021.

Viruses from a new species of piscichuvirus were strongly associated with severe lymphocytic meningoencephalomyelitis in several free-ranging aquatic turtles from 3 coastal US states during 2009-2021. Sequencing identified 2 variants (freshwater turtle neural virus 1 [FTuNV1] and sea turtle neural virus 1 [STuNV1]) of the new piscichuvirus species in 3 turtles of 3 species. In situ hybridization localized viral mRNA to the inflamed region of the central nervous system in all 3 sequenced isolates and in 2 of 3 additional nonsequenced isolates. All 3 sequenced isolates phylogenetically clustered with other vertebrate chuvirids within the genus Piscichuvirus. FTuNV1 and STuNV1 shared ≈92% pairwise amino acid identity of the large protein, which narrowly places them within the same novel species. The in situ association of the piscichuviruses in 5 of 6 turtles (representing 3 genera) with lymphocytic meningoencephalomyelitis suggests that piscichuviruses are a likely cause of lymphocytic meningoencephalomyelitis in freshwater and marine turtles.

Divergent Serpentoviruses in Free-Ranging Invasive Pythons and Native Colubrids in Southern Florida, United States.

Burmese python (Python bivittatus) is an invasive snake that has significantly affected ecosystems in southern Florida, United States. Aside from direct predation and competition, invasive species can also introduce nonnative pathogens that can adversely affect native species. The subfamily Serpentovirinae (order Nidovirales) is composed of positive-sense RNA viruses primarily found in reptiles. Some serpentoviruses, such as shingleback nidovirus, are associated with mortalities in wild populations, while others, including ball python nidovirus and green tree python nidovirus can be a major cause of disease and mortality in captive animals. To determine if serpentoviruses were present in invasive Burmese pythons in southern Florida, oral swabs were collected from both free-ranging and long-term captive snakes. Swabs were screened for the presence of serpentovirus by reverse transcription PCR and sequenced. A total serpentovirus prevalence of 27.8% was detected in 318 python samples. Of the initial swabs from 172 free-ranging pythons, 42 (24.4%) were positive for multiple divergent viral sequences comprising four clades across the sampling range. Both sex and snout-vent length were statistically significant factors in virus prevalence, with larger male snakes having the highest prevalence. Sampling location was statistically significant in circulating virus sequence. Mild clinical signs and lesions consistent with serpentovirus infection were observed in a subset of sampled pythons. Testing of native snakes (n = 219, 18 species) in part of the python range found no evidence of python virus spillover; however, five individual native snakes (2.3%) representing three species were PCR positive for unique, divergent serpentoviruses. Calculated pairwise uncorrected distance analysis indicated the newly discovered virus sequences likely represent three novel genera in the subfamily Serpentovirinae. This study is the first to characterize serpentovirus in wild free-ranging pythons or in any free-ranging North America reptile. Though the risk these viruses pose to the invasive and native species is unknown, the potential for spillover to native herpetofauna warrants further investigation.

PULMONARY AND COELOMIC MYCOSES DUE TO METARHIZIUM AND BEAUVERIA SPECIES IN REPTILES.

This report documents cases of fatal pulmonary mycosis caused by entomopathogenic fungi in the genera Metarhizium and Beauveria (Order Hypocreales) in a loggerhead sea turtle (Caretta caretta), a Chinese alligator (Alligator sinensis), two gopher tortoises (Gopherus polyphemus), a Cuvier's dwarf caiman (Paleosuchus palpebrosus), a false gharial (Tomistoma schlegelii), a green sea turtle (Chelonia mydas), and a Kemp's ridley sea turtle (Lepidochelys kempii), and a case of granulomatous coelomitis in a hawksbill sea turtle (Eretmochelys imbricata). Fungi identified in these cases included Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, Metarhizium robertsii, and one case of infection by a novel Metarhizium species. The animals were either housed at zoos or brought into rehabilitation from the wild. Although the majority of animals had comorbidities, the fungal infections were believed to be the primary cause of death. Fungal susceptibility testing was performed on two Beauveria spp. isolates, and revealed lower minimum inhibitory concentrations for itraconazole and voriconazole when compared to terbinafine and fluconazole. This case series demonstrates that a variety of reptile species from different orders are vulnerable to infection with Metarhizium, and multiple species of sea turtle are susceptible to infection with Beauveria.

Infection with the fox lungworm (Crenosoma vulpis) in two dogs from New England - Two clinical reports and updated geographic distribution in North America.

Crenosoma vulpis, the fox lungworm, is a helminth parasite endemic to the fox population of New England. Domestic dogs are susceptible to infection via ingestion of snails and slugs. Two dogs from New England were diagnosed with C. vulpis. The predominant clinical sign in both dogs was a chronic cough. Treatment with steroids and antibiotics only temporarily relieved clinical signs. Thoracic radiographs in both dogs revealed bronchial patterns. Endotracheal washes were performed in each dog revealing marked, mixed inflammation consisting mainly of neutrophils with eosinophils in lesser numbers. Helminth larvae could also be visualized on cytology. A fecal flotation revealed helminth larvae in one dog but failed to identify larvae in the second dog. The diagnosis of C. vulpis was confirmed via PCR analysis and sequencing of samples from both endotracheal washes. One dog was treated with fenbendazole (50 mg/kg PO q24h for 14 days), enrofloxacin (13 mg/kg PO q 24 h for 5 days), and a tapering protocol of prednisone (20 mg PO q12h for 5 days, 20 mg PO q24h for 5 days, then 20 mg PO q48h for 10 days). The second dog was treated with fenbendazole (50 mg/kg PO q24h for 10 days) with an additional 7 days of febantel and two doses of milbemycin, achieving complete resolution of clinical signs. This lungworm is becoming increasingly more prevalent in domestic dogs worldwide and may be more prevalent in New England than previously thought. Veterinary practitioners of New England should include this respiratory helminth as a differential in dogs with respiratory signs, and respiratory washes and Baermann fecal examinations are warranted in dogs presenting with non-specific respiratory clinical signs.

A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

Physiological effects of capture and short-term captivity in an invasive snake species, the Burmese python (Python bivittatus) in Florida.

It is important to evaluate the role of captivity as a potential stressor. An understanding of stress responses to capture and transition to captivity may inform the limitations of laboratory studies on wild animals, aid in understanding the consequences of introducing animals into captive environments, and help predict which species may be successful invasives. We investigated physiological effects of captivity by comparing at-capture blood variables in wild Burmese pythons (Python bivittatus) in Florida to pythons recently brought into captivity (1-109 days). We conducted an acute restraint test by collecting samples at baseline (immediately at handling) and one hour post-restraint across wild field-sampled (n = 19) and recently-captive (n = 33) pythons to evaluate fluctuations in plasma corticosterone, bacterial killing ability, antibody response, leukogram, and serpentovirus infection. We observed higher baseline plasma corticosterone and monocytes in recently captive compared to wild snakes, which both subsided in snakes held for a longer time in captivity, and a mild decrease in lymphocytes in the middle of the captivity period. Functional immunity and viral infection were not affected by captivity, and pythons maintained restraint-induced responses in corticosterone, heterophil to lymphocyte ratio, and monocyte counts throughout captivity. Prevalence for serpentovirus was 50%, though infection status was related to sampling date rather than captivity, indicating that viral infection may be seasonal. The history of Burmese python as a common captive animal for research and pet trade, as well as its general resilience to effects of capture and short-term captivity, may contribute to its invasion success in Florida.

A NEW COCCIDIAN (APICOMPLEXA: EIMERIIDAE) IN THE CRITICALLY ENDANGERED CENTRAL AMERICAN RIVER TURTLE (DERMATEMYS MAWII) IN BELIZE.

As part of a biannual health examination, coprological samples from 3-mo-old Central American river turtles, Dermatemys mawii (Gray, 1847) in a breeding program in Belize, Central America, revealed a previously undescribed coccidian (Apicomplexa) in 17 of 46 (37%) samples. Of 3 positive fecal samples transported to the University of Florida, coccidian oocysts were observed in 1 sample. Sporulated oocysts were measured and described, and using polymerase chain reaction (PCR), an approximately 400-base pair (bp) region of both the small subunit (18S) ribosomal RNA gene and 1,200-bp region of the internal transcribed spacer (ITS) gene were amplified in all 3 samples and their products were sequenced. For comparative value, the same PCR reactions and amplifications were performed on a fecal sample containing oocysts of Eimeria mitraria obtained from a red-eared slider, Trachemys scripta elegans. Results indicated a new eimerian in D. mawii, Eimeria grayi n. sp.

Sustained Ranavirus Outbreak Causes Mass Mortality and Morbidity of Imperiled Amphibians in Florida.

A persistent 2-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and disease signs in N. perstriatus continued for 5 weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a frog virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

Rat Lungworm (Angiostrongylus cantonensis) in the Invasive Cuban Treefrog (Osteopilus septentrionalis) in Central Florida, USA.

Cuban treefrogs, Osteopilus septentrionalis, were grossly examined for parasites and parasite species confirmed by PCR. Angiostrongylus cantonensis larvae were recovered from the hind leg muscle of O. septentrionalis. This is the first report of the zoonotic rat lungworm in the Cuban treefrog and new geographic location (Volusia County) in Florida, US.