Charles J. Deutsch

CLINICAL BIOCHEMISTRY IN HEALTHY MANATEES (TRICHECHUS MANATUS LATIROSTRIS)

Abstract 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, γ-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.

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.

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.

Validation of a serum immunoassay to measure progesterone and diagnose pregnancy in the West Indian manatee (Trichechus manatus)

The objective was to validate a high-sensitivity chemiluminescent assay of serum progesterone concentrations for pregnancy diagnosis in manatees. Assay analytical sensitivity was 0.1 ng/mL, with mean intra- and inter-assay coefficients of variation of 9.7 and 9.2%, respectively, and accuracy had a mean adjusted R(2) of 0.98. Methods comparison (relative to Siemens Coat-A-Count RIA) demonstrated r=0.98, Deming regression slope of 0.95, and an intercept of 0.01. Based on ROC analysis, a progesterone concentration >or=0.4 ng/mL was indicative of pregnancy. Assay results were not significantly altered by two freeze-thaw cycles of samples. Characteristic progesterone concentrations during pregnancy were Months 1-4 (1.7-4.7 ng/mL), 5-8 ( approximately 1.0 ng/mL), and 10 and 11 (0.3-0.5 ng/mL), whereas two late-pregnant females with impending abortion had progesterone concentrations of 0.1 ng/mL. Among pregnant females, maximum progesterone concentrations occurred in autumn (3.9+/-1.8 ng/mL), and were greater during all seasons than concentrations in non-pregnant females (0.1-0.2 ng/mL). Progesterone concentrations were also significantly higher in pregnant females than in non-pregnant females and males. This highly sensitive, specific, and diagnostic assay will be valuable for monitoring pregnancy and abortion in manatees.

Monitoring oral temperature, heart rate, and respiration rate of West Indian manatees (Trichechus manatus) during capture and handling in the field

West Indian manatees (Trichechus manatus) are captured, handled, and transported to facilitate conservation, research, and rehabilitation efforts. Monitoring manatee oral temperature (OT), heart rate (HR), and respiration rate (RR) during outof-water handling can assist efforts to maintain animal well-being and improve medical response to evidence of declining health. To determine effects of capture on manatee vital signs, we monitored OT, HR, and RR continuously for a 50-min period in 38 healthy, awake, juvenile and adult Florida manatees (T. m. latirostris) and 48 similar Antillean manatees (T. m. manatus). We examined creatine kinase (CK), potassium (K

A quantitative framework for investigating risk of deadly collisions between marine wildlife and boats

Summary Speed regulations of watercraft in protected areas are designed to reduce lethal collisions with wildlife but can have economic consequences. We present a quantitative framework for investigating the risk of deadly collisions between boats and wildlife. We apply encounter rate theory to demonstrate how marine mammal–boat encounter rate can be used to predict the expected number of deaths associated with management scenarios. We illustrate our approach with management scenarios for two endangered species: the Florida manatee Trichechus manatus latirostris and the North Atlantic right whale Eubalaena glacialis. We used a Monte Carlo simulation approach to demonstrate the uncertainty that is associated with our estimate of relative mortality. We show that encounter rate increased with vessel speed but that the expected number of encounters varies depending on the boating activities considered. For instance, in a scenario involving manatees and boating activities such as water skiing, the expected number of encounters in a given area (in a fixed time interval) increased with vessel speed. In another scenario in which a vessel made a transit of fixed length, the expected number of encounters decreases slightly with boat speed. In both cases, the expected number of encounters increased with distanced travelled by the boat. For whales, we found a slight reduction (˜0·1%) in the number of encounters under a scenario where speed is unregulated; this reduction, however, is negligible, and overall expected relative mortality was ˜30% lower under the scenario with speed regulation. The probability of avoidance by the animal or vessel was set to 0 because of lack of data, but we explored the importance of this parameter on the model predictions. In fact, expected relative mortality under speed regulations decreases even further when the probability of avoidance is a decreasing function of vessel speed. By applying encounter rate theory to the case of boat collisions with marine mammals, we gained new insights about encounter processes between wildlife and watercraft. Our work emphasizes the importance of considering uncertainty when estimating wildlife mortality. Finally, our findings are relevant to other systems and ecological processes involving the encounter between moving agents.

Influence of Manatees' Diving on Their Risk of Collision with Watercraft

Watercraft pose a threat to endangered Florida manatees (Trichechus manatus latirostris). Mortality from watercraft collisions has adversely impacted the manatee population’s growth rate, therefore reducing this threat is an important management goal. To assess factors that contribute to the risk of watercraft strikes to manatees, we studied the diving behavior of nine manatees carrying GPS tags and time–depth recorders in Tampa Bay, Florida, during winters 2002–2006. We applied a Bayesian formulation of generalized linear mixed models to depth data to model the probability (Pt) that manatees would be no deeper than 1.25 m from the water’s surface as a function of behavioral and habitat covariates. Manatees above this threshold were considered to be within striking depth of a watercraft. Seventy-eight percent of depth records (individual range 62–86%) were within striking depth (mean = 1.09 m, max = 16.20 m), illustrating how vulnerable manatees are to strikes. In some circumstances manatees made consecutive dives to the bottom while traveling, even in areas >14 m, possibly to conserve energy. This is the first documentation of potential cost-efficient diving behavior in manatees. Manatees were at higher risk of being within striking depth in shallow water (<0.91 m), over seagrass, at night, and while stationary or moving slowly; they were less likely to be within striking depth when ≤50 m from a charted waterway. In shallow water the probability of a manatee being within striking depth was 0.96 (CI = 0.93–0.98) and decreased as water depth increased. The probability was greater over seagrass (Pt = 0.96, CI = 0.93–0.98) than over other substrates (Pt = 0.73, CI = 0.58–0.84). Quantitative approaches to assessing risk can improve the effectiveness of manatee conservation measures by helping identify areas for protection.

Serum proteins in healthy and diseased Florida manatees (Trichechus manatus latirostris)

A major goal of this study was to determine whether serum protein fractions of healthy Florida manatees differ with age, sex, or living environments (wild versus housed). A second goal was to determine which serum protein fractions vary in diseased versus healthy manatees. Serum protein fractions were determined using agarose gel electrophoresis. Healthy adults had slightly higher total serum protein and total globulin concentrations than younger animals. This largely resulted from an increase in gamma globulins with age. Total serum protein, albumin, alpha-1 globulin, beta globulin, and total globulin concentrations were slightly higher in housed manatees compared to wild manatees, but there was no significant difference in the albumin/globulin (A/G) ratio, suggesting a difference in hydration between these groups. No significant differences were attributable to sex or pregnancy. Serum albumin concentrations and A/G ratios were significantly lower for manatees with boat trauma, entanglements, emaciation, or cold stress compared to healthy manatees. Variable increases were seen in alpha-1globulins, alpha-2 globulins, beta globulins, and gamma globulins. These globulin fractions contain positive acute-phase proteins and immunoglobulins, and their increases may reflect acute or chronic active inflammation. Changes in serum protein fractions were not consistent enough to justify the use of serum protein electrophoresis as a routine diagnostic test for manatees. However, serum (or plasma) protein electrophoresis is required when accurate values for albumin and globulins are needed in manatees and in determining which protein fractions may account for a hyperproteinemia or hypoproteinemia reported in a clinical chemistry panel.

An expert elicitation process to project the frequency and magnitude of Florida manatee mortality events caused by red tide (Karenia brevis)

Table summarizing results from expert elicitation process to project the frequency and magnitude of Florida manatee mortality events caused by red tide (Karenia brevis).