Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

Abstract

Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.

Cytokine RT-qPCR and ddPCR for immunological investigations of the endangered Australian sea lion (Neophoca cinerea) and other mammals

“…The broad cross-reactivity suggests that these primer sets are likely to cross react across several other mammalian species. Studies of basal cytokine levels in marine mammals are forthcoming but still limited (Beineke et al, 2007;Beineke et al, 2004;Ferrante, Hunter & Wellehan, 2018;Hofstetter et al, 2017) and, consistent with our study, others have encountered challenges with sensitivity. Sitt et al (2016) determined that IL-4 transcripts were typically absent in killer whales (Orcinus orca) and levels of IL-4 in domestic pig (Sus scrofa) remained low even after lymphocyte stimulation (Murtaugh et al, 1996).…”

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

“…It has been demonstrated that ConA (a selective T-cell mitogen) induces proliferative activity and gene expression of cytokines in bottlenose dolphins, but no information is available regarding the time course ( Hofstetter et al, 2017 ; Segawa et al, 2013 ; Sitt et al, 2008 ). Previous studies on ConA-induced cytokine mRNA expression levels of cPBMCs only presented one or two time points ( Segawa et al, 2013 ; Sitt et al, 2008 ).…”

Dopamine D2 and Serotonin 5-HT1A Dimeric Receptor-Binding Monomeric Antibody scFv as a Potential Ligand for Carrying Drugs Targeting Selected Areas of the Brain

“…Experiments were conducted on RAW 264.7 cells, which are mouse macrophage cells. Cytokine production tests are considered to be reliable in immunogenicity estimation [ 71 ].…”