Virulence factors of Salmonella spp. isolated from free-living grass snakes Natrix natrix.

Salmonellosis associated with reptiles is a well-researched topic, particularly in China and the United States, but it occurs less frequently in Europe. The growth of the human population and changes in the environment could potentially increase the interaction between humans and free-living reptiles, which are an unidentified source of Salmonella species. In this study, we sought to explore this issue by comparing the microbiota of free-living European grass snakes, scientifically known as Natrix natrix, with that of captive banded water snakes, or Nerodia fasciata. We were able to isolate 27 strains of Salmonella species from cloacal swabs of 59 N. natrix and 3 strains from 10 N. fasciata. Our findings revealed that free-living snakes can carry strains of Salmonella species that are resistant to normal human serum (NHS). In contrast, all the Salmonella species strains isolated from N. fasciata were sensitive to the action of the NHS, further supporting our findings. We identified two serovars from N. natrix: Salmonella enterica subspecies diarizonae and S. enterica subspecies houtenae. Additionally, we identified three different virulotypes (VT) with invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA genes, and ß-galactosidase synthesised by 23 serovars. The identification of Salmonella species in terms of their VT is a relatively unknown aspect of their pathology. This can be specific to the serovar and pathovar and could be a result of adaptation to a new host or environment.

Host microbiome responses to the Snake Fungal Disease pathogen (Ophidiomyces ophidiicola) are driven by changes in microbial richness.

Dermatophytic pathogens are a source of disturbance to the host microbiome, but the temporal progression of these disturbances is unclear. Here, we determined how Snake Fungal Disease, caused by Ophidiomyces ophidiicola, resulted in disturbance to the host microbiome. To assess disease effects on the microbiome, 22 Common Watersnakes (Nerodia sipedon) were collected and half were inoculated with O. ophidiicola. Epidermal swabs were collected weekly for use in microbiome and pathogen load characterization. For the inoculated treatment only, we found a significant effect of disease progression on microbial richness and Shannon diversity consistent with the intermediate disturbance hypothesis. When explicitly accounting for differences in assemblage richness, we found that ß-diversity among snakes was significantly affected by the interaction of time and treatment group, with assemblages becoming more dissimilar across time in the inoculated, but not the control group. Also, differences between treatments in average microbiome composition became greater with time, but this interactive effect was not evident when accounting for assemblage richness. These results suggest that changes in composition of the host microbiome associated with disease largely occur due to changes in microbial richness related to disease progression.

EPIDEMIOLOGY OF OPHIDIOMYCOSIS IN LAKE ERIE WATERSNAKES (NERODIA SIPEDON INSULARUM).

Ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, is an infectious disease of wild and managed snakes worldwide. Lake Erie watersnakes (LEWS; Nerodia sipedon insularum) were listed as threatened under the US Endangered Species Act from 1999 to 2011 and were first diagnosed with ophidiomycosis in 2009. Our objective was to characterize the epidemiology of ophidiomycosis in LEWS. We hypothesized that the prevalence of skin lesions, O. ophidiicola DNA, and ophidiomycosis disease categories would show spatial and temporal variation and clustering, with higher prevalence at sites with greater human disturbance and prevalence increasing over time. Snakes were captured via visual encounter surveys at five sites across four islands and visually inspected for skin lesions suggestive of ophidiomycosis; then body swabs were collected to detect O. ophidiicola DNA using quantitative PCR. Each snake was assigned an ophidiomycosis category based on the presence of skin lesions and O. ophidiicola. We evaluated 837 LEWS between 2017 and 2020 and detected ophidiomycosis at all five sites. Logistic regression analysis showed temporal and spatial variation in disease, with higher risk of apparent ophidiomycosis (lesions present and O. ophidiicola detected) at Kelleys Island State Park, compared to all other sites; in May, compared to July; and in 2019, compared to 2018. The presence of emerging herbaceous wetlands, urban land change, and certain soil types increased the odds of both lesion presence and quantitative PCR detection of O. ophidiicola. Overall, ophidiomycosis epidemiology varied among sites: the disease appeared to be endemic at most sites and emerging at one site. Ongoing efforts to monitor population health and mortality associated with disease prevalence are needed to inform mitigation aimed at reducing the impact of ophidiomycosis in LEWS.

Testing the febrile response of snakes inoculated with Ophidiomyces ophidiicola, the causative agent of snake fungal disease.

Snake Fungal Disease (SFD) negatively impacts wild snake populations in the eastern United States and Europe. Ophidiomyces ophidiicola causes SFD and manifests clinically by the formation of heterophilic granulomas around the mouth and eyes, weight loss, impaired vision, and sometimes death. Field observations have documented early seasonal basking behaviors in severely infected snakes, potentially suggesting induction of a behavioral febrile response to combat the mycosis. This study tested the hypothesis that snakes inoculated with Ophidiomyces ophidiicola would seek elevated basking temperatures to control body temperature and behaviorally induce a febrile response. Eastern ribbon snakes (Thamnophis saurita, n = 29) were experimentally or sham inoculated with O. ophidiicola. Seven days after inoculation, snakes were tested on a thermal gradient and the internal body temperature and substrate temperature of each snake was recorded over time. Quantitative PCR was used when snakes arrived, during pre-inoculation, and post-inoculation to test snakes for the presence of O. ophidiicola. Some snakes arrived with O. ophidiicola and were subsequently inoculated, allowing for an assessment of secondary exposure effects. Snake thermoregulatory behavior was compared between 1) O. ophidiicola inoculated vs. sham inoculated treatments, 2) infected vs. disease negative groups, and 3) disease naïve vs. pre-exposed immune response categories. Neither internal nor substrate temperatures differed among initially prescribed, and qPCR recovered disease states, although infected snakes tended to reach a preferred body temperature faster than disease negative snakes. Snakes experiencing their first exposure (disease naïve) sought higher substrate temperatures than snakes experiencing their second exposure (pre-exposed). Here, we recover no evidence for behaviorally induced fever in snakes with SFD but do elucidate a febrile immune response associated with secondary exposure.

Taxonomy, not locality, influences the cloacal microbiota of two nearctic colubrids: a preliminary analysis.

BACKGROUND: The gut microbiota is an emerging frontier in wildlife research and its importance to vertebrate health and physiology is becoming ever more apparent. Reptiles, in particular snakes, have not received the same attention given to other vertebrates and the composition of their wild gut microbiome remains understudied. The primary goal of this work was to describe the cloacal microbiota of two Colubrids, the Eastern Gartersnake (Thamnophis sirtalis sirtalis) and the Northern Watersnake (Nerodia sipedon sipedon), and if their cloacal microbiota differed as well as if it did between a wetland and upland population of the former species. METHODS AND RESULTS: We utilized next-generation sequencing of cloacal swabs-a non-destructive proxy for the gut microbiota. The cloacal microbiome of Eastern Gartersnakes (N = 9) was like those of other snakes being comprised of Proteobacteria, Bacteroidetes, and Firmicutes, while that of Northern Watersnakes (N = 6) was dominated by Tenericutes. Seven microbial operational taxonomic units (OTUs), all members of Proteobacteria, were shared among all individuals and were indicative of a core microbiome in Eastern Gartersnakes, but these OTUs were not particularly relevant to Northern Watersnakes. The latter had greater OTU richness than did Eastern Gartersnakes, and habitat did not have any apparent effect on the microbial community composition in Eastern Gartersnakes. CONCLUSIONS: Our findings suggest host taxonomy to be a determining factor in the cloacal microbiota of snakes and that Tenericutes are associated with aquatic habitats. This is the first report to examine the cloacal microbiome of these species and provides a useful foundation for future work to build upon.

Snake Fungal Disease in Colubridae Snakes in Connecticut, USA in 2015 and 2017.

Snake fungal disease (SFD), caused by the fungus Ophidiomyces ophiodiicola, is an emerging threat to wild snake populations in the US. Data regarding its distribution, prevalence, and population-level impacts are sparse, and more information is needed to better manage SFD in the wild. In this study, we captured 38 wild snakes of five species in Connecticut in the summers of 2015 and 2017. Skin lesions were biopsied and evaluated histologically for fungal dermatitis. At least one individual from each species was positive for SFD, and 48% of snakes sampled in 2015 and 39% of snakes sampled in 2017 were positive for SFD. A Dekay's brownsnake (Storeria dekayi dekayi) with SFD lesions, captured in the summer of 2017, extended the host range of the disease. Thus, SFD was present in wild Connecticut snakes in 2015 and 2017, which demonstrated a wide-spread distribution throughout the state.

Cloacal aerobic bacterial flora and absence of viruses in free-living slow worms (Anguis fragilis), grass snakes (Natrix natrix) and European Adders (Vipera berus) from Germany.

Disease problems caused by viral or bacterial pathogens are common in reptiles kept in captivity. There is no information available on the incidence of viral pathogens or the physiological cloacal bacterial flora of common free-living reptiles in Germany. Therefore, 56 free-living reptiles including 23 European adders (Vipera berus), 12 grass snakes (Natrix natrix) and 21 slow worms (Anguis fragilis) were investigated on the island Hiddensee in northeastern Germany. Pharyngeal and cloacal swabs were taken immediately after capture. Bacteriological examination was performed from the cloacal swabs to study the aerobic cloacal flora. Molecular biological examination included amplification of DNA or RNA from adeno-, rana- and ferlaviruses as well as culturing on Russell's viper heart cells for virus isolation. Salmonella spp. were isolated from European adders but not from the other reptiles examined. The minimal inhibitory concentration was determined from the isolated Salmonella spp. However, some potentially human pathogenic bacteria, such as Proteus vulgaris, Aeromonas hydrophila, Klebsiella pneumoniae and Escherichia coli were isolated. Viruses were not detected in any of the examined reptiles. To the authors' best knowledge, the present study is the first survey of viral pathogens in free-living snakes and slow worms in Germany and the first survey of cloacal aerobic bacterial flora of slow worms.

Heterosporis anguillarum infection in a garter snake (Thamnophis sirtalis).

A captive garter snake (Thamnophis sirtalis) showed marked circumferential thickening of the body wall due to a chronic fibrous to necrotizing inflammatory reaction extending from the serosa to the skin. The lesions were associated with the presence of intra- and extracellular microsporidian organisms. With ultrastructural and molecular analyses the microsporidia were identified as Heterosporis anguillarum. This organism causes a morphologically similar disease in the Japanese eel (Anguilla japonica), but this is the first time it has been identified in an infected reptile.

Reptile infection with Anaplasma phagocytophilum, the causative agent of granulocytic anaplasmosis.

Granulocytic anaplasmosis (GA) is a potentially fatal tick-borne rickettsial disease that occurs sporadically in the far western United States. We evaluated the prevalence of Anaplasma phagocytophilum in multiple species of lizards and snakes from enzootic sites in northern California, described the infestation prevalence of its tick vector Ixodes pacificus on reptiles, and conducted an experimental challenge of western fence lizards (Sceloporus occidentalis) and Pacific gopher snakes (Pituophis catenifer) with A. phagocytophilum delivered via needle inoculation or tick bite. Both serologically and polymerase-chain reaction (PCR)-positive lizards (seroprevalence = 10.8%, PCR prevalence = 10.2%) and snakes (seroprevalence = 5.8%, PCR prevalence = 11.7%) were detected among wild-caught animals. A DNA sequence of the A. phagocytophilum groESL gene from a PCR-positive snake was 100% homologous to that of the human-derived A. phagocytophilum. Experimental attempts to infect naïve animals were unsuccessful for snakes (n = 2), but 1 of 12 lizards became infected for 1 wk only by tick bite. Xenodiagnostic I. pacificus larvae that fed on a PCR-positive lizard did not acquire or transmit rickettsiae. Our findings suggest that lizards and snakes are exposed to A. phagocytophilum by infected ticks, but that they do not serve as primary reservoir hosts of this rickettsia.

Isolation and characterization of a new fungal species, Chrysosporium ophiodiicola, from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta).

Isolation and characterization of the new species Chrysosporium ophiodiicola from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta) are reported. Analysis of the sequences of different fragments of the ribosomal genes demonstrated that this species belongs to the Onygenales and that this species is genetically different from other morphologically similar species of Chrysosporium. This new species is unique in having both narrow and cylindrical-to-slightly clavate conidia and a strong, pungent odor.

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae).

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.

Fatal cutaneous mycosis in tentacled snakes (Erpeton tentaculatum) caused by the Chrysosporium anamorph of Nannizziopsis vriesii.

The fungus Chrysosporium anamorph of Nannizziopsis vriesii was identified as the cause of fatal, multifocal, heterophilic dermatitis in four freshwater aquatic captive-bred tentacled snakes (Erpeton tentaculatum). Pale, 1- to 4-mm focal lesions involving individual scales, occurred primarily on the head and dorsum. Histology showed multifocal coagulation necrosis of the epidermis, with marked heterophilic infiltration without involvement of the underlying dermis. Septate, irregularly branched hyphae, and clusters of 4- to 8- by 2- to 3-microm rod-shaped cells (arthroconidia) were present within the lesions and in a superficial crust. Failure to maintain an acidic environment was likely a predisposing factor in the development of these lesions.

Physicochemical properties and cytopathogenicity of an adenovirus-like agent isolated from corn snake (Elaphe guttata).

A virus isolated from the internal organs of a moribund corn snake (Elaphe guttata) replicated in reptilian cell cultures (IgH-2, TH-1 cells) between 10 and 30 degrees C. Highest infectivity titers of 10(5.5) TCID50/ml were obtained in IgH-2 cells at 25 degrees C. Infected IgH-2 cells showed the development of three morphologically different intranuclear inclusion bodies. During viral assembly the particles formed typical crystalline aggregates in the nucleus. About 64 h after infection progressive desintegration of the nuclear membrane was evident and virus particles were released into the cytoplasm. Different fish cell lines (CLC, CHSE-214, BF-2, PG, RTG-2) were not capable of propagating the virus. The DNA containing agent proved to be stable at pH3, more or less at pH 12 and to treatment with chloroform, but it was rapidly inactivated at 56 degrees C. Electron microscopy revealed nonenveloped icosahedral particles with a diameter of 65-70 nm.