Microscopic Findings in the Cardiac Muscle of Stranded Extreme Deep-Diving Cuvier's Beaked Whales (Ziphius cavirostris).

Considerable information has been gained over the last few decades on several disease processes afflicting free-ranging cetaceans from a pathologist's point of view. Nonetheless, there is still a dearth of studies on the hearts of these species. For this reason, we aimed to improve our understanding of cardiac histological lesions occurring in free-ranging stranded cetaceans and, more specifically, in deep-diving Cuvier's beaked whales. The primary cardiac lesions that have been described include vascular changes, such as congestion, edema, hemorrhage, leukocytosis, and intravascular coagulation; acute degenerative changes, which consist of contraction band necrosis, wavy fibers, cytoplasmic hypereosinophilia, and perinuclear vacuolization; infiltration of inflammatory cells; and finally, the presence and/or deposition of different substances, such as interstitial myoglobin globules, lipofuscin pigment, polysaccharide complexes, and intra- and/or extravascular gas emboli and vessel dilation. This study advances our current knowledge about the histopathological findings in the cardiac muscle of cetaceans, and more specifically, of Cuvier's beaked whales.

Ultrastructural characterization of normal Merkel cells in the dog.

BACKGROUND: Involvement of Merkel cells (MKs) in different cutaneous diseases as well as in the growth, differentiation and homeostasis of the skin has been previously documented. HYPOTHESIS/OBJECTIVES: The aim was to assess the ultrastructural features of MKs in canine skin, including morphometrics, highlighting their similarities with and differences from those described for other mammals. ANIMALS: Hard palate, nasal planum, lower lip and whisker pad samples were taken from two healthy young dogs destined for academic purposes. METHODS: Ultrathin sections of samples fixed in osmium tetroxide and embedded in Epon 812 resin were stained with uranyl acetate and lead citrate and examined using a JEOL JEM 2010 transmission electron microscope. RESULTS: Ultrastructural characteristics included the following: (i) arrangement in clusters in the basal layer of the epidermis, oral mucosa and external follicular root sheath; (ii) inconstant link with nerve terminal; (iii) oval (10.27 ± 1.64 µm major axis) cell shape with large lobulated nuclei (5.98 ± 1.16 µm major axis); (iv) spine-like and thick cytoplasmic processes interdigitating with surrounding keratinocytes; (v) presence of desmosomes in the cell body or at the base of spine-like processes attaching to neighbouring keratinocytes; and (vi) cytoplasm containing loosely arranged intermediate filaments (10.04 ± 1.17 nm) and numerous dense-core granules (100.1 ± 17.12 nm) arranged in the basal portion of the cytoplasm. CONCLUSIONS AND CLINICAL IMPORTANCE: This study provides the first complete description of the ultrastructural characteristics of MKs in the dog, enhancing our knowledge of the skin structure in this species and providing a basis for future physiological and pathological studies of the role of these cells in normal and damaged canine tissues.

Fatal systemic morbillivirus infection in bottlenose dolphin, canary islands, Spain.

A systemic morbillivirus infection was diagnosed postmortem in a juvenile bottlenose dolphin stranded in the eastern North Atlantic Ocean in 2005. Sequence analysis of a conserved fragment of the morbillivirus phosphoprotein gene indicated that the virus is closely related to dolphin morbillivirus recently reported in striped dolphins in the Mediterranean Sea.

Disseminated avian mycobacteriosis in a free-living grey heron (Ardea cinerea).

Wild birds share with humans the capacity for moving fast over large distances. During migratory movements, birds carry pathogens that can be transmitted between species. One of these concerning pathogens is Mycobacterium spp. A necropsy was performed in a grey heron (Ardea cinerea) that had been medically treated for a polyarthritic process. Grossly, firm white-yellowish nodules of various size, resembling granulomas, were observed in right carpal joint, both patellar joints, neck musculature, palate, pharynx, larynx, nasal sinuses, pericardial sac, air sacs, proventriculus and intestinal serosa, pancreas, kidneys, adrenal glands, and oviduct. Microscopically, these lesions were composed of multinucleated giant cells, large macrophages, and lymphocytes, with a central zone of necrosis and in some cases with peripheral fibrosis. Acid-fast bacilli were detected within these lesions. Lesions were cultured and Mycobacterium avium subsp. avium was identified. To the best of our knowledge this is the first description of mycobacteriosis in grey herons. Although the grey heron is currently considered a least-concern species, avian mycobacteriosis remains a threat on the conservation management of endangered avian species, and constitutes a public health concern as well.

Pathology and causes of death of stranded cetaceans in the Canary Islands (1999-2005).

Between 1999 and 2005, 233 stranded cetaceans (comprising 19 species) were reported in the waters of the Canary Islands. Of these, 138/233 (59.2%) were subjected to a complete or partial standardized necropsy, including 4 Balaenopteridae, 9 Physeteridae, 8 Kogiidae, 27 Ziphiidae and 90 Delphinidae. Of these, 46/138 (33.3%) cetaceans were diagnosed with anthropogenic pathological categories (i.e. the cause of death was anthropogenic). These included fishing interaction (bycatch) (19 individuals), 'atypical' mass stranding events linked to naval exercises (13), ship collisions (8) and other anthropogenic-related pathology (6). 'Natural' (i.e. non-anthropogenic) causes of death accounted for another 82/138 (59.4%) cases, including infectious and non-infectious diseases (63), neonatal pathology (8), intra- and interspecific interactions (6) and mass strandings (5). The cause(s) of death could not be determined in 10/138 (7.3%) necropsied animals. The most common causes of death were ship collisions in 6/9 (66.6%) Physeteridae, 'atypical' mass stranding linked to naval exercises in 13/27 (48.1%) Ziphiidae, and 'natural' infectious and non-infectious diseases in 55/90 (61.1%) Delphinidae. Interaction with fishing activities was established as cause of death in 15/90 (16.7%) Delphinidae. These data show that a range of anthropogenic and natural single and mass mortality events occur in multiple cetacean species stranded in the Canary Islands.

Morphological Assessment of Concomitant Lesions Detected in Goat Herds Naturally Infected with Paratuberculosis (Johne's Disease).

Paratuberculosis (PTB), caused by Mycobacterium avium subspecies paratuberculosis (MAP), causes significant financial losses in the ruminant industry. The aim of this study is to describe the concomitant pathological findings as well as PTB-induced lesions in 39 naturally infected goats (15 vaccinated and 24 non-vaccinated). All animals exhibited MAP-induced microscopic lesions affecting target organs, although only 62% of those were detected grossly. Mainly concomitant inflammatory pathologies were recognized affecting the hemolymphatic, respiratory and gastrointestinal systems. Non-vaccinated animals exhibited both moderate and marked granulomatous enteritis in contrast with vaccinated ones which presented mild intestinal affection. Our results demonstrate that non-vaccinated animals presented pneumonia in all age groups studied (from 12 up to >48 months old). A significantly higher prevalence of ileocecal valve PTB lesions was detected in non-vaccinated animals with pneumonic lesions (p = 0.027). Furthermore, a reduction of gastrointestinal non-PTB processes was described in vaccinated goats. In conclusion, a PTB infected goat herd can be affected by a wide range of concomitant pathologies, mostly inflammatory in origin. Anatomic pathology is of crucial importance for correct herd diagnosis and histopathology is an indispensable tool for lesion detection. Additionally, anti-MAP vaccination could have a beneficial effect on the reduction of respiratory and gastrointestinal non-PTB diseases.

Herpes virus infection associated with interstitial nephritis in a beaked whale (Mesoplodon densirostris).

BACKGROUND: The capacity for herpesvirus to cause disease in cetaceans is unclear and may be varied depending on the different conditions of individuals and between different species. Kidney pathology and intralesional virus-associated infection have been rarely reported in cetaceans. RESULT: On April 2004, an old adult male Blainville's beaked whale (Mesoplodon densirostris) 420 cm long with a poor body condition was stranded on Tenerife Island. During necropsy, no gross lesions were observed in the kidneys. However, membranous glomerulonephritis, multifocal interstitial lymphoplasmacytic nephritis and acute multifocal necrotizing tubulointerstitial nephritis with intranuclear inclusion bodies was diagnosed by histological analysis. Tissue samples were submitted for bacteriological analysis and molecular viral screening. CONCLUSION: A novel alpha herpesvirus associated with interstitial nephritis was identified in an old adult male Blainville's beaked whale (M. densirostris) with a poor body condition stranded in the Canary Islands. This report suggests that identification of herpesvirus infection could be used as a differential diagnosis for interstitial nephritis in cetaceans.

Toothed Whales Have Black Neurons in the Blue Spot.

Neuromelanin (NM) is a dark polymer pigment that is located mostly in the human substantia nigra, and in the locus ceruleus, referred to as "the blue spot". NM increases linearly with age, and has been described mainly in the human brain; however, it also occurs in the neurons of monkeys, horses, giraffes, cattle, sheep, goats, dogs, rats, and even in frogs. While in most of these mammals NM shows the histochemical and ultrastructural features typical of lipofuscins, human NM is confined within cytoplasmic organelles that are surrounded by a double membrane, suggesting an autophagic origin. In a study on the morphology of the locus ceruleus of the family Delphinidae, the presence of a variable quantity of NM in the interior of locus ceruleus neurons was observed for the first time; meanwhile, nothing is known about its ultrastructure and composition. Transmission electron microscopy demonstrated in two toothed whales-an Atlantic spotted dolphin (Stenella frontalis; family Delphinidae) and a Blainville's beaked whale (Mesoplodon densirostris; family Ziphiidae)-the presence of melanin granules associated with lipid droplets and membranes that were very similar to that of human NM. The relationship between NM and neuronal vulnerability must be studied in depth, and cetaceans may offer a new natural-spontaneous comparative model for the study of NM and its implication in neurodegenerative diseases.

Effect of heat-inactivated Mycobacterium avium subspecies paratuberculosis (MAP) vaccine on the lesions and immunopathology developed in target tissues of naturally MAP-infected goats.

Paratuberculosis (PTB) is a disease caused by Mycobacterium avium subspecies paratuberculosis (MAP), which affects a broad range of hosts, including domestic and wild animals. PTB is a chronic granulomatous enteritis and lymphadenitis that compromises animal welfare and causes economic losses. The objective of this study was to evaluate the effect of a commercial heat-inactivated MAP vaccine on lesions and immunopathology developed in the target tissues of goats naturally infected with MAP. Lesions compatible with PTB in the intestine and regional lymph nodes (LNs), as well as local immune response to MAP, were evaluated and compared in Gudair®-vaccinated (n = 14) and unvaccinated (n = 11) goats from a MAP-infected farm. The percentage of animals with multifocal granulomatous lesions in the jejunal (p = 0.05) and ileocecal (p = 0.02) LNs was higher in unvaccinated animals, while a lesion score reduction of 50% was found in the LNs of vaccinated animals. Unvaccinated animals showed increased numbers and wider distribution of macrophages (MΦs, CD68 +) in histiocytic infiltrate (p = 0.0003), associated with increased numbers of mycobacteria. Increased inducible nitric oxide synthase (iNOS) expression was also reported in these animals, while M2 MΦs (CD163 +) were scarce in both groups. Vaccinated animals showed an increase in CD3 + lymphocytes, although differences in interferon gamma (IFNγ) were negligible. These results support the hypothesis that heat-inactivated MAP vaccination could reduce the severity of PTB lesions and mycobacterial load in target tissues in vaccinated adult goats.

Budd-Chiari-like pathology in dolphins.

Nearly two decades ago, pathologic examination results suggested that acoustic factors, such as mid-frequency active naval military sonar (MFAS) could be the cause of acute decompression-like sickness in stranded beaked whales. Acute systemic gas embolism in these whales was reported together with enigmatic cystic liver lesions (CLL), characterized by intrahepatic encapsulated gas-filled cysts, tentatively interpreted as "gas-bubble" lesions in various other cetacean species. Here we provide a pathologic reinterpretation of CLL in odontocetes. Among 1,200 cetaceans necropsied, CLL were only observed in four striped dolphins (Stenella coeruleoalba), with a low prevalence (2%, N = 179). Together, our data strongly suggest that CLL are the result of the combination of a pre-existing or concomitant hepatic vascular disorder superimposed and exacerbated by gas bubbles, and clearly differ from acute systemic gas embolism in stranded beaked whales that is linked to MFAS. Budd-Chiari-like syndrome in dolphins is hypothesized based on the present pathologic findings. Nonetheless, further researched is warranted to determine precise etiopathogenesis(es) and contributing factors for CLL in cetaceans.

Decompressive Pathology in Cetaceans Based on an Experimental Pathological Model.

Decompression sickness (DCS) is a widely known clinical syndrome in human medicine, mainly in divers, related to the formation of intravascular and extravascular gas bubbles. Gas embolism and decompression-like sickness have also been described in wild animals, such as cetaceans. It was hypothesized that adaptations to the marine environment protected them from DCS, but in 2003, decompression-like sickness was described for the first time in beaked whales, challenging this dogma. Since then, several episodes of mass strandings of beaked whales coincidental in time and space with naval maneuvers have been recorded and diagnosed with DCS. The diagnosis of human DCS is based on the presence of clinical symptoms and the detection of gas embolism by ultrasound, but in cetaceans, the diagnosis is limited to forensic investigations. For this reason, it is necessary to resort to experimental animal models to support the pathological diagnosis of DCS in cetaceans. The objective of this study is to validate the pathological results of cetaceans through an experimental rabbit model wherein a complete and detailed histopathological analysis was performed. Gross and histopathological results were very similar in the experimental animal model compared to stranded cetaceans with DCS, with the presence of gas embolism systemically distributed as well as emphysema and hemorrhages as primary lesions in different organs. The experimental data reinforces the pathological findings found in cetaceans with DCS as well as the hypothesis that individuality plays an essential role in DCS, as it has previously been proposed in animal models and human diving medicine.

Contribution to Herpesvirus Surveillance in Beaked Whales Stranded in the Canary Islands.

Herpesviruses (HVs) (Alpha- and Gammaherpesvirinae subfamilies) have been detected in several species of cetaceans with different pathological implications. However, available information on their presence in beaked whales (BWs) is still scarce. In this study, a total of 55 BWs (35 Ziphius cavirostris and 20 animals belonging to the Mesoplodon genus) were analyzed. Samples (n = 294) were obtained from BWs stranded along the coasts of the Canary Islands (1990-2017). Molecular detection of HV was performed by means of a conventional nested PCR based on the DNA polymerase gene. Herpesvirus was detected in 14.45% (8/55) of the analyzed BWs, including 2 positive animals from a previous survey. A percentage positivity of 8.57% was found within the Cuvier's BW group, while the percentage of positivity rose to 25% within the Mesoplodon genus group (three M. densirostris, one M. europaeus, and one M. bidens). All the obtained sequences from this study belonged to the Alphaherpesvirinae subfamily, from which three are considered novel sequences, all of them within the Mesoplodon genus group. In addition, to our knowledge, this is the first description of HV infection in Gervais' and Sowerby's BWs. Three out of eight HV-positive BWs displayed histopathological lesions indicative of active viral replication.

Assessment of Lung Disease in Finishing Pigs at Slaughter: Pulmonary Lesions and Implications on Productivity Parameters.

Swine respiratory disease is associated with productive losses. We evaluated the prevalence of lung lesions with an emphasis on Mycoplasma hyopneumoniae (Mh), porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as the impact on productive parameters in 108 finishing pigs at slaughter. Pathologic, immunohistochemical (IHC) and serologic analyses were performed. Pneumonic processes were observed in 73.1% of the animals. They mainly consisted of cranioventral bronchopneumonia (CBP) (46.3%) and pleuritis (17.6%). Microscopically, bronchointerstitial pneumonia (67.4%) was common and was occasionally combined (27.9%) with interstitial pneumonia (IP). Mh and PCV2-antigens were detected in bronchointerstitial pneumonia (70.7%) and IP cases (33.3%). There were low titers against Mh (18%) and high titers against PRRSV (100%) and PCV2 (65%). Animals with CBP remained at the farm longer; those with >10% of lung parenchyma involvement were sent later (208.8 days old) and had a lower average carcass weight (74.1 kg) and a lower daily weight gain (500.8 gr/day) compared with animals without lesions (567.2 gr/day, 77.7 kg, 200.8 days old). We suggest that animals that do not reach the weight at slaughter should be sent to slaughter regardless to avoid further negative impacts of respiratory disease in productive parameters.

Comparison of Methods for the Histological Evaluation of Odontocete Spiral Ganglion Cells.

Cetaceans greatly depend on their hearing system to perform many vital activities. The spiral ganglion is an essential component of the auditory pathway and can even be associated with injuries caused by anthropogenic noise. However, its anatomical location, characterized by surrounding bony structures, makes the anatomical and anatomopathological study of the spiral ganglion a difficult task. In order to obtain high-quality tissue samples, a perfect balance between decalcification and the preservation of neural components must be achieved. In this study, different methodologies for spiral ganglion sample preparation and preservation were evaluated. Hydrochloric acid had the shortest decalcification time but damaged the tissue extensively. Both formic acid and EDTA decalcification solutions had a longer decalcification time but exhibited better preservation of the neurons. However, improved cell morphology and staining were observed on ears pretreated with EDTA solution. Therefore, we suggest that decalcifying methodologies based on EDTA solutions should be used to obtain the highest quality samples for studying cell morphology and antigenicity in cetacean spiral ganglion neurons.

Increased Plasma Cardiac Troponin I in Live-Stranded Cetaceans: Correlation with Pathological Findings of Acute Cardiac Injury.

Capture myopathy (CM), is a syndrome that occurs as the result of the stress during and after capture, handling, restraint, and transport of wild animals. Although CM has been described for many species of cetaceans, characterization of the acute cardiac injury - an important component of this syndrome - are still scarce. In this study, we firstly estimated a normal range for cardiac troponin I (cTnI) on cetaceans. Here, through biochemical analysis (especially of cTnI) and histopathological, histochemical, and immunohistochemical correlations with decreased troponin immunolabelling, we studied the cardiac injury in live-stranded cetaceans. Nine cetaceans which stranded alive on the Canary Islands (January 2016 - June 2019) were included in this study. Sampled individuals presented elevated values of plasma cTnI, which were correlated to histopathological lesions comprised of vascular changes and acute degenerative lesions. Immunohistochemically, injured cardiomyocytes showed a decreased intrafibrillar troponin immunoreaction. This is the first attempt to establish a normal baseline range for cTnI in cetaceans, and the first study comparing plasma biomarkers values with histopathological and immunohistochemical findings. This approach allowed us to demonstrate the degree of cardiac damage as a result of injury, consistent with ischemia-reperfusion lesions. The knowledge gained here could improve decision-making procedures during stressful situations, mainly in live-strandings, handling, and rehabilitation, thereby reducing the mortality of cetaceans.

Retrospective Study of Traumatic Intra-Interspecific Interactions in Stranded Cetaceans, Canary Islands.

Aggressive encounters involving cetacean species are widely described in the literature. However, detailed pathological studies regarding lesions produced by these encounters are scarce. From January 2000 to December 2017, 540 cetaceans stranded and were necropsied in the Canary Islands, Spain. Of them, 24 cases of eight species presented social traumatic lesions produced by cetaceans of the same or different species. All the cases presented severe multifocal vascular changes, 50% (12/24) presented fractures affecting mainly the thoracic region, 41.7% (10/24) acute tooth-rake marks, 37.5% (9/24) undigested food in the stomach, 33.3% (8/24) tracheal edema, and 12.5% (3/24) pulmonary perforation. In 10 cases with tooth-rake marks, the distance between the teeth, allowed us to further identify the aggressor species: four cases were compatible with killer whales (Orcinus orca) affecting three species [pigmy sperm whale (Kogia breviceps), Cuvier's beaked whale (Ziphius cavirostris), and short-finned pilot whale (Globicephala macrorhynchus)] and four cases compatible with common bottlenose dolphins (Tursiops truncatus) affecting two species [short-beaked common dolphin (Delphinus delphis) and Atlantic spotted dolphin (Stenella frontalis)]. We also described two cases of intraspecific interaction in stripped dolphin (Stenella coeruleoalba). Microscopically, 70.8% (17/24) of the cases presented acute degenerative myonecrosis, 66.7% (14/21) presented vacuoles in the myocardiocytes, 36.8% (7/19) pigmentary tubulonephrosis, 31.6% (6/19) cytoplasmic eosinophilic globules within hepatocytes, 21.4% (3/14) hemorrhages in the adrenal gland, and 17.3% (4/23) bronchiolar sphincter contraction. The statistical analysis revealed that deep divers, in good body condition and nearby La Gomera and Tenerife were more prone to these fatal interactions. Additionally, in this period, three animals died due to an accident during predation: a false killer whale (Pseudorca crassidens) died because of a fatal attempt of predation on a stingray, and two Risso's dolphins (Grampus griseus) died as a consequence of struggling while predating on large squids.

Comparative morphology, histology, and cytology of odontocete cetaceans prostates.

The prostate is the only male accessory gland in cetaceans. However, little is known about this organ in these species. Anatomical and histological characteristics of the prostate have been described in only a few cetacean species, further, one study reported a high incidence of prostatic pathologies in cetaceans that may impair reproduction. The objective of this work was to describe and compare the morphological, histological, and cytological characteristics of the prostate in different odontocete cetaceans. To this end, the prostate glands of 47 animals from nine different species of cetaceans were macroscopically and microscopically studied. Members of the families Delphinidae, Ziphiidae, and Physeteridae were included. In general, the prostate appeared as a musculo-glandular organ with two distinct parts-the Corpus prostatae and the Pars disseminata prostatae. In the pygmy sperm whale (Kogia breviceps) and the Cuvier's beaked whale (Ziphius cavirostris), the prostate was a discrete gland with a small Corpus prostatae. Microscopically, the prostates of different delphinids species shared similarities; however, the prostate of the pygmy sperm whale revealed significant histological differences compared to those of the delphinids. Immunohistochemical analysis was performed using low- and high-molecular-weight cytokeratin, vimentin, and prostatic specific antigen commercial antibodies. Electron microscopy analysis was performed on the prostate of a bottlenose dolphin and the cytomorphological differences among the major epithelial components of the prostatic epithelium were described. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:2036-2053, 2020. © 2019 American Association for Anatomy.

Fat embolism secondary to yellow fat disease in an Appaloosa horse.

A 4-year-old female Appaloosa horse was referred to the Department of Comparative Pathology, Veterinary Faculty, with a history of colic and acute respiratory distress. At necropsy, gross lesions consisted of extensive firm, multinodular, hemorrhagic foci in fat tissues with yellow-brown discoloration. The most affected areas were peritoneal fat and perirenal, epicardial, and subcutaneous adipose tissues. Other findings were hepatic lipidosis and multiple 1-1.5 cm hemorrhagic foci scattered in both lungs. Histopathological examination revealed severe degeneration and necrosis of adipose tissue with dystrophic calcification. The necrotic fat was infiltrated by abundant foamy macrophages and multinucleated giant cells. Based on these histopathological changes, yellow fat disease, also called nutritional panniculitis, was diagnosed. In addition, the microscopic examination of lung and kidney sections stained with osmium tetroxide and oil red O revealed numerous lipid droplets within glomerular and alveolar septal capillaries. To the authors' knowledge, this is the first description of the development of fat embolism in horses affected by yellow fat disease.

A semi-defined medium without serum for small ruminant mycoplasmas.

The composition of the medium used to cultivate Mycoplasma species is very important. Serum is one of the most important additives as it contains lipids (cholesterol) and serum proteins, which are essential for the growth of the organisms. This work reports the development of a semi-defined medium, called MWS (Medium Without Serum) produced without animal serum and bovine serum albumin. MWS seems to be suitable for cultivating several species of caprine mycoplasma, especially M. mycoides subsp. mycoides (LC) and M. mycoides subsp. capri.