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.

COMPARING THE EFFECTS OF LITHIUM HEPARIN AND DIPOTASSIUM ETHYLENEDIAMINETETRAACETIC ACID ON HEMATOLOGIC VALUES IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Anticoagulants are employed to prevent clotting and preserve cellular morphology for clinical pathology tests. Lithium heparin (LH) is the most frequently used anticoagulant in chelonians; however, dipotassium ethylenediaminetetraacetic acid (EDTA) may be superior in some species. Although eastern box turtles' (Terrapene carolina carolina) hematologic parameters are well studied, the effects of different anticoagulants on hematology in this species are unknown. This study evaluated the effects of LH and EDTA on hematologic values in free-living eastern box turtles (N = 59). Blood samples were collected from eastern box turtles in Illinois and immediately divided between LH and EDTA microtainers, and complete blood counts were performed on each sample. Grossly, plasma from EDTA blood samples was frequently and significantly hemolyzed. Blood mixed with LH had higher packed cell volume (PCV) (P = 0.04), white blood cell count (WBC) determined by Leukopet (P < 0.0001), WBC determined by blood film estimate (P < 0.0001), absolute heterophils (P = 0.007), absolute lymphocytes (P < 0.0001), and lower total solids (P < 0.0001) and absolute monocytes (P = 0.0001) than blood mixed with EDTA. All relative leukocyte counts were significantly different between the anticoagulants (P < 0.0001). EDTA apparently lysed turtle erythrocytes in this study, making it difficult to accurately count white blood cells and artificially lowering PCV. These findings demonstrate that EDTA should not be used in eastern box turtles.

PREVALENCE AND ANTIMICROBIAL RESISTANCE PATTERNS OF SALMONELLA SPP. IN TWO FREE-RANGING POPULATIONS OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Salmonellosis is an important zoonotic infection, and exposure to pet reptiles has been implicated in several human outbreaks. Although several studies report a low prevalence of salmonellae in free-ranging chelonians, they may serve as a reservoir. In spring and summer of 2013 and 2019, free-ranging eastern box turtles (Terrapene carolina carolina) from populations in Illinois (rural) and Tennessee (urban) were collected through canine and visual search. Cloacal swab samples were collected from each turtle, selectively enriched with tetrathionate broth, then plated on selective and differential media to isolate Salmonella spp. Genus was confirmed via MALDI-TOF MS and antibiotic sensitivities were performed. Isolates were serotyped by the National Veterinary Services Laboratory. Of the 341 turtles sampled, Salmonella spp. were detected in nine individuals (2.64%; 95% CI: 1.2-5.0%). The isolates included five different serovars: Anatum (n = 2), Newport (n = 2), Thompson (n = 1), Bareilly (n = 2), and Hartford (n = 2). Salmonella spp. were detected from six animals in 2013 (3.19%, 95% CI: 1.2-6.8%) and three in 2019 (1.96%, 95% CI: 0.4-5.6%). There was no significant difference in prevalence between state, (P = 0.115), Illinois locations (P = 0.224), season (P = 0.525), year (P = 0.297), sex (P = 0.435), or age class (P = 0.549). The health of Salmonella-positive and -negative turtles was not significantly different, as assessed through hematology and plasma biochemistry (P > 0.05), indicating asymptomatic carrier status. The low prevalence detected in this study likely concludes that free-ranging eastern box turtles play a minimal role in the spread of salmonellae. However, the identified serotypes are potentially human- and animal-pathogenic. Documenting the prevalence of Salmonella serotypes in animal indicators furthers our understanding of their spread between humans, animal agriculture, and the environment.

A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum.

Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in CryptosporidiumIMPORTANCECryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.