Manatee Trichechus manatus latirostris calf mortality in Florida: a retrospective review of pathology data from 2009-2017.

High maternal investment and extended inter-calving intervals in Florida manatees Trichechus manatus latirostris make calf survivorship critical to overall population growth. However, detailed patterns of causes of mortality in calves have not been reported and state agency statistics report portions of perinatal mortality based on body length rather than actual cause of death (COD). The objectives of this study were to categorize COD based on necropsy data and geographical location in Florida for 1209 manatee calf carcasses (<236 cm total length) examined between January 2009 and December 2017 and to describe factors contributing to calf mortality. Results indicated COD was attributed to natural causes (47%, n = 573), cold stress syndrome (38%, n = 457), watercraft injury (13%, n = 155), or other human-related causes (2%, n = 24). Natural causes were the leading COD for small calves <151 cm, with death due to stillbirth or dystocia most frequent (48%, n = 273/573). Enteric trematodiasis contributed to a large proportion of deaths from natural causes in large calves within the southwest region of Florida, with an increasing annual trend. Brevetoxicosis contributed substantially to natural causes within the southwest region exclusively and was commonly comorbid with enteric trematodiasis. Cold stress syndrome was the leading cause of death for large calves (151-235 cm), with the Atlantic region having the highest proportion of cases. Watercraft injury was a sustained threat to large calves, especially within the southwest region. This report provides details on specific health threats and patterns of mortality among manatee calves.

Clinicopathological prognostic indicators of survival and pathological findings in cold-stressed Florida manatees Trichechus manatus latirostris.

Cold-stress syndrome (CSS) is a leading natural cause of mortality in free-ranging Florida manatees Trichechus manatus latirostris, but comprehensive investigations into blood analyte derangements and prognostic indicators in CSS are lacking. The objectives of this study were to (1) compare admission blood analyte data of manatees pre and post rehabilitation for CSS to identify clinicopathological derangements, (2) identify blood analyte prognostic indicators for survival, and (3) correlate post-mortem anatomic pathological changes with clinicopathological findings to improve the understanding of CS pathophysiology. CSS manatees admitted to a rehabilitation facility between 2007 and 2017 were included: 59 manatees with data for clinicopathological analysis (7 non-survivors and 49 survivors) and 14 manatees with necropsy data (7 with and 7 without blood analyte data). Main interpretive clinicopathological findings indicated systemic inflammation, bone marrow damage, diuresis, malnutrition, tissue necrosis, fat mobilization, hepatic impairment, acid-base imbalances, and gastrointestinal ulceration. The best diagnostically performing prognostic indicators for survival included platelet concentration, aspartate aminotransferase, calcium, and blood urea nitrogen. The main anatomic pathological findings were cutaneous lesions (n = 14), lipid depletion (n = 12), upper gastrointestinal ulceration and/or hemorrhage (n = 9), and pneumonia (n = 5). Based on the identified blood prognostic indicators interpreted in the context of anatomic pathological findings, multi-organ tissue injury, gastrointestinal ulceration and/or hemorrhage, and hemodynamic and platelet derangements are the presumptive major factors of CSS manatee mortality. These results contribute to the understanding of the complex CSS pathophysiology and offer the use of blood analyte prognostic indicators as a clinically applicable tool for the medical care of manatees during rehabilitation, thereby contributing to increased rehabilitation success and conservation of the Florida manatee.

Disseminated toxoplasmosis Toxoplasma gondii in a wild Florida manatee Trichechus manatus latirostris and seroprevalence in two wild populations.

Marine mammals are important indicators for ecosystem health and serve as sentinel species for infectious agents including zoonoses. Histological examination of tissues from a stranded Florida manatee Trichechus manatus latirostris revealed protozoal cysts in the cerebrum and intrahistiocytic tachyzoites in the liver and caudal mesenteric lymph node. Disseminated Toxoplasma gondii infection was confirmed by immunohistochemistry and sequencing of the nuclear ribosomal internal transcribed spacer region of formalin-fixed tissues. The lack of baseline information on Florida manatees' exposure to this pathogen prompted a study into the seroprevalence of T. gondii in 2 separate geographic habitats in Florida, USA, during the winters from 2011-2014. Serum was collected during routine health assessments of 44 apparently healthy manatees from Crystal River (n = 26) on the west central coast of Florida and Brevard County (n = 18) on the east coast of Florida. Serum was screened for detection of T. gondii immunoglobulin G (IgG) antibodies via the modified agglutination test. Two animals from Crystal River from 2011 and 2012 (7.7%) and one animal from Brevard County from 2011 (5.6%) tested positive for T. gondii antibodies. Overall seroprevalence for T. gondii was low in the 2 sampled populations and may reflect a low seroprevalence or animal susceptibility. However, continued monitoring of this pathogen in aquatic ecosystems is warranted due to both possible anthropogenic sources and zoonotic potential.

REPRODUCTIVE NEOPLASMS IN WILD AND LONG-TERM CAPTIVE FEMALE FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

Few reports of neoplastic diseases in manatees exist in the veterinary literature. This case series presents reproductive neoplasia noted in eight wild and long-term captive female Florida manatees (Trichechus manatus latirostris) obtained through carcass recovery and animal rehabilitation programs between April 2009 and May 2014. All cases were evaluated histologically, and diagnoses of uterine carcinoma (n = 1), granulosa cell tumor (n = 2), ovarian adnexal tumor (n = 1), and leiomyoma (n = 5) were made. The underlying cause of tumor development and effects on reproductive success is currently unknown, but possible asymmetric reproductive aging and/or a correlation between obesity and reproductive disorder in long-term nonreproductive female manatees are of interest and warrant further investigation.

ESTABLISHMENT OF ECHOCARDIOGRAPHIC PARAMETERS OF CLINICALLY HEALTHY FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

A standardized echocardiographic technique was recently established for the Florida manatee (Trichechus manatus latirostris). There are no available published data on normal echocardiographic parameters in any Sirenian species. The purpose of this study was to report reference parameters for various echocardiographic measurements. These parameters are intended to serve as a comparison for future research into the prevalence of cardiac diseases in the manatee and to aid in diagnosing animals with suspected cardiac disease in rehabilitation facilities. Annual health assessments of free-ranging manatees in Crystal River National Wildlife Refuge, Florida, and pre-release health assessments of rehabilitated manatees at Tampa's Lowry Park Zoo permitted comparison of echocardiographic measurements in adult (n=14), subadult (n=7), and calf (n=8) animals under manual restraint.

Differences in fecal androgen patterns of breeding and nonbreeding kori bustards (Ardeotis kori).

To better understand breeding conditions to promote reproduction in captive kori bustards, fundamental endocrine studies measuring fecal androgen metabolites in male and female kori bustards were conducted. Feces collected weekly from males and females were analyzed for testosterone using enzyme-linked immunoassay. Results from adult males (n = 5), adult females (n = 10), immature males (n = 10), and immature females (n = 10) revealed seasonally elevated testosterone concentrations in fertile, but not nonfertile adult males and females (P > 0.05). Adult females that were not maintained in a breeding group, or that did not produce eggs, did not demonstrate increases in testosterone compared to egg laying counterparts. In males, but not females, seasonal testosterone increases were accompanied by weight gain. Peaks in male fecal androgen metabolites ranged from 10- to 22-fold higher than nonbreeding season (181.5 ± 19.1 vs. 17.0 ± 0.94 ng/g; P < 0.05). Mean breeding season values for adult males were 83.6 ± 6.1 ng/g vs. nonbreeding season values of 12.3 ± 0.73 ng/g (P < 0.05). In females, average breeding season testosterone concentrations were approximately 4-fold higher than nonbreeding season (55.9 ± 6.0 vs. 14.5 ± 1.8 ng/g), with peaks 10- to 30-fold higher. Results show that noninvasive fecal androgen metabolite analysis can provide a means of predicting fertility potential of male and female kori bustards and might be utilized to assess effects of modifying captive environments to promote reproduction in this species.

Echocardiographic evaluation of clinically healthy Florida manatees (Trichechus manatus latirostris).

Antemortem studies pertaining to the manatee cardiovascular and cardiopulmonary systems are limited despite reports of cardiac disease in postmortem specimens. The objective of this project was to develop a technique for echocardiography in the Florida manatee (Trichechus manatus latirostris). Because of their unique anatomy, a ventral approach was employed by use of an echocardiography table designed specifically for this study. Fourteen clinically healthy, free-ranging and captive Florida manatees underwent echocardiography between the fall of 2011 and winter of 2012. Eight females and six males of various age categories were included in the study. Clear visualization of all valves and chambers was accomplished, and length and width measurements of the left atrium, peak aortic flow velocity, and ejection fraction percentage were calculated in most animals. Abnormalities observed during the study included atrioventricular regurgitation and severe right-atrial enlargement. Based on the results of this study, echocardiography in the Florida manatee is possible, which has both clinical and research implications in larger epidemiologic studies evaluating diseases of the cardiopulmonary and cardiovascular systems.

Cytochemical staining of leukocytes and platelets in the Florida manatee (Trichechus manatus latirostris): identification of a bilobed monocyte similar to other members of the Paenungulata.

Manatees (Antillean-, Amazonian, and African-) and dugongs belong to the Order Sirenia, and when combined with elephants and rock hyraxes, form the Paenungulata. A bilobed mononuclear cell has previously been identified in elephants and rock hyraxes, but not in manatees and dugongs, with cytochemical staining identifying these cells as bilobed monocytes in elephants. The objective of this study was to characterize leukocytes (white blood cells, WBC) and platelets in blood films of Florida manatees (Trichechus manatus latirostris; n = 8) using one routine hematological (Wright-Giemsa) and eight cytochemical stains: alkaline phosphatase (ALP), α-naphthyl butyrate esterase (ANBE), chloroacetate esterase (CAE), Luna, myeloperoxidase (MPx), periodic acid-Schiff (PAS), Sudan black B (SBB), and toluidine blue (TB). Heterophils and lymphocytes comprised most of the WBC, with low numbers of eosinophils, basophils, and monocytes. Additionally, 1-3% of the WBC were bilobed mononuclear cells. Bilobed mononuclear cell proportions were similar to rock hyraxes, but lower than elephants (approximate range 20-60%). Heterophils and eosinophils were positive for MPx, ALP, SBB, and PAS, with heterophils also being positive for CAE. Most of the lymphocytes were positive for ANBE and they were variably positive for CAE. Monocytes and bilobed mononuclear cells had similar cytochemical staining reactions (variably positive for all stains, except Luna and TB), supporting a monocytic origin, like elephants. Platelets were ANBE- and PAS-positive. Luna stain was useful for identifying eosinophils and TB was uninformative. This study provides new information on the morphological features and cytochemical staining characteristics of WBC and platelets and will aid in obtaining accurate hematological data of Florida manatees.

Disseminated Histiocytic Sarcoma in Two Free-Living Florida Manatees (Trichechus manatus latirostris).

During necropsy of two manatees, masses in multiple organs including liver and spleen were identified. They were composed of neoplastic round to spindle-shaped cells, positive for vimentin, Iba-1, lysozyme, and Mac387, consistent with histiocytic sarcoma. One manatee also had an undifferentiated sarcoma that may have arisen from the histiocytic sarcoma.

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.

Cold-related Florida manatee mortality in relation to air and water temperatures.

Many tropical and subtropical species are sensitive to sudden temperature changes, especially drops in temperature. During winters 2009-2010 and 2010-2011, unusually cold temperatures occurred in many parts of Florida, USA, resulting in increased mortality of Florida manatees, sea turtles, fish, corals, and other species. The Florida manatee, in particular, is highly susceptible to cold stress and death when water temperatures drop below 20°C. We sought to characterize the magnitude and timing of reports of cold-related manatee carcasses in relation to fluctuations in water and air temperatures in central-east and central-west Florida during the six winters from 2008 to 2014. We used a generalized linear model to predict counts of manatee carcasses with a cold-related cause of death reported over 7-day bins in relation to various short-term (two weeks or less) and cumulative (incrementally summed from the start of the winter) heating-degree-day effects (HDD; < 20°C) and a categorical winter variable. Using water temperature data, the top-ranked model in both regions included a short-term temperature effect (14-day HDD sum) that preceded increases in reports of cold-related manatee carcasses by 7 days. Cumulative exposure to cold weather over the winter amplified effects on mortality in the central-east region. Quantifying the relationship between cold events and manatee mortality helps us prepare for rescue and salvage operations when extremely cold weather is forecast. This is especially important because anticipated loss or degradation of warm-water refuges due to human activities and sea level rise could potentially impact the manatee population in the future. These methods could also be applied to other species susceptible to cold-related mortality.

Accuracy, precision, and error in age estimation of Florida manatees using growth layer groups in earbones.

Ages of Florida manatees (Trichechus manatus latirostris) can be estimated by counting annual growth layer groups (GLGs) in the periotic dome portion of the tympanoperiotic complex of their earbones. The Florida Fish and Wildlife Conservation Commission manages an archive of more than 8,700 Florida manatee earbones collected from salvaged carcasses from 1989 to 2017. Our goal was to comprehensively evaluate techniques used to estimate age, given this large sample size and changes to processing protocols and earbone readers over time. We developed new standards for estimating ages from earbones, involving two independent readers to obtain measurements of within- and between-reader precision. To quantify accuracy, precision, and error, 111 earbones from manatees with approximately known ages (first known as calves: "KAC") and 69 earbones from manatees with minimum known ages ("MKA," based on photo-identification sighting histories) were processed, and their ages were estimated. There was greater precision within readers (coefficient of variation, CV: 2.4-8.5%) than between readers (CV: 13.1-13.3%). The median of age estimates fell within the true age range for 63.1% of KAC cases and was at least the sighting duration for 75.0% of MKA cases. Age estimates were generally unbiased, as indicated by an average raw error ± SD of -0.05 ± 3.05 years for the KAC group. The absolute error (i.e., absolute value of raw error) of the KAC data set averaged 1.75 ± 2.50 years. Accuracy decreased and error increased with increasing known age, especially for animals over 15 years old, whose ages were mostly underestimated due to increasing levels of resorption (the process of bone turnover that obscures GLGs). Understanding the degree of uncertainty in age estimates will help us assess the utility of age data in manatee population models. We emphasize the importance of standardizing and routinely reviewing age estimation and processing protocols to ensure that age data remain consistent and reliable.

Fatal Systemic Salmonellosis in a Florida Manatee ( Trichechus manatus latirostris).

A subadult male Florida manatee ( Trichechus manatus latirostris) stranded dead on Florida's Atlantic coast in January 2015. Necropsy and histopathologic findings confirmed chronic systemic bacterial infection caused by Salmonella enterica serotype IV 50:z4,z23,:- involving renal, respiratory, lymphatic, and skeletal systems. This was a unique case of systemic salmonellosis in a 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.

Hypercalcemia in two Amazon parrots with malignant lymphoma.

Malignant lymphoma is a common malignancy in birds. Paraneoplastic syndromes, which are commonly observed in domestic animals, have not been reported in association with lymphoma in birds. Hypercalcemia and hyperglobulinemia were found on plasma chemistry in two Amazon parrots, which were presented with aspecific symptoms. In both cases radiography and ultrasound demonstrated signs of hepatomegaly, which proved to be due to malignant lymphoma on postmortem examination. The hypercalcemia was found to be most consistent with a paraneoplastic effect of the malignant lymphoma in these birds. The exact origin of the hyperglobulinemia remains unclear.

Diaphragmatic hernia and right-sided heart enlargement in a Florida manatee (Trichechus manatus latirostris).

Postmortem evaluation of a Florida manatee (Trichechus manatus latirostris) revealed cold stress lesions and previous watercraft trauma that included broken ribs, a diaphragmatic hernia, an enlarged vena cava, and right-sided cardiomegaly. We discuss these findings and present a possible pathogenesis for the cardiomegaly.