Furthering the development of the Jamaican fruit bat as an animal model for immunology
Date
2023
Authors
Burke, Bradly E., author
Schountz, Tony, advisor
Henao-Tamayo, Marcela, advisor
Zabel, Mark, committee member
Bosco-Lauth, Angela, committee member
Hansen, Tod, committee member
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Abstract
Bats are the only flying mammals capable of powered sustained flight and encompass over 1,400 species. Bats interconnect important ecological services, agricultural crop services, agricultural husbandry health, and human health. The order of chiroptera is largely understudied for immunological research even though bats are reservoirs for viruses that are transmissible to livestock and humans including, henipaviruses, filoviruses, coronaviruses, and lyssaviruses. In 2006 Colorado State University established a breeding colony of Jamaican fruit bats (Artibeus jamaicensis) for use in the study of bat-borne viral infections. Establishment of a bat colony is only the first step to the development of an animal model. The next step to develop an animal model is to elucidate the immunological systems and functionality of them. To elucidate the immunological systems, bat specific reagents are needed to characterize and perform hypothesis driven research. However, the paucity of bat specific reagents largely limits hypothesis driven research. Antibodies and cell lines are foundational reagents to immunology and virology. Antibodies are integral reagents used for many immunological and biochemical assays: ELISAs, ELISPOTs, western blots, cytometric bead assays, magnetic bead pull-down, surface plasmon resonance, microscopy, in vivo cellular depletion, and flow cytometry. Cell lines allow for in vitro assays and the propagation of viruses to be performed. The production and validation of Jamaican fruit bat specific antibodies targeting CD3γ, CD4, and CD8α epitopes for the investigation of cytotoxic CD8 T cells, and CD4 T helper cells are described in this study. Furthermore, the identification and validation of commercially available cross-reactive antibodies targeted to epitopes for various proteins and glycolipids: asialo GM1, CD3ε, CD8α, CD19, CD34, CD40, CD44, CD45, CD80, CD104, CD154, CD161, and MHC-II. This work has described their use in highly quantitative flow cytometric analysis, enrichment of cell populations by fluorescently activated cell sorting, and highly qualitative anatomical data by microscopy. This study has identified a novel CD19+CD3+ T cell population under homeostatic conditions of the Jamaican fruit bat immune system that has not been identified in humans, mice, or other bat species under a homeostatic state. The use of anti-asialo GM1 in vivo treatment of Jamaican fruit bats to target natural killer cells is also described in this work. Lastly the construction of plasmids for the production of Jamaican fruit bat growth factors: epidermal growth factor, wnt3a, R-Spondin-2, noggin, and gastrin are described for their use in culturing Jamaican fruit bat primary cells – especially gastrointestinal crypt stem cells. Validation of cellular markers and reagents is a crucial first step in the investigative process that allows for the generation of informed conclusions – in any study. Furthermore, validation of cellular markers holds a higher level of imminent need and accuracy in underdeveloped animal models. This work provides a framework for other researchers in the advancement of underdeveloped animal models for immunology to more rigorously test antibody cross-reactivity. Furthermore, this work highlights the need to build a robust body of literature of cross-reactive antibodies for underdeveloped animal models.
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Embargo Expires: 05/26/2025
Subject
coronavirus
flow cytometry
immunology
cross-reactive antibodies
bats
hybridoma