PRELIMINARY EVALUATION OF SYMMETRIC DIMETHYLARGININE AS A BIOMARKER OF RENAL INSUFFICIENCY IN WILD AND REHABILITATED FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

Currently, there are limited antemortem tests available to evaluate renal function in Florida manatees (Trichechus manatus latirostris). Although few reports of renal pathology in manatees exist in the veterinary literature, debilitated animals that present to rehabilitation centers are frequently dehydrated, may have sustained renal trauma secondary to watercraft injury, and may experience ischemic events related to coagulation disorders leading to renal compromise. Clinicians remain limited to the analysis of blood urea nitrogen, creatinine levels, and urinalysis (if urine is obtained) to determine the extent of renal insufficiency, which may not accurately reflect renal function. This poses a diagnostic challenge for clinicians in discerning how critical renal compromise is to the animal's overall health and prognosis. For the first phase of this study, retrospective symmetric dimethylarginine (SDMA) values were determined from banked serum or plasma samples from 14 wild Florida manatees that had been collected while the animals were under rehabilitation in zoological institutions prior to their deaths. Values of SDMA for nine samples from eight manatees with known renal disease, as determined by histopathology, were compared to SDMA values for seven samples from six manatees with no reported renal lesions on histopathology. Values of SDMA from wild Florida manatees with known renal disease (mean 33.56 µg/dl ± 13.15, P= 0.017) were statistically significantly elevated when compared to SDMA values from manatees with no reported renal lesions (mean = 18.71 µg/dl ± 6.9) on histopathology. For the second phase of the study, serum or plasma samples were obtained from wild manatee populations from two geographically separate presumed-healthy wild manatee populations (n = 57). Although the upper limit was higher, serum SDMA concentrations from presumed-healthy wild manatees were comparable to those reported in small animal and equine medicine at 5.88-16.97 µg/dl.

Internal parasites of the two subspecies of the West Indian manatee Trichechus manatus.

The West Indian manatee Trichechus manatus is divided into 2 subspecies: the Antillean (T. m. manatus) and Florida (T. m. latirostris) manatees. This study reports sample prevalence of manatee parasites from populations of these 2 subspecies in different geographical locations. Although necropsy is a valuable diagnostic tool for parasite infections, the need for antemortem diagnostic techniques is important. Fecal samples collected during necropsies of Antillean manatees (n = 3) in Puerto Rico and Florida manatees (n = 10) in Crystal River, Florida, as well as from live-captured Florida manatees (n = 11) were evaluated using centrifugal flotation with sucrose and ethyl acetate sedimentation to compare parasites from each of the populations. Although both fecal examination methods provided similar results, the centrifugal flotation method required less time for diagnosis. The most common parasite eggs found in both populations included the trematodes Pulmonicola cochleotrema and Nudacotyle undicola, oocysts of the coccidian Eimeria spp., and eggs of the ascarid Heterocheilus tunicatus. Eggs of the trematode Chiorchis groschafti were found in both populations of manatees; however, eggs of a related species, Chiorchis fabaceus, were abundant in the Florida samples, but not found in Puerto Rico populations. Trematode eggs of Moniligerum blairi were found in both populations, but were more common in the Florida manatee (42%) than the Antillean manatee (33%). To our knowledge, this is the first report of both Eimeria manatus and Eimeria nodulosa oocysts in Antillean manatees from Puerto Rico.

Investigating seagrass in Toxoplasma gondii transmission in Florida (Trichechus manatus latirostris) and Antillean (T. m. manatus) manatees.

Toxoplasma gondii is a feline protozoan reported to cause morbidity and mortality in manatees and other marine mammals. Given the herbivorous nature of manatees, ingestion of oocysts from contaminated water or seagrass is presumed to be their primary mode of infection. The objectives of this study were to investigate oocyst contamination of seagrass beds in Puerto Rico and determine the seroprevalence of T. gondii in Antillean (Trichechus manatus manatus) and Florida (T. m. latirostris) manatees. Sera or plasma from Antillean (n = 5) and Florida (n = 351) manatees were tested for T. gondii antibodies using the modified agglutination test. No T. gondii DNA was detected via PCR in seagrass samples (n = 33) collected from Puerto Rico. Seroprevalence was 0%, suggesting a lower prevalence of T. gondii in these manatee populations than previously reported. This was the first study to investigate the potential oocyst contamination of the manatee diet, and similar studies are important for understanding the epidemiology of T. gondii in herbivorous marine mammals.

Blood mineral concentrations in manatees (Trichechus manatus latirostris and Trichechus manatus manatus).

Limited information is available regarding the role of minerals and heavy metals in the morbidity and mortality of manatees. Whole-blood and serum mineral concentrations were evaluated in apparently healthy, free-ranging Florida (Trichechus manatus latirostris, n = 31) and Belize (Trichechus manatus manatus, n = 14) manatees. Toxicologic statuses of the animals and of their environment had not been previously determined. Mean mineral whole-blood (WB) and serum values in Florida (FL) and Belize (BZ) manatees were determined, and evaluated for differences with respect to geographic location, relative age, and sex. Mean WB and serum silver, boron, cobalt, magnesium, molybdenum, and WB cadmium concentrations were significantly higher in BZ versus FL manatees (P < 0.05). Mean WB aluminum, calcium, manganese, sodium, phosphorus, vanadium, and serum zinc concentrations were significantly lower in BZ versus FL manatees. Adult manatees had significant and higher mean WB aluminum, manganese, sodium, antimony, vanadium, and serum manganese and zinc concentrations compared to juvenile animals. Significant and lower mean WB and serum silver, boron, cobalt, and serum copper and strontium concentrations were present in adults compared to juveniles (P < or = 0.05). Females had significant and higher mean WB nickel and serum barium compared to males (P < or = 0.05). Mean WB arsenic and zinc, and mean serum iron, magnesium, and zinc concentrations fell within toxic ranges reported for domestic species. Results reveal manatee blood mineral concentrations differ with location, age, and sex. Influence from diet, sediment, water, and anthropogenic sources on manatee mineral concentration warrant further investigation.

Disseminated toxoplasmosis in Antillean manatees Trichechus manatus manatus from Puerto Rico.

Necropsies were conducted on 4 Antillean manatees Trichechus manatus manatus that were stranded in single events on the coastal beaches of Puerto Rico from August 2010 to August 2011. Three manatees were emaciated and the gastrointestinal tracts were devoid of digesta. Microscopically, all manatees had severe widespread inflammatory lesions of the gastrointestinal tract and heart with intralesional tachyzoites consistent with Toxoplasma gondii identified by histological, ultrastructural and immunohistochemical techniques. The gastrointestinal lesions included severe, multifocal to diffuse, chronic-active enteritis, colitis and/or gastritis often with associated ulceration, necrosis and hemorrhage. Enteric leiomyositis was severe and locally extensive in all cases and associated with the most frequently observed intralesional protozoans. Moderate to severe, multifocal, chronic to chronic-active, necrotizing myocarditis was also present in all cases. Additionally, less consistent inflammatory lesions occurred in the liver, lung and a mesenteric lymph node and were associated with fewer tachyzoites. Sera (n = 30) collected from free-ranging and captive Puerto Rican manatees and a rehabilitated/released Puerto Rican manatee from 2003 to 2012 were tested for antibodies for T. gondii. A positive T. gondii antibody titer was found in 2004 in 1 (3%) of the free-ranging cases tested. Disease caused by T. gondii is rare in manatees. This is the first report of toxoplasmosis in Antillean manatees from Puerto Rico. Additionally, these are the first reported cases of disseminated toxoplasmosis in any sirenian. The documentation of 4 cases of toxoplasmosis within one year and the extremely low seroprevalence to T. gondii suggest that toxoplasmosis may be an emerging disease in Antillean manatees from Puerto Rico.

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris).

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra- and inter-assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R(2)≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free-ranging or short-term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long-term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free-ranging, short-term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate.

African manatee (Trichechus senegalensis) habitat suitability at Lake Ossa, Cameroon, using trophic state models and predictions of submerged aquatic vegetation.

The present study aims at investigating the past and current trophic status of Lake Ossa and evaluating its potential impact on African manatee health. Lake Ossa is known as a refuge for the threatened African manatees in Cameroon. Little information exists on the water quality and health of the ecosystem as reflected by its chemical and biological characteristics. Aquatic biotic and abiotic parameters including water clarity, nitrogen, phosphorous, and chlorophyll concentrations were measured monthly during four months at each of 18 water sampling stations evenly distributed across the lake. These parameters were then compared with historical values obtained from the literature to examine the dynamic trophic state of Lake Ossa. Results indicate that Lake Ossa's trophic state parameters doubled in only three decades (from 1985 to 2016), moving from a mesotrophic to a eutrophic state. The decreasing nutrient gradient moving from the mouth of the lake (in the south) to the north indicates that the flow of the adjacent Sanaga River is the primary source of nutrient input. Further analysis suggests that the poor transparency of the lake is not associated with chlorophyll concentrations but rather with the suspended sediments brought-in by the Sanaga River. Consequently, our model demonstrated that despite nutrient enrichment, less than 5% of the lake bottom surface sustained submerged aquatic vegetation. Thus, shoreline emergent vegetation is the primary food available for the local manatee population. During the dry season, water recedes drastically and disconnects from the dominant shoreline emergent vegetation, decreasing accessibility for manatees. The current study revealed major environmental concerns (eutrophication and sedimentation) that may negatively impact habitat quality for manatees. The information from the results will be key for the development of the management plan of the lake and its manatee population. Efficient land use and water management across the entire watershed may be necessary to mitigate such issues.

Diet composition of the African manatee: Spatial and temporal variation within the Sanaga River Watershed, Cameroon.

The present study aimed to investigate the diet of African manatees in Cameroon to better inform conservation decisions within protected areas. A large knowledge gap on diet and seasonal changes in forage availability limits the ability to develop informed local management plans for the African manatee in much of its range. This research took place in the Sanaga River Watershed, which includes two protected areas in the Littoral Region of Cameroon: the Douala-Edea National Park and the Lake Ossa Wildlife Reserve. We analyzed 113 manatee fecal samples and surveyed shoreline emergent and submerged vegetation within the Sanaga River Watershed. We used microhistological analyses to determine the relative contribution of each plant species to African manatee diets and compared across locations and across seasons (wet vs. dry season). We found that the shoreline vegetation is diverse with over 160 plant species, unevenly distributed across space and season, and dominated by emergent vegetation mostly represented by the antelope grass (Echinochloa pyramidalis). We recorded a total of 36 plant species from fecal samples with a spatial and temporal distribution mostly reflecting that of the corresponding shoreline vegetation. African manatees appear to be primarily opportunistically feeding on available vegetation across the seasons and habitat. This work documents the current, but changing, state of plant availability in the Sanaga River Watershed and reports the African manatee diet in Cameroon for the first time. This information can play a critical role in successfully managing the species and these protected areas. If we wish to protect the African manatee and the aquatic ecosystems within the Sanaga River Watershed, we must understand how forage availability changes over time, especially as its waters become nutrient enriched, eutrophic, and exposed to invasive species of plants in a changing world.

Hematology of healthy Florida manatees (Trichechus manatus).

BACKGROUND: Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals. OBJECTIVES: The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. METHODS: Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. RESULTS: Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. CONCLUSIONS: Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.

Per- and polyfluoroalkyl substances (PFAS) in plasma of the West Indian manatee (Trichechus manatus).

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous, synthetic anthropogenic chemicals known to infiltrate and persist in biological systems as a result of their stability and bioaccumulation potential. This study investigated 15 PFAS, including short-chain carboxylic and sulfonic acids, and their presence in a threatened herbivore, the West Indian manatee (Trichechus manatus). Seven of the 15 PFAS examined were detected in manatee plasma. Perfluorooctanesulfonic acid (PFOS) (ranging from 0.13 to 166 ng/g ww) and perfluorononanoic acid (PFNA) (ranging from 0.038 to 3.52 ng/g ww) were detected in every manatee plasma sample examined (n = 69), with differing medians across sampling sites in Florida, Crystal River (n = 39), Brevard County (n = 18), Everglades National Park (n = 8), and four samples (n = 4) from Puerto Rico. With an herbivorous diet and long life-span, the manatee provides a new perspective to monitoring PFAS contamination.

COASTAL HABITAT CHANGE AND MARINE MEGAFAUNA BEHAVIOR: FLORIDA MANATEES ENCOUNTERING REDUCED FOOD PROVISIONS IN A PROMINENT WINTER REFUGE.

A decline in submerged aquatic vegetation (SAV) within Florida's spring-fed, thermal refuges raises questions about how these systems support winter foraging of Florida manatees (Trichechus manatus latirostris). We analyzed telemetry data for 12 manatees over seven years to assess their use of Kings Bay, a winter refuge with diminished SAV. After accounting for the effect of water temperature, we hypothesized that the number of trips out of Kings Bay would increase and the time wintering manatees spent in Kings Bay would decrease. Trips out of and into Kings Bay also were compared to assess potential influences on exiting or entering. There were no detectable differences in the number of trips out of the bay or overall time manatees spent in Kings Bay across winters. The percentage of time water temperatures were below 20°C was the single best predictor of increased time spent in Kings Bay. Trips out of Kings Bay were more likely to occur after 12:00 h and during a high but ebbing tide, compared to trips into the bay. Nine manatees tracked for longer than 75 days in winter spent 7-57% of their time in the Gulf of Mexico, and three of these manatees spent 7-65% of the winter >80 km from the mouth of Kings Bay. Results suggest the low amount of SAV in Kings Bay does not obviate its use by manatees, though there are likely tradeoffs for manatees regularly foraging elsewhere. Accounting for movements of Florida manatees through a network of habitats may improve management strategies and facilitate desirable conservation outcomes.

Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.

The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V subgroup synteny and chain-specific evolution.

The Florida manatee (Trichechus manatus latirostris) has limited diversity in the immunoglobulin heavy chain. We therefore investigated the antigen receptor loci of the other arm of the adaptive immune system: the T cell receptor. Manatees are the first species from Afrotheria, a basal eutherian superorder, to have an in-depth characterization of all T cell receptor loci. By annotating the genome and expressed transcripts, we found that each chain has distinct features that correlates to their individual functions. The genomic organization also plays a role in modulating sequence conservation between species. There were extensive V subgroup synteny blocks in the TRA and TRB loci between T. m. latirostris and human. Increased genomic locus complexity correlated to increased locus synteny. We also identified evidence for a VHD pseudogene for the first time in a eutherian mammal. These findings emphasize the value of including species within this basal eutherian radiation in comparative studies.

Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals.

Mammals diversified by colonizing drastically different environments, with each transition yielding numerous molecular changes, including losses of protein function. Though not initially deleterious, these losses could subsequently carry deleterious pleiotropic consequences. We have used phylogenetic methods to identify convergent functional losses across independent marine mammal lineages. In one extreme case, Paraoxonase 1 (PON1) accrued lesions in all marine lineages, while remaining intact in all terrestrial mammals. These lesions coincide with PON1 enzymatic activity loss in marine species' blood plasma. This convergent loss is likely explained by parallel shifts in marine ancestors' lipid metabolism and/or bloodstream oxidative environment affecting PON1's role in fatty acid oxidation. PON1 loss also eliminates marine mammals' main defense against neurotoxicity from specific man-made organophosphorus compounds, implying potential risks in modern environments.

Clinical biochemistry in healthy manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) are endangered aquatic mammals living in coastal and riverine waterways of Florida and adjacent states. Serum or plasma biochemical analyses are important tools in evaluating the health of free-ranging and captive manatees. The purpose of this study was to measure diagnostically important analytes in the plasma of healthy manatees and to determine whether there was significant variation with respect to location (free-ranging versus captive), age class (small calves, large calves, subadults, adults), and gender. No significant differences in plasma sodium, potassium, bilirubin, glucose, alanine aminotransferase, or creatine kinase were found among these classes of animals. Compared to free-ranging manatees, captive animals had significantly lower mean concentrations of plasma chloride, phosphate, magnesium, triglycerides, anion gap, and lactate. Captive manatees had significantly higher mean values of total CO2, calcium, urea, creatinine, alkaline phosphatase, gamma-glutamyltransferase, total protein, albumin, and albumin/globulin ratio than did free-ranging animals. Differences in the environments of these two groups, including diet, temperature, salinity, and stress, might account for some of these results. The higher plasma lactate and anion gap concentrations and lower total CO2 concentrations of free-ranging manatees were probably due to greater exertion during capture, but the lack of elevated plasma creatine kinase activity relative to captive animals indicates that there was no serious muscle injury associated with capture. Plasma phosphate decreased and total globulins increased with age. Plasma cholesterol and triglyceride concentrations were highest in small calves. Plasma aspartate aminotransferase was higher in large calves than in adults and subadults, and the albumin/ globulin ratio was higher in subadults than in adults. Plasma total CO2 was higher and chloride was slightly lower in females than in males.

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.

Molecular identification of a papilloma virus from cutaneous lesions of captive and free-ranging Florida manatees.

Cutaneous papillomatous lesions were biopsied from three captive Florida manatees (Trichechus manatus latirostris) at Homosassa Springs State Wildlife Park (HSSWP), Homosassa, Florida, USA, and from six free-ranging Florida manatees from Crystal and Homosassa rivers, Florida. Total DNA extracted from these lesions was assayed for the presence of papilloma virus genomes using the polymerase chain reaction (PCR) with primers that target the L1 capsid protein gene. The amplification generated DNA fragments 458 base pairs in length that encompassed a highly conserved domain within the L1 capsid protein and translated into identical polypeptides of 152 amino acids, suggesting the involvement of a single papilloma virus genotype. Multiple amino acid sequence and phylogenetic analyses of the L1 fragment indicated that the Florida manatee papilloma virus is a unique and quite distinct papillomavirus from other known papilloma viruses. The emergence of this new pathogen raises concerns about its potential impact on the already endangered Florida manatee.

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.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida.

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through PERMANOVA, also indicated significant differences in bacterial communities based on the season.