Development and testing of species-specific ELISA assays to measure IFN-γ and TNF-α in bottlenose dolphins (Tursiops truncatus)

Abstract

Monitoring the immune status of cetaceans is important for a variety of health conditions. Assays to quantify cytokines, especially pro-inflammatory cytokines, could be employed, in addition to currently available diagnostic assays, to screen for alterations in the health status of an animal. Though a number of immunological assays are readily available for humans and mice, specific assays for many veterinary species, including cetaceans such as bottlenose dolphins (Tursiops truncatus), are more limited. Herein, we describe the development of IFN-gamma (IFN-γ) and TNF-alpha (TNF-α) enzyme-linked immunosorbent assays (ELISAs) specific to bottlenose dolphins. Utilizing these assays, we monitored the immune status of bottlenose dolphins from a managed population over a period of eleven months. The ELISA assays developed for bottlenose dolphins were used to measure IFN-γ and TNF-α in serum or in culture supernatants from peripheral blood mononuclear cells (PBMCs) stimulated with varying concentrations of mitogens concanavalin A (ConA) or phytohemagglutinin (PHA). Induction of TNF-α in PBMC cultures was consistently highest with 1 μg/mL ConA, while 1 μg/mL PHA induced the highest secretion of IFN-γ. Serum levels of TNF-α and IFN-γ remained relatively constant for each animal over the time period examined. CBC and plasma chemistry variables measured concurrently in the bottlenose dolphins were then examined as independent predictors of cytokine levels. We found these clinical variables were more likely to predict linear changes in serum IFN-γ and TNF-α levels compared to concentrations of these cytokines in mitogen-stimulated PBMC culture supernatants. Cytokine assays developed will be of substantial benefit in monitoring bottlenose dolphin health as an adjunct to currently available diagnostic tests.

Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus)

“…Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 µg/ml C-AgNP 20 after 24 h of culture. TNF-α is a cytokine specifically useful to measure the inflammatory state of an animal and it is primarily produced by macrophages and both Th1 and Th2 cells in response to both acute and chronic conditions ( Eberle et al, 2018 ). Previous studies have demonstrated that TNF-α is a contributing factor in the inflammatory response against infection of intracellular micropathogens such as Plasmodium spp., T. gondii , Leishmania major , and Trypanosoma spp.…”

T Helper Cell Subsets and Their Functions in Common Bottlenose Dolphins (Tursiops truncatus)

“…While informative on the distribution of the cell types secreting cytokines in different tissues, this method is limited to the use of tissues from dead animals, or biopsies in live animals, which would be a rather invasive procedure. ELISAs have been developed to measure killer whale-specific IL-6 (57) and dolphin-specific IFNγ and TNFα (58) in serum and tissue culture medium. While sensitive and reproducible under stringent laboratory conditions, ELISAs are relatively time consuming and labor-intensive.…”