Emydomyces testavorans Surveillance in Multiple Free-Ranging Terrestrial and Aquatic Chelonian Species in Illinois, USA.

The order Onygenales contains multiple fungal pathogens that affect free-ranging and zoo-housed reptilian species. Emydomyces testavorans, an onygenalean fungus associated with skin and shell disease, has been sporadically detected in aquatic chelonians. Because of the recent discovery of this organism, little is known about its prevalence in free-ranging chelonians. The objective of this study was to perform surveillance for E. testavorans in six free-ranging aquatic and terrestrial chelonian species in Illinois, USA: Blanding's turtles (n=437; Emydoidea blandingii), painted turtles (n=199; Chrysemys picta), common snapping turtles (n=35; Chelydra serpentina), red-eared sliders (n=62; RES; Trachemys scripta elegans), eastern box turtles (n=73; Terrapene carolina carolina) and ornate box turtles (n=29; Terrapene carolina ornata). Combined cloacal-oral swabs (COSs) or shell (carapace and plastron surfaces) swabs were collected from 2019 to 2021 and tested for E. testavorans using quantitative PCR. The PCR detected E. testavorans in COSs of an adult male, subadult female, and juvenile male Blanding's turtle (0.6%; 95% confidence interval [CI], 0.2-1.9%) and a shell swab from an adult female RES (1.6%; 95% CI, 0-8.7%). Shell lesions consistent with E. testavorans infection were present in two of the positive Blanding's turtles. These results document the rarity of this pathogen on the landscape in Illinois. Additional studies should determine this pathogen's impact on individuals and clarify its significance for conservation efforts of Blanding's turtle, in which E. testavorans has not been reported previously.

Serosurvey for Canine Influenza in Coyotes (Canis latrans) in Illinois, USA, 2000-23.

Coyotes (Canis latrans) share urban habitats with domestic dogs (Canis lupus familiaris), providing opportunities for pathogen transmission. In Chicago, Illinois, USA, canine influenza virus (CIV) is prevalent in dogs. Serologic investigation for exposure in 101 coyote samples collected 2000-23 did not detect any antibodies against CIV H3N2 and H3N8.

Development and analytical validation of a novel quantitative PCR assay for the detection of Trachemys herpesvirus 1.

Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 107 to 1.0 × 101 copies per reaction with an R2 of 0.999, slope of -3.386, and efficiency of 97.39%. The limit of detection was 101 copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.

Otariid gammaherpesvirus 1 in South American fur seals (Arctocephalus australis) and a novel related herpesvirus in free-ranging South American sea lions (Otaria byronia): Prevalence and effects of age, sex, and sample type.

Otariid gammaherpesvirus 1 (OtGHV1) is associated with high rates of urogenital carcinoma in free-ranging California sea lions (Zalophus californianus; CSL), and until recently was reported only in the Northern Hemisphere. The objective of this study was to survey free-ranging South American sea lions (Otaria byronia; SASL) and South American fur seals (Arctocephalus australis: SAFS) in Punta San Juan, Peru for OtGHV1 and to determine prevalence characteristics. Twenty-one percent (14/67) of urogenital swabs collected over three years (2011, 2014, 2015) from live pinnipeds of both species tested positive with a pan-herpesvirus conventional PCR. Sequencing of SAFS amplicons revealed 100% homology to OtGHV1 at the DNA polymerase, glycoprotein B, and viral bcl2-like genes. Sequencing of SASL amplicons revealed a novel related virus, herein called Otariid gammaherpesvirus 8 (OtGHV8). For comparison of sample sites, urogenital, conjunctival, and oropharyngeal swabs collected from 136 live pinnipeds of both species at Punta San Juan between 2011-2018 were then assayed using quantitative PCR for a segment of the OtGHV1/8 DNA polymerase gene using a qPCR assay now determined to cross-react between the two viruses. In total, across both species, 38.6% (51/132) of urogenital swabs, 5.6% (4/71) of conjunctival swabs, and 1.1% (1/90) of oropharyngeal swabs were positive for OtGHV1/8, with SASL only positive on urogenital swabs. Results from SASL were complicated by the finding of OtGHV8, necessitating further study to determine prevalence of OtGHV1 versus OtGHV8 using an alternate assay. Results from SAFS suggest a potential relationship between OtGHV1 in SAFS and CSL. Though necropsy surveillance in SAFS is very limited, geographic patterns of OtGHV1-associated urogenital carcinoma in CSL and the tendency of herpesviruses to cause more detrimental disease in aberrant hosts suggests that it is possible that SAFS may be the definitive host of OtGHV1, which gives further insight into the diversity and phyogeography of this clade of related gammaherpesviruses.

COMPARING THE EFFECTS OF DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID AND LITHIUM HEPARIN ON HEMATOLOGIC VALUES IN BLANDING'S TURTLES (EMYDOIDEA BLANDINGII), PAINTED TURTLES (CHRYSEMYS PICTA), AND COMMON SNAPPING TURTLES (CHELYDRA SERPENTINA).

Hematology is a routine component of clinical management in veterinary patients. Anticoagulant choice can profoundly influence morphologic assessment of erythrocytes, leukocytes, thrombocytes, and their subsequent quantification. Previous chelonian studies suggest that lithium heparin (LH) is a superior anticoagulant due to hemolysis resulting from dipotassium ethylenediaminetetraacetic acid (dEDTA) in some species. The aim of this study was to compare the effects of dEDTA and LH on hematologic values in Blanding's turtles (Emydoidea blandingii, n = 35), painted turtles (Chrysemys picta, n = 34), and common snapping turtles (Chelydra serpentina, n = 36). We collected samples from free-ranging turtles and immediately divided whole blood into LH and dEDTA tubes. Packed cell volume, total solids, erythrocyte sedimentation rate, white blood cell counts, and differential leukocyte counts were determined. Hemolysis was observed macro- and microscopically in dEDTA samples from painted turtles and common snapping turtles. Packed cell volume and heterophil:lymphocyte was lower and erythrocyte sedimentation rate was higher in LH samples from painted turtles (p, 0.05). In snapping turtles, the PCV, number of monocytes, and number of eosinophils was lower in LH samples (p, 0.05). In Blanding's turtles, the number of eosinophils and basophils was higher in LH samples, while heterophil counts were lower (p, 0.05). Anticoagulant choice created constant and proportional bias for multiple analytes in a species-dependent fashion. LH is the recommended anticoagulant for hematology in painted turtles and common snapping turtles. Either LH or dEDTA may be used in Blanding's turtles, though anticoagulant-specific reference intervals may be necessary.

Development and analytical characteristics of a quantitative real-time PCR assay for detection of spheniscid alphaherpesvirus 1 in penguins.

Herpesviruses are associated with disease in many penguin species. Herpesvirus-associated lesions can cause significant morbidity and mortality in penguins and have been identified in African penguins (Spheniscus demersus), Humboldt penguins (Spheniscus humboldti), and a little blue penguin (Eudyptula minor) infected with spheniscid alphaherpesvirus 1 (SpAHV1). Further investigation is necessary to understand the impact of herpesviruses on penguin health, but there are no rapid, sensitive, and specific methods for detecting and quantifying herpesviral load. We therefore developed a quantitative real-time PCR (qPCR) assay for the detection of SpAHV1 in penguins. TaqMan primer-probes targeting the DNA polymerase gene were designed using a commercial software program. Inter- and intra-assay variability, dynamic range, limit of detection, and analytical specificity were assessed. We used our assay to analyze previously collected field samples from Punta San Juan, Peru, in which conventional consensus PCR had detected one SpAHV1-positive penguin sample. Our qPCR assay was highly specific for SpAHV1. It had a dynamic range of 107-101 target copies per reaction and performed with high efficiency and low intra- and inter-assay variability. Reaction efficiency was not impacted by penguin DNA from SpAHV1-negative tracheal swabs. We detected an additional field sample as positive with our newly developed qPCR assay, and although this likely represents detection of another infected penguin, the true disease status of this population is currently uncharacterized given that no gold-standard test exists for SpAHV1. Our qPCR assay may provide a valuable tool in the surveillance and characterization of SpAHV1 in penguins.

COMPARING THE EFFECTS OF LITHIUM HEPARIN AND DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID ON HEMATOLOGIC VALUES IN PRAIRIE RATTLESNAKES (CROTALUS VIRIDIS) AND LAKE ERIE WATER SNAKES (NERODIA SIPEDON INSULARUM).

Anticoagulants prevent clotting of blood samples and preserve cellular morphology for hematologic evaluations, but studies comparing anticoagulants are limited in snakes. The objective of this study was to evaluate the effects of lithium heparin (LH) and dipotassium ethylenediaminetetraacetic acid (EDTA) on hematologic values in prairie rattlesnakes (PR; Crotalus viridis, n = 16) and Lake Erie water snakes (LEWS; Nerodia sipedon insularum, n = 21). Venipuncture was performed and blood samples were immediately aliquoted into LH and EDTA microtainers. Packed cell volume (PCV), total solids (TS), 100-cell differential counts, and Avian Leukopet white blood cell counts (WBC) were determined for each anticoagulant. Passing-Bablok regression and Bland-Altman plots revealed that anticoagulant choice did not constantly or proportionally bias the values of any WBC parameter. Mixed models demonstrated that blood anticoagulated with EDTA had higher PCV in PR (P = 0.04) and TS in both species (P < 0.05). However, the magnitude of the differences attributable to anticoagulant choice was relatively small and likely not clinically important. Hemolysis was not appreciated in any samples. Our findings demonstrate that LH and EDTA are equally appropriate for use in PR and LEWS, but may require separate reference values.

CUTANEOUS MYIASIS AND ITS RELATIONSHIP TO WELLNESS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) IN CAPE COD, MASSACHUSETTS.

Eastern box turtles (Terrapene carolina carolina) face a variety of anthropogenic, infectious, and environmental threats and have been affected by high morbidity and mortality disease events. Wellness parameters in free-ranging eastern box turtles with a high prevalence of myiasis on Cape Cod, MA, were documented to identify epidemiologic trends or associations with several health parameters. There were 109 samples collected from 59 individual box turtles over the course of 4 mon. Six turtles died over the course of this study. Fly larvae infestations varied in severity and were observed in the cutaneous and subcutaneous tissue (n = 18; 30.5%). Animals with myiasis had fewer plastron abnormalities than those without (P = 0.034), and all turtles found in bogs had evidence of fly larvae infections (P < 0.0001). Individuals with myiasis also had lower body condition index (P = 0.014), lower total white blood cells (P = 0.031), lower PCV (P < 0.0001), lower total solids (P < 0.0001), higher erythrocyte sedimentation rate (P < 0.0001), lower calcium (P = 0.018), and lower phosphorus (P = 0.017). Three turtles tested positive for terrapene herpesvirus 1, but presence was not associated with myiasis. Heavy metal analysis revealed no significant differences between turtles with and without myiasis. This study examined the health of a population of eastern box turtles, and continued health assessments will be beneficial in determining the impact of myiasis on future conservation plans.

CONTROLLED CLINICAL TRIAL USING TERBINAFINE NEBULIZATION TO TREAT WILD LAKE ERIE WATERSNAKES (NERODIA SIPEDON INSULARUM) WITH OPHIDIOMYCOSIS.

Ophidiomycosis (snake fungal disease) is an important infectious disease caused by the fungus Ophidiomyces ophidiicola. To mitigate the disease's impact on individual snakes, a controlled clinical trial was conducted using terbinafine nebulization to treat snakes with ophidiomycosis. Fifty-three wild-caught Lake Erie watersnakes (Nerodia sipedon insularum) with apparent ophidiomycosis (skin lesions present, qPCR positive for O. ophidiicola) were divided into treatment and control groups: treatment snakes were nebulized with a 2 mg/ml terbinafine solution for 30 min daily for 30 d; control snakes received nebulization with 0.9% saline or no nebulization. Weekly physical exams were conducted to assign disease severity scores based on the number, type, location, and size of lesions, and qPCR was repeated after each 30-d course of treatment. Persistently qPCR-positive snakes received multiple nebulization courses. Terbinafine nebulization showed mixed results as a treatment for ophidiomycosis: 29.2% of animals treated with terbinafine showed molecular resolution of external disease, based on antemortem swabbing, following 3-6 mon of daily nebulization; this was significantly more than with saline nebulization (5%), but molecular resolution also occurred in 11.1% of snakes that received no treatment. Terbinafine nebulization did not significantly decrease clinical disease, as measured by disease severity scores. Evaluating molecular response to treatment using fungal quantities, terbinafine nebulization significantly reduced fungal quantity after three or more courses of treatment. These results indicate that, although terbinafine nebulization is a promising treatment for ophidiomycosis, snakes may require multiple nebulization courses and disease may not always resolve completely, despite treatment. This treatment may be most useful in snakes from managed populations that can be treated for several months, rather than wild snakes who are not releasable after multiple months in captivity.