Investigating the gene expression profiles of rehabilitated Florida manatees (Trichechus manatus latirostris) following red tide exposure.

To investigate a Florida manatee (Trichechus manatus latirostris) mortality event following a red tide bloom in Southwest Florida, an RNA sequencing experiment was conducted. Gene expression changes in white blood cells were assessed in manatees rescued from a red tide affected area (n = 4) and a control group (n = 7) using RNA sequencing. The genes with the largest fold changes were compared between the two groups to identify molecular pathways related to cellular and disease processes. In total, 591 genes (false discovery rate <0.05) were differentially expressed in the red tide group. Of these, 158 were upregulated and 433 were downregulated. This suggests major changes in white blood cell composition following an exposure to red tide. The most highly upregulated gene, Osteoclast associated 2C immunoglobulin-like receptor (OSCAR), was upregulated 12-fold. This gene is involved in initiating the immune response and maintaining a role in adaptive and innate immunity. The most highly downregulated gene, Piccolo presynaptic cytomatrix protein (PCLO), was downregulated by a factor of 977-fold. This gene is associated with cognitive functioning and neurotransmitter release. Downregulation of this gene in other studies was associated with neuronal loss and neuron synapse dysfunction. Among the cellular pathways that were most affected, immune response, including inflammation, wounds and injuries, cell proliferation, and apoptosis were the most predominant. The pathway with the most differentially expressed genes was the immune response pathway with 98 genes involved, many of them downregulated. Assessing the changes in gene expression associated with red tide exposure enhances our understanding of manatee immune response to the red tide toxins and will aid in the development of red tide biomarkers.

Lack of cross-reactivity of human and porcine reagents to quantify manatee (Trichechus manatus) cytokines.

Veterinary medical examinations, including both physical examination and diagnostic tests, are important to monitor the health of both managed-care and wild marine mammals. However, limited species-specific reagents and assays are available that may contribute to a broader medical examination. This project evaluated if commercially available human and porcine antibodies and reagents would cross-react with manatee (Trichechus manatus) cytokines as the first step to validate a new diagnostic tool for manatees. Overall, as a result of limited cross-reactivity, human and porcine commercial reagents did not allow for the quantification of manatee cytokines. At this point, caution must be exercised when using human or porcine immunoassay reagents to quantify manatee cytokines if the reagents have not been fully validated. Future efforts will continue to explore and test the cross-reactivity of reagents to measure manatee cytokines as new species-specific and commercial reagents become available.

The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity.

Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee-specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity.

Sublethal red tide toxin exposure in free-ranging manatees (Trichechus manatus) affects the immune system through reduced lymphocyte proliferation responses, inflammation, and oxidative stress.

The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected by exposure to blooms of the toxic dinoflagellate, Karenia brevis. K. brevis blooms are common in manatee habitats of Florida's southwestern coast and produce a group of cyclic polyether toxins collectively referred to as red tide toxins, or brevetoxins. Although a large number of manatees exposed to significant levels of red tide toxins die, several manatees are rescued from sublethal exposure and are successfully treated and returned to the wild. Sublethal brevetoxin exposure may potentially impact the manatee immune system. Lymphocyte proliferative responses and a suite of immune function parameters in the plasma were used to evaluate effects of brevetoxin exposure on health of manatees rescued from natural exposure to red tide toxins in their habitat. Blood samples were collected from rescued manatees at Lowry Park Zoo in Tampa, FL and from healthy, unexposed manatees in Crystal River, FL. Peripheral blood leukocytes (PBL) isolated from whole blood were stimulated with T-cell mitogens, ConA and PHA. A suite of plasma parameters, including plasma protein electrophoresis profiles, lysozyme activity, superoxide dismutase (SOD) activity, and reactive oxygen/nitrogen (ROS/RNS) species, was also used to assess manatee health. Significant decreases (p<0.05) in lymphocyte proliferation were observed in ConA and PHA stimulated lymphocytes from rescued animals compared to non-exposed animals. Significant correlations were observed between oxidative stress markers (SOD, ROS/RNS) and plasma brevetoxin concentrations. Sublethal exposure to brevetoxins in the wild impacts some immune function components, and thus, overall health, in the Florida manatee.

Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris.

The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected each year by exposure to cold weather or harmful algal blooms (red tide; Karenia brevis). Exposures can be sublethal, resulting in stressed animals that are rescued and taken to authorized facilities for rehabilitation, or lethal if exposures are prolonged or unusually severe. To investigate whether sublethal environmental exposures can impair immune function in manatees, rendering animals vulnerable to disease or death, mitogen-induced proliferation was assessed in lymphocytes from manatees exposed to cold temperatures (N=20) or red tide (N=19) in the wild, and compared to lymphocyte responses from healthy free-ranging manatees (N=32). All animals sampled for this study were adults. Lymphocytes were stimulated in vitro with either concanavalin A (ConA) or phytohemagglutinin (PHA) and proliferation was assessed after 96 h using incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into newly synthesized DNA. Proliferation of lymphocytes from manatees rescued from exposure to red tide or cold-stress was approximately one-third that of lymphocytes from healthy free-ranging manatees. To examine the direct effects of red tide toxins on lymphocyte function, mitogen-induced proliferation was assessed following co-culture of lymphocytes with K. brevis toxin extracts. Stimulation indices decreased with increasing toxin concentration, with a significant decrease in proliferation occurring in the presence of 400 ng red tide toxins/ml. When lymphocytes from cold-stressed manatees were co-cultured with red tide toxin extracts, proliferative responses were reduced even further, suggesting multiple stressors may have synergistic effects on immune function in manatees.

Viral papillomatosis in Florida manatees (Trichechus manatus latirostris).

The Florida manatee (Trichechus manatus latirostris) is one of the most endangered marine mammals in American coastal waters. Naturally resistant to infectious disease, the manatee immune system appears highly developed to protect it against the harsh marine environment and the effects of human-related injury. In 1997, seven captive Florida manatees developed multiple, cutaneous, pedunculated papillomas over a period of 6 months. Approximately 3 years later, four of the seven manatees developed multiple, cutaneous, sessile papillomas topically and clinically distinct from the initial lesions, some of which are still present. Histologic, ultrastructural, and immunohistochemical features indicated that the two distinct phenotypic lesions were caused by papillomaviruses (PVs). Preliminary immunologic data correlated with daily clinical observations suggested that the manatees were immunologically suppressed and that the papillomas were caused by activation of latent PV infections and reinoculation from active infections. The emergence of PV-induced papillomas in captive manatees, the possibility of activation of latent infection or transmission of active infection to free-ranging manatees, and the underlying cause of immune suppression predisposing manatees to develop viral papillomatosis are serious concerns for the future management of this highly endangered species.