Quantitative Measurement of Cytokine MRNA in Manatee (Trichechus manatus) Blood Leukocytes for Identifying Pro- and Anti-inflammatory States
IAAAM Archive
Brett Smith1; Federica Riva1,2; Christina Funke1; Jeffrey Stott1
1Laboratory for Marine Mammal Immunology; Department of Pathology/Microbiology/Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA; 2Department of Animal Pathology, Faculty of Veterinary Medicine, University of Milan, Italy


The immune system of the Florida manatee is not well-defined. Development and application of advanced techniques for measuring manatee immunologic parameters would be most beneficial in health assessment of free-ranging and captive manatees. The intent of the current study was to develop quantitative assays for identifying the expression of cytokines in manatee peripheral blood leukocytes. Cytokines are low-molecular-weight regulatory proteins or glycoproteins that serve as soluble messengers of the immune system. They are differentially produced by leukocytes, and various other cells, and play a pivotal role in leukocyte development, response, effector function, traffic and inflammation. Cytokines, or their mRNA transcripts, are ideal targets for providing quantitative data indicative of animal health.

Initial efforts were directed at establishing partial genetic sequences for a variety of pro-and anti-inflammatory manatee cytokine genes including TNFα, IL-1β, IL-6 and IL-10. Genes encoding manatee IL-4, inducible nitric oxide synthetase (iNOS) and a housekeeping gene (ribosomal subunit gene, S9) were also identified. Manatee peripheral blood leukocytes were non-specifically stimulated with mitogens and lipopolysaccharide (LPS) followed by extraction of the induced mRNA. Degenerate primers for bovine IL1β and S9, orcinus IL1β, TNFα, iNOS and S9, and tursiops iNOS and IL-10 were used in PCR to generate cDNA for sequencing. Manatee-specific primers were subsequently designed and successfully used to develop quantitative PCR's (Q-PCR) for measuring relative amounts of message in peripheral blood leukocytes.

Whole blood samples have been collected from a variety of manatees including: i) free-ranging animals in Tampa Bay, ii) captive-display animals from Disney, iii) animals in rehabilitation just prior to release, iv) animals in Homosassa Springs (before and after placement in warmer water, artificial pools) and v) more recently an animal suffering from cold-stress syndrome (CSS). Blood was collected in PaxGene vacutainers tubes to negate the rapid degradation of mRNA, a feature that is especially problematic for cytokine transcripts. Blood was frozen at -70 C for storage and mRNA extracted within six months. cDNA was synthesized from the mRNA using random-primers in PCR and then subjected to Q-PCR.

To date, 49 bloods samples have been analyzed, many representing multiple time points from the same animal. While the generation of cytokine expression data is in its infancy relative to establishing clinical utility, several observations are worth noting. A comparison of the free-ranging (n=12) with the pre-release rehabilitated animals (n=10) identified significantly lower levels of TNFα (p <0.05) and higher levels of IL-1β (p <0.05) in the latter group; differences in IL-10 and iNOS were not detected. A temporal (7 time points/animal over 9 months) analysis of two manatees moved from the relatively cold waters of Homosassa Springs to adjacent artificial pools, followed by transport to the Columbus Zoo, suggested transient increases in the pro-inflammatory cytokines, TNFα and IL-1β; levels of IL-10 and iNOS were variable. Lastly, analysis of the CSS manatee demonstrated some of the lowest levels of TNFα and IL-1β recorded to date.

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Brett Smith