Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

PLoS One. 2017 Dec 22;12(12):e0189437. doi: 10.1371/journal.pone.0189437. eCollection 2017.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bottle-Nosed Dolphin / blood*
  • Cluster Analysis
  • Cytokines / blood
  • Cytokines / genetics*
  • Leukocytes / metabolism*
  • RNA, Messenger / genetics*
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • Cytokines
  • RNA, Messenger

Grants and funding

This work was supported by the Office of Naval Research grant N0001412IP20029 (https://www.onr.navy.mil/Contracts-Grants). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.