Cold Stress Syndrome Research Articles

Sirenian genomes illuminate the evolution of fully aquatic species within the mammalian superorder afrotheria

Sirenians of the superorder Afrotheria were the first mammals to transition from land to water and are the only herbivorous marine mammals. Here, we generated a chromosome-level dugong (Dugong dugon) genome. A comparison of our assembly with other afrotherian genomes reveals possible molecular adaptations to aquatic life by sirenians, including a shift in daily activity patterns (circadian clock) and tolerance to a high-iodine plant diet mediated through changes in the iodide transporter NIS (SLC5A5) and its co-transporters. Functional in vitro assays confirm that sirenian amino acid substitutions alter the properties of the circadian clock protein PER2 and NIS. Sirenians show evidence of convergent regression of integumentary system (skin and its appendages) genes with cetaceans. Our analysis also uncovers gene losses that may be maladaptive in a modern environment, including a candidate gene (KCNK18) for sirenian cold stress syndrome likely lost during their evolutionary shift in daily activity patterns. Genomes from nine Australian locations and the functionally extinct Okinawan population confirm and date a genetic break ~10.7 thousand years ago on the Australian east coast and provide evidence of an associated ecotype, and highlight the need for whole-genome resequencing data from dugong populations worldwide for conservation and genetic management.

EVALUATION OF THE PHARMACOKINETIC BEHAVIOR OF TULATHROMYCIN (DRAXXIN) IN FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS) UNDERGOING MEDICAL REHABILITATION.

Florida manatees (Trichechus manatus latirostris) frequently present to rehabilitation care facilities for various conditions, including boat strike trauma, cold stress syndrome, and brevetoxicosis. Throughout the course of treatment, antimicrobial use to treat respiratory disease is frequently warranted. To date, clinicians have extrapolated dosages based on established information available in bovine and equine medicine. The routes of administration, efficacy, and treatment intervals are considerations in dealing with critical wild animals. The use of tulathromycin, a triamilide antibiotic, has been studied in multiple domestic species of economic importance, including cattle, small ruminants, and swine, and has revealed efficacy against respiratory diseases. Given this information, this antibiotic has also been used in manatees with positive clinical outcomes. This study employed sparse sampling and evaluated banked plasma samples at various time intervals post-tulathromycin administration obtained during the clinical treatment course of nine animals during their rehabilitation. Preliminary pharmacokinetic analysis following administration of a single dose estimated a half-life of 33.75 h and volume of distribution per fraction absorbed (Vz/F = 4.29 L/kg). The pharmacokinetic behavior of tulathromycin in Florida manatees can be used to optimize dosage regimens in this species.

Lipidomics reveals specific lipid molecules associated with cold stress syndrome in the Florida manatee (Trichechus manatus latirostris)

Cold stress syndrome (CSS) in the Florida manatee (Trichechus manatus latirostris) results in perturbations to many physiological pathways, often leading to further illness or death. In this study, we applied a non-targeted lipidomics approach with ultra-high performance liquid chromatography and high-resolution tandem mass spectrometry to characterize changes related to CSS in the lipidomic profiles of manatee plasma. Lipidomic analyses were conducted on healthy manatee (control) and cold-exposed manatee plasma samples with varying concentrations of Serum Amyloid A (SAA), an acute-phase protein that is associated with inflammatory disease. Control manatees (n = 10) were compared to all manatees exposed to cold temperatures (n = 17), and a subset of those manatees with SAA values > 120 μg/mL (n = 9). Increased SAA values were associated with higher levels of various acylcarnitine lipids, while several triacylglycerols and oxidized triacylglycerols were significantly lower in manatees with cold exposure. These identified lipids are critical molecules involved in the maintenance of energy homeostasis and could potentially be examined in conjunction with current physical parameters to characterize cold stress. The ability to detect such differences highlights the addition of lipidomics as a valuable tool in understanding cold stress and potentially other illnesses in manatees. Further investigation into the function of the altered lipids could greatly increase our understanding of lipid metabolism in physiologically stressed manatees as well as other marine mammals and inform future management recovery strategies.

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.

ESOPHAGEAL MEASUREMENT OF CORE BODY TEMPERATURE IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS)

Cold-stress syndrome (CSS) is one of the leading natural threats to free-ranging Florida manatees (Trichechus manatus latirostris). Cold water exposure below the species' acceptable physiologic range is a frequent occurrence for manatees during cold weather months causing CSS-induced systemic illness and significant annual mortality. Although CSS is a commonly presented condition at manatee rehabilitation facilities, the core body temperatures in CSS manatees are currently unknown due to the lack of clinically applicable and accurate temperature measurement methodologies. Our objective was to establish a clinically applicable measurement methodology of core body temperature in manatees. A novel, minimally invasive temperature technique to obtain esophageal temperature by placing a temperature sensor through an oro-gastric tube was compared to current oral and nasal methods in 20 clinically healthy manatees. Results identified the esophageal measurement as the best performing and most precise temperature methodology. The superior performance of esophageal temperature measurements differed significantly from both nasal and oral measurements, while nasal and oral measurements did not differ when compared with each other. The esophageal measurements were consistent with manatee core body temperature, facilitating generation of a reference interval for core body temperature in healthy manatees (35.0-35.8 C). Four CSS medical cases were evaluated with the newly validated esophageal temperature method, facilitating diagnosis of hypothermia. The application of this temperature measurement technique to CSS manatees in field or rehabilitation settings will help in understanding CSS pathophysiology, improve medical assessments during rehabilitation, and contribute to conservation efforts.

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.

Heat flux in manatees: an individual matter and a novel approach to assess and monitor the thermal state of Florida manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) possess an unusual suite of adaptations to accommodate both a fully aquatic lifestyle and an herbivorous diet, including a low metabolic rate and a very limited thermoneutral zone. Their relatively high lower critical temperature of around 20°C suggests strong sensitivity to cold, thereby limiting their distribution to tropical and subtropical waters. "Cold stress syndrome" affects and kills Florida manatees every year during intense or prolonged cold weather, posing one of the major threats to manatees. However, knowledge regarding manatee thermoregulation is sparse, but essential for effective conservation and management of this threatened species. We measured heat flux in two captive Florida manatees at multiple times of the year, at 41 sites distributed across the entire body surface of each manatee. Heat flux differed significantly between individuals, and among body sites and times of the year. The pectoral flippers and axillae were identified as areas with highest heat exchange. Despite exposure to constant water temperature throughout the year, the manatees in this study had significantly lower heat flux in winter than in summer. We used the measured heat flux values to calculate total heat dissipation in individual manatees. The values estimated this way correspond well with the low metabolic rates estimated in previous studies, confirming the reliability of our novel approach. Our method provides simple and useful options for enhancing manatee welfare by monitoring the animals' thermal state during potentially stressful activities such as during medical treatment, capture restraints and transportation.

Thromboelastography in wild Florida manatees (Trichechus manatus latirostris).

Thromboelastography (TEG) provides a comprehensive evaluation of blood clot formation. This test can be used to identify abnormalities in coagulation by assessing multiple aspects of the clotting cascade, including the speed of clot initiation and formation, clot strength, and ultimately fibrinolysis. Thromboembolic disease has been hypothesized to play a role in the pathophysiology of cold stress syndrome (CSS), an important cause of mortality in the threatened Florida manatee (Trichechus manatus latirostris). The objective of this study was to establish thromboelastography RIs using the TEG 5000 with citrated whole blood samples and kaolin activation in wild, healthy manatees. In December 2014 and January 2015, 29 wild manatees (17 male and 12 female) were blood sampled as part of the annual wild manatee health assessments organized by United States Geological Survey (USGS). TEG was performed using citrated kaolin-activated samples. The samples were obtained from manatees caught in Crystal River, Citrus County, and used to identify the mean±SD of normal TEG parameters: R=reaction time 2.1minutes (0.8), K=clot formation time 0.8min (0), α angle=83.1° (2), MA=maximum amplitude 75mm (7.6), and LY30=clot lysis 0.41% (0.68). No significant differences were found between manatee size, sex, or time between sampling and running the test. Manatee TEG parameters demonstrate a relatively hypercoagulable condition when compared to other mammals. This information will facilitate detection of changes in hemostasis during injury and disease and provide a valuable reference range.

Identifying coagulopathies in the pathophysiology of cold stress syndrome in the Florida manatee Trichechus manatus latirostris

Cold stress syndrome (CSS) in the Florida manatee Trichechus manatus latirostris has been defined as morbidity and mortality resulting from prolonged exposure to water temperatures <20°C. The pathophysiology is described as multifactorial, involving nutritional, immunological and metabolic disturbances; however, the exact mechanisms are unknown. We hypothesized that thromboembolic complications contribute to the pathophysiology of CSS in addition to the previously described factors. During the winter of 2014-2015, 10 Florida manatees with clinical signs of CSS were presented to Lowry Park Zoo, Tampa, FL, USA. Thromboelastography (TEG) and coagulation panels were performed at admission. In addition, coagulation panel data from 23 retrospective CSS cases were included in the analyses. There were numerous differences between mean values of TEG and coagulation parameters for healthy manatees and those for CSS cases. Among TEG parameters, reaction time (R), clot formation time (K) and percentage of clot lysed after 30 min (LY30) values were significantly different (p < 0.05) between the 2 groups. CSS cases also had significantly higher mean D-dimer concentration and coagulation factor XI activity, prolonged mean activated partial thromboplastin time (aPTT) and significantly decreased mean antithrombin activity. These combined abnormalities include clinicopathologic criteria of disseminated intravascular coagulation, indicating an increased risk of thromboembolic disease associated with manatee CSS.

PROPOSAL AND APPLICATION OF A NOVEL DISSEMINATED INTRAVASCULAR COAGULATION SCORING SYSTEM IN THE FLORIDA MANATEE (TRICHECHUS MANATUS LATIROSTRIS).

Disseminated intravascular coagulopathy (DIC) is an acquired disorder of hemostasis resulting in activation of the coagulation and fibrinolytic pathways. It is reported secondarily to multiple disease processes and can be associated with increased mortality. Previous research at Tampa's Lowry Park Zoo (LPZ) demonstrated that Florida manatees ( Trichechus manatus latirostris) with cold stress syndrome (CSS) demonstrated thromboembolic disease. The object of this retrospective study was to establish the presence and clinical relevance of DIC in Florida manatees admitted to LPZ for rehabilitation from 07 March 2010 to 15 August 2015. A coagulation panel, including prothrombin time, partial thromboplastin time, platelet count, fibrinogen level, and D-dimer level was used to diagnose DIC. There were 100 cases identified in the study period: 35 trauma, 43 CSS, 17 secondary to harmful algae blooms (HAB), and five miscellaneous. Manatees with CSS had the highest incidence of DIC with 24 of 43 cases (56%) affected, followed by trauma with 18 of 35 cases (52%) affected. None of the manatees with HAB were found to have DIC. Manatees that developed DIC during rehabilitation or when DIC progressed did not survive. Due to the clinical implications of DIC, identifying its presence and recognizing its severity could improve clinical outcomes by enabling more intensive treatment protocols.

Comment on “Insulative capacity of the integument of the dugong (Dugong dugon): thermal conductivity, conductance and resistance measured by in vitro heat flux” by Horgan, Booth, Nichols and Lanyon (2014)

Cold stress syndrome is an incompletely understood but clearly very complicated, multisystemic entity that has been described as affecting Florida manatees chronically exposed to cold water (Bossart et al. 2002). Florida and south-east Queensland (QLD) are very similar in climate, species composition and marine environment. In 2013, our group published an article comparing the lesions described in manatees with those we were seeing in many of the dugongs presenting to our post-mortem room during the colder months of the year in Queensland. This description was combined with presentation of supporting environmental data (Owen et al. 2013). Horgan, Booth, Nichols and Lanyon have made statements contradicting our argument in their article, namely: “with no credible pathological evidence of CSS (cf. Owen et al. 2013)” and “Skin lesions suggested as characteristic of cold stress and reported for dugongs by Owen et al. (2013) are grossly and histologically different to those reported for the Florida manatee, are found in dugongs throughout the tropics (Woolford and Lanyon unpublished data) and are also found in dugongs in cooler waters outside the winter period (the latter point also reported by Owen et al. 2013). Each of these factors casts serious doubt as to these skin characteristics being thermally related”. These statements are not based on sound scientific principles. In the former instance, this statement has been made without offering any supporting or qualifying evidence and is therefore an unsubstantiated opinion. The dugong samples used for Owen et al. (2013) were assessed and compared by experienced pathologists and experts in marine population health, one of whom (MF) is in the unique position of having first-hand experience with disease in both manatees in Florida and dugongs in southeast QLD. Data and the resulting interpretations in this article were also deemed as credible by multiple reviewers. The qualifying This article presents original research to investigate the relationship between integumentary composition and predictive thermal capacity. The authors suggest that cold stress syndrome (CSS) is unlikely to occur in dugongs, a claim that is at odds with a recent study from our group where we described the local appearance of mortality cases consistent with this syndrome. Horgan et al. (2014) made two statements, in particular, which disregarded a diagnosis following observations by specialist veterinary pathologists, yet without, to our knowledge, accessing the material themselves and instead quoting ‘unpublished data’ to support their conclusions. Further, and importantly, Horgan et al. (2014) ignore the pathological changes we described in other organ systems, which were consistent with changes noted during CSS in manatees and led to our diagnosis.

Evidence of sirenian cold stress syndrome in dugongs Dugong dugon from southeast Queensland, Australia

Cold stress syndrome (CSS) is the term used to describe the range of clinical signs and chronic disease processes that can occur in Florida, USA, manatees Trichechus manatus latirostris exposed to water temperatures below 20°C for extended periods. Although no cold-related adverse events have been described in the closely related dugong Dugong dugon thus far, it has been established that they make movements in response to water temperatures lower than about 17 to 18°C. In this study, archive reports for dugong carcasses submitted to The University of Queensland School of Veterinary Science for post mortem examination during 2010 to 2012 were examined. These animals had been recovered from Moreton Bay, southeast Queensland, Australia, and 10 out of 14 fulfilled the criteria for 'potential cold stress cases.' Epidermal hyperplasia and secondary bacterial infection, serous atrophy of pericardial adipose tissue, and multisystem abscessation were features commonly noted in these cases. Water temperature data were correlated with the time of year that carcasses were submitted for examination. Higher numbers of carcasses diagnosed with potential CSS were noted during sustained periods in which water temperature was below 20°C. Given the pattern of increased submission of non-specifically, chronically unwell animals in the colder months and evidence that environmental conditions known to precipitate CSS occur in southeast Queensland, it is probable that, like manatees, dugongs in this area are affected by CSS. Further investigation to confirm and to better characterize the syndrome is recommended to refine management practices and improve treatment of affected animals.

COMPARISON OF METHODS USED TO DIAGNOSE GENERALIZED INFLAMMATORY DISEASE IN MANATEES (TRICHECHUS MANATUS LATIROSTRIS)

Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as "cold stress syndrome" and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha, acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10-50 microg/ml with an equivocal interval of 51-70 microg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7-1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4-2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100-400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.

Pathologic Findings in Florida Manatees (&lt;I&gt;Trichechus manatus latirostris&lt;/I&gt;)

This report describes pathologic findings associated with mortality in Florida manatees (Trichechus manatus latirostris) (n = 68) between January 1996 and January 2004. The most frequent causes of death among these Florida manatees were trauma (47%), cold stress syndrome (CSS) (18%), inflammatory/infectious disease (12%), and suspected brevetoxicosis (9%). There were few perinatal deaths (7%). Probably all deaths due to trauma, as well as some, and perhaps many, cases of CSS, may be regarded as anthropogenic, reinforcing the need for conservation and management strategies to mitigate these impacts on this endangered species. Cause of death was determinable in a high proportion (94%) of sample cases, demonstrating the importance of performing timely gross and microscopic necropsy examinations on marine mammals.

Pathological features of the Florida manatee cold stress syndrome

Pathological features of the Florida manatee cold stress syndrome