Peripheral blood hematology, plasma biochemistry, and the optimization of an in vitro immune-based assay in the brown watersnake (Nerodia taxispilota)

ABSTRACT

Reptiles represent a phylogenetic lineage that provides a unique link between ectothermic anamniotes and endothermic amniotes. Compared to mammalian and avian species, our understanding of the reptilian immune system is greatly lacking. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In the present study, brown watersnakes (Nerodia taxispilota) were live-captured in the wild, sexed, weighed, measured, bled via the caudal vein, and released. At 24 hr post-collection, peripheral blood leukocytes were enriched and evaluated with an established mammalian in-vitro lymphocyte proliferation assay. Snake peripheral blood leukocyte enrichment yielded >90% lymphocytes with viabilities averaging 81.5%. Baseline physiologic data for N. taxispilota, including hematology and total solids, leukocyte differentials, cell recovery, and plasma biochemistry, were also collected. Cells cultured with Concanavalin A exhibited significantly increased proliferation at both 72 and 96 hr. These preliminary results show that enriched peripheral blood from wild-caught N. taxispilota provides a sufficient yield of leukocytes that can be cultured and functionally evaluated using a standard mammalian in-vitro immune-based assay.

KEYWORDS:

• Snake
• peripheral blood collection
• hematology
• lymphocyte separation
• mitogen
• proliferation

Acknowledgments

All snake handling and blood collection procedures were reviewed and approved by the University of Georgia’s Institutional Animal Care and Use Committee (AUP# A2019 05-024-Y1-A0). The authors thank Dr. Nicole Stacy for her assistance with interpretation of blood smear data. The authors would also like to thank the anonymous reviewers who provided valuable feedback for this manuscript. This project was partially funded by the Department of Energy under award number DE-EM0004391 to the University of Georgia Research Foundation and by the Savannah River Nuclear Solutions – Area Completions Project. Data collection and analysis were also assisted by funds from the American Society of Ichthyologists and Herpetologists (ASIH). Preparation of this manuscript was also supported by an assistantship through the University of Georgia’s Interdisciplinary Toxicology Program.

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Additional information

Funding

This work was supported by the University of Georgia’s Interdisciplinary Toxicology Program; American Society of Ichthyologists and Herpetologists; U.S. Department of Energy [DE-EM0004391].