The main objective of wildlife forensic investigation is to recognize pathologic changes and cause of death. Even though it may not always be possible to determine the specific illness and/or etiology, the description and subsequent interpretation of the injuries provide an invaluable understanding of pathology in cetacean post-mortem investigations. Although pathological studies have been previously reported in various cetacean species, such descriptions of the infraorder Mysticeti remain rare. A live-stranded neonatal Bryde's whale (Balaenoptera edeni) which subsequently died soon after the stranding, was assessed by physical exam, blood examination, gross necropsy evaluation, histopathology, and immunohistochemistry. It presented with elevated serum levels of creatine kinase, cardiac troponin I, urea, and creatinine. Microscopically, we observed keratin spicules (squamous epithelial cells) and areas of atelectasis in the lungs. Acute degeneration in the myocytes and cardiomyocytes were comparable to the findings previously described in cases of capture myopathy in live-stranded cetaceans. Immunohistochemistry biomarkers such as myoglobin, fibrinogen, and troponin were analyzed. Skeletal and myocardial damage has been documented in several cetacean species. However, this is the first reported case of skeletal and cardiac rhabdomyolysis associated with live-stranding in a newborn Bryde's whale that suffered from fetal distress.

/pdf/skeletal-and-cardiac-rhabdomyolysis-in-a-live-stranded-20cd0bvgxp.pdf

Skeletal and Cardiac Rhabdomyolysis in a Live-Stranded Neonatal Bryde's Whale With Fetal Distress

Plasma biochemical analysis remains one of the established ways of monitoring captive marine mammal health. More recently, complementary plasma lipidomic analysis has proven to be a valid tool in disease diagnosis and prevention, with the potential to validate and complement common biochemical analysis, providing a more integrative approach. In this study, we thoroughly characterized the plasma polar lipid content of Tursiops truncatus, the most common cetacean species held under human care. Our results showed that phosphatidylcholine, lysophosphatidylcholine, and sphingomyelins (CerPCho) are the most represented phospholipid classes in T. truncatus plasma. Palmitic, oleic, and stearic acids are the major fatty acid (FA) present esterified to the plasma polar lipids of this species, although some n-3 species are also remarkably present, namely eicosapentaenoic and docosahexaenoic acids. The polar lipidome identified by HILIC LC-MS allowed identifying 304 different lipid species. These species belong to the phosphatidylcholine (103 lipid species), lysophosphatidylcholine (35), phosphatidylethanolamine (71), lysophosphatidylethanolamine (20), phosphatidylglycerol (13), lysophosphatidylglycerol (5), phosphatidylinositol (15), lysophosphatidylinositol (3), phosphatidylserine (6) lysophosphatidylserine (1), and sphimgomyelin (32) classes. This was the first time that the dolphin plasma phospholipid profile was characterized, providing a knowledge that will be important to further understand lipid metabolism and physiological regulation in small cetaceans. Furthermore, this study proved the practicability of the use of plasma lipid profiling for health assessment in marine mammals under human care.

The plasma phospholipidome of Tursiops truncatus: From physiological insight to the design of prospective tools for managed cetacean monitorization.

Applications for acute phase reactants (APRs) in nondomesticated mammals include identifying inflammatory disease, monitoring the course of specific disease processes and recovery during rehabilitation, detecting preclinical or subclinical disease, being used as bioindicators for monitoring population and ecosystem health, and as markers of stress and animal welfare. Serum amyloid A, haptoglobin, C-reactive protein, fibrinogen, albumin, and iron are most commonly measured. The procedure for evaluating an APR in a nondomesticated mammalian species should follow a stepwise approach beginning with an assessment of analytical performance, followed by an evaluation of overlap performance, clinical performance, and impact on patient outcomes and management. The lack of species-specific standards and antibodies for nondomesticated mammals presents a challenge, and more attention needs to be focused on assessing cross-reactivity and ensuring adequate analytical performance of APR assays. Sample selection for the initial evaluation of APRs should consider preanalytical influences and should originate from animals with confirmed inflammatory disease and healthy animals. Reference intervals should be generated according to published guidelines. Further evaluation should focus on assessing the diagnostic utility of APRs in specific disease scenarios relevant to a species. Greater attention should be paid to assay performance and uniformity of methods when using APRs for population and ecosystem surveillance. Veterinary clinical pathologists should work closely with zoo veterinarians and wildlife researchers to optimize the accuracy and utility of APR measurements in these various conservation medicine scenarios. 

Acute phase reactants in nondomesticated mammals—A veterinary clinical pathology perspective

Aspergillosis remains difficult to diagnose in animals. Laboratory-based assays are far less developed than those for human medicine, and only few studies have been completed to validate their utility in routine veterinary diagnostics. To overcome the current limitations, veterinarians and researchers have to propose alternative methods including extrapolating from human diagnostic tools and using innovative technology. In the present overview, two specific examples were complementarily addressed in penguins and dolphins to illustrate how is challenging the diagnosis of aspergillosis in animals. Specific focus will be made on the novel application of simple testing in blood based on serological assays or protein electrophoresis and on the new information garnered from metabolomics/proteomics to discover potential new biomarkers. In conclusion, while the diagnostic approach of aspergillosis in veterinary medicine cannot be directly taken from options developed for human medicine, it can certainly serve as inspiration.

/pdf/challenges-to-establish-the-diagnosis-of-aspergillosis-in-3539dhxw.pdf

Challenges to establish the diagnosis of aspergillosis in non-laboratory animals: looking for alternatives in veterinary medicine and demonstration of feasibility through two concrete examples in penguins and dolphins

Simple Summary The presence of round to oval, single to multiple, hyaline eosinophilic globules inside the cytoplasmic of the hepatocytes of cetaceans are unknown. Therefore, this article aims to describe their occurrence and characterize their features using different laboratorial techniques. A total of 95 out of 115 cetaceans (83%) from 16 different species exhibited intracytoplasmic eosinophilic globules (IEGs) within the hepatocytes. These globules were positive for fibrinogen (FB, 97%), albumin (Alb, 85%), and α1-antitrypsine (A1AT, 53%), with the positivity for FB and A1AT correlated with live-stranding, hepatic congestion and a good nutritional status. The IEGs in 36 cetaceans that died due to bycatch were, all except one, FB-positive and A1AT-negative. The IEGs exhibited morphologic and compositional variations at the ultrastructural level, suggesting various stages of development. The proteomic analyses confirmed an association between the IEGs and acute phase proteins, suggesting a relationship between acute stress (i.e., bycatch), disease, and cellular protective mechanisms. Abstract The nature, etiopathogenesis, and clinicopathologic relevance of the prevalent intracytoplasmic eosinophilic globules (IEGs) within hepatocytes of cetaceans are unknown. This study aims to evaluate the presence and characterize the IEGs in the hepatocytes of cetaceans using histochemical and immunohistochemical electron microscopy, Western blot, lectin histochemistry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry techniques. A total of 95/115 (83%) animals (16 species) exhibited histologically evident intracytoplasmic round to oval, single to multiple, hyaline eosinophilic globules within the hepatocytes. These globules were largely PAS-positive, diastase resistant, and were immunopositive for fibrinogen (FB, 97%), albumin (Alb, 85%), and α1-antitrypsine (A1AT, 53%). The IEG positivity for FB and A1AT were correlated with live-stranding, hepatic congestion and a good nutritional status. The cetaceans lacking IEGs were consistently dead stranded and had poor body conditions. The IEGs in 36 bycaught cetaceans were, all except one, FB-positive and A1AT-negative. The IEGs exhibited morphologic and compositional variations at the ultrastructural level, suggesting various stages of development and/or etiopathogenesis(es). The glycocalyx analysis suggested an FB- and A1AT-glycosylation pattern variability between cetaceans and other animals. The proteomic analyses confirmed an association between the IEGs and acute phase proteins, suggesting a relationship between acute stress (i.e., bycatch), disease, and cellular protective mechanisms, allowing pathologists to correlate this morphological change using the acute hepatocytic cell response under certain stress conditions.

/pdf/cetacean-intracytoplasmic-eosinophilic-globules-a-15m85g3e.pdf

Cetacean Intracytoplasmic Eosinophilic Globules: A Cytomorphological, Histological, Histochemical, Immunohistochemical, and Proteomic Characterization

https://hal.archives-ouvertes.fr/hal-02439539/document

Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus)

Acute-phase proteins (APP) are the foundation to the innate immune response and valuable biomarkers that increase with inflammation, infection, neoplasia, stress, and trauma.2,4,16 Little is known about the acute-phase response in cetaceans and if these proteins can be used for health monitoring in individuals and free-ranging populations. The purpose of this study was to characterize serum concentrations of haptoglobin (Hp) and serum amyloid A (SAA), as well as electrophoretic profiles of common bottlenose dolphins (Tursiops truncatus) in free-ranging (n = 33) and professional care (n = 27) settings. Results were correlated to commonly utilized inflammatory indices including erythrocyte sedimentation rate (ESR), fibrinogen, total white blood cell count (WBC), and absolute neutrophil count. SAA levels, measured with a dolphin-specific enzyme linked immunosorbent assay (ELISA), were significantly higher (P = 0.05) in free-ranging dolphins (mean = 4.26; SE = 1.12) when compared with those under professional care (mean = 1.82; SE = 0.45). For dolphins under professional care, a statistically significant correlation was identified between ESR and Hp (P < 0.001; r = 0.69), ESR and SAA (P < 0.001; r = 0.67), fibrinogen and Hp (P = 0.001; r = 0.58), and fibrinogen and SAA (P = 0.002; r = 0.56). In addition, there was a significant correlation between WBC and SAA (P = 0.01; r = 0.38) and absolute neutrophil count and SAA (P = 0.04; r = 0.32). There were no significant correlations between study variables observed in free-ranging dolphins. The variable correlation of APPs with commonly utilized inflammatory indices demonstrates that these proteins are independent measures of inflammation with unique sensitivity, specificity, and timeline of expression. The results of this study contribute to improved health monitoring of dolphins and have the potential to assist in identification of compromised health.

Serum acute-phase proteins in bottlenose dolphins (tursiops truncatus) and correlation with commonly utilized inflammatory indices.

Neonatal Critical Care and Hand-Rearing of a Bottlenose Dolphin (Tursiops truncatus) Calf

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Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus)

Serum acute-phase proteins in bottlenose dolphins (tursiops truncatus) and correlation with commonly utilized inflammatory indices.

Neonatal Critical Care and Hand-Rearing of a Bottlenose Dolphin (Tursiops truncatus) Calf