Bacterial septicemia and herpesvirus infection in Antarctic fur seals (Arctocephalus gazella) stranded in the São Paulo coast, Brazil.

In August 2021, two juvenile male Antarctic fur seals (Arctocephalus gazella) stranded in the southeastern Brazilian coast and were referred to rehabilitation centers. The animals presented increased body temperature, prostration, respiratory distress and despite treatment died. A necropsy following a standardized protocol was performed, and formalin-fixed tissues were processed for microscopic examination. Samples were screened for morbillivirus, herpesvirus, and Brucella spp. by molecular analyses (PCR, RT-PCR). Bacteriological culture was performed in samples collected from the lungs, trachea, and lymph nodes of both cases. The main histopathologic findings were of infectious nature, including multifocal necrotizing and fibrinous mixed interstitial pneumonia, bronchiolitis, and bronchitis, with intralesional myriad bacteria associated with vascular fibrinoid necrosis. Pseudomonas aeruginosa was isolated from tracheal and lung swabs of Case 1, and Klebsiella oxytoca was found in nostril swabs, tracheobronchial lymph nodes, and lung of Case 2. Gammaherpesvirus infection was detected in both cases, and the sequences retrieved were classified into the genus Percavirus. All tested samples were PCR-negative for Brucella spp. and morbillivirus. We hypothesize that the deficient immunological status in association with starvation predisposed the reactivation of herpesvirus and secondary bacterial co-infections. To the authors' knowledge, this is the first molecular detection of herpesvirus in an Antarctic pinniped. These findings reinforce that Otariid gammaherpesvirus circulating in the Southern Hemisphere are likely endemic in the Arctocephalus genus. This report contributes to the current knowledge of health aspects affecting wild pinnipeds, especially in the poorly studied Antarctic species.

Survey of selected viral agents (herpesvirus, adenovirus and hepatitis E virus) in liver and lung samples of cetaceans, Brazil.

Hepatic and pulmonary lesions are common in cetaceans, despite their poorly understood viral etiology. Herpesviruses (HV), adenoviruses (AdV) and hepatitis E virus (HEV) are emerging agents in cetaceans, associated with liver and/or pulmonary damage in mammals. We isolated and molecularly tested DNA for HV and AdV (n = 218 individuals; 187 liver and 108 lung samples) and RNA for HEV (n = 147 animals; 147 liver samples) from six cetacean families. All animals stranded or were bycaught in Brazil between 2001 and 2021. Positive-animals were analyzed by histopathology. Statistical analyses assessed if the prevalence of viral infection could be associated with the variables: species, family, habitat, region, sex, and age group. All samples were negative for AdV and HEV. Overall, 8.7% (19/218) of the cetaceans were HV-positive (4.8% [9/187] liver and 11.1% [12/108] lung), without HV-associated lesions. HV-prevalence was statistically significant higher in Pontoporiidae (19.2%, 10/52) when compared to Delphinidae (4.1%, 5/121), and in southeastern (17.1%, 13/76)-the most industrialized Brazilian region-when compared to the northeastern region (2.4%, 3/126). This study broadens the herpesvirus host range in cetaceans, including its description in pygmy sperm whales (Kogia breviceps) and humpback whales (Megaptera novaeangliae). Further studies must elucidate herpesvirus drivers in cetaceans.

Cardiac disease in the Spix Macaw (Cyanopsitta spixii): two cases.

Cardiovascular disease in avian species, other than poultry, is being increasingly reported. In psittacine birds, atherosclerosis and congestive heart failure are the leading cardiovascular diseases, often resulting in multiorgan dysfunction and demise. The Spix's macaw (Cyanopsitta spixii) is arguably the most endangered psittacine species worldwide. We aimed to describe the gross and microscopic findings in two adult Spix's macaws wherein severe cardiovascular pathology resulted in sudden death. Bird 1 had pathologic findings consistent with fibrinoheterophilic vegetative pulmonic valvular endocarditis with luminal obliterative thrombosis, myocarditis and epicarditis, myocardial fibrofatty infiltration and cardiomyocyte loss, as well as generalized septicaemia. Microbiological analysis yielded Pantoea septica from the intestines and Acinetobacter baylyi from the cerebrum. Bird 2 had changes suggestive of right brachiocephalic coarctation-like obliterative arteriopathy. The latter is a novel cardiovascular pathology in avian species, and its severity and extent likely led to acute decompensation of pre-existing cardiac disease. These results add to the body of knowledge on avian cardiovascular pathology and may aid in veterinary medical decisions on caged birds, including those part of ex situ conservation efforts.

Pathological Findings in Leatherback Sea Turtles (Dermochelys coriacea) During an Unusual Mortality Event in São Paulo, Brazil, in 2016.

The leatherback sea turtle (LST; Dermochelys coriacea) is highly migratory and is primarily distributed throughout tropical and subtropical regions. The LST populations found in Brazil are critically endangered. An unusual mortality event occurred between August and November 2016 with 23 LSTs stranded along a 100 km coastal segment in Iguape, Ilha Comprida and Ilha do Cardoso in southern São Paulo state. This study investigated the pathological findings and most likely causes of death of 10 LSTs. Male (n = 9) and adult (n = 9) animals predominated. All but one animal was in good body condition and all were found dead. The most prevalent gross findings were suggestive of bycatch, namely cutaneous erosions, abrasions and/or lacerations around the neck and flippers (n = 9), generalized congestion (n = 8) and pulmonary oedema (n = 6). Other prevalent gross findings were: cutaneous epibiosis by Stomatolepas elegans (n = 7); ileocecal diverticulitis (n = 7); distal oesophagitis (n = 5); and fibrinous coelomitis (n = 5). Microscopically, the most prevalent findings were: hepatic melanomacrophage centre hypertrophy or hyperplasia (n = 9); interstitial pneumonia (n = 8); multisystemic congestion (n = 6); pulmonary oedema with or without aspirated material (n = 5); adrenal coccidiosis (n = 5) with variable multiorgan involvement; and multiorgan bacterial disease (n = 5). Five animals had food ingesta (cnidarians, crustaceans) in the oesophagus or stomach; only one had evident plastic foreign bodies. Asphyxia due to entanglement in nets was the most frequently identified cause of death (n = 8); a cause of death was not identified in two animals. Our findings provide evidence of the severe negative impact of entanglement in fishing nets in LSTs, raising concerns for conservation. These findings also contribute to knowledge of the pathology of LSTs in South American populations.

Facial Squamous Cell Carcinoma and Abdominal Peripheral Nerve Sheath Tumour with Rhabdomyoblastic Differentiation in a Rough-toothed Dolphin (Steno bredanensis).

We report the pathological features of a facial squamous cell carcinoma (SCC) and an abdominal peripheral nerve sheath tumour (PNST) with rhabdomyoblastic differentiation in an aged free-ranging rough-toothed dolphin (Steno bredanensis). The animal was found stranded dead in poor body condition. On external examination, there was a 25 × 7 × 3 cm extensively ulcerated area on the right maxillary region of the rostrum, involving the oral mucocutaneous junction with prominent nodular edges, severe soft tissue loss and extensive maxillary and premaxillary bone lysis. On abdominal dissection, a 5 × 4 × 3.5 cm pale tan to red, raised mass expanded the inner aspect of the right transverse abdominis muscle. Microscopically, the aggressive facial lesion was an acantholytic SCC with extensive osteolysis; there was no evidence of metastasis in the tissues examined. The abdominal mass had cytohistomorphological features compatible with a localized PNST, including whorling, Antoni A and Antoni B areas and Verocay bodies intermixed with rhabdomyoblastic components, as suggested by phosphotungstic acid haematoxylin stain. This neoplasm was locally infiltrative, yet no metastases were observed in the tissues examined. No immunohistochemical investigations could be performed due to lack of tissue availability. Total DNA from the formalin-fixed and paraffin wax-embedded SCC was extracted and tested by polymerase chain reaction for herpesvirus and papillomavirus genetic material. There was no amplification for either of these genera. Other pathological findings observed in this animal were related to the 'live-stranding stress response'. The severity and extent of the facial SCC likely related to anorexia and poor body condition and might have played a role in the stranding and death of this dolphin. These two tumour subtypes add to the relatively uncommon reports of neoplasia in cetaceans. Specifically, these appear to be the first neoplasia records for rough-toothed dolphins, including the first documentation of a PNST with features compatible with rhabdomyoblastic differentiation in a marine mammal.

Pathological Findings in Lowland Tapirs (Tapirus terrestris) Killed by Motor Vehicle Collision in the Brazilian Cerrado.

The lowland tapir (Tapirus terrestris) is the largest land mammal in South America. The species faces steady population decline due to poaching, habitat loss and fragmentation, road-kill, pesticide pollution, competition with domestic livestock and fires, among other threats. The lowland tapir is currently listed as vulnerable to extinction. Little information is available about natural disease processes for the species. This study aimed to report the pathological findings recorded in a cohort of 35 lowland tapirs killed by motor vehicle collision (MVC) on highways of Mato Grosso do Sul State, Brazil, between 2015 and 2018. The main gross pathological findings were those associated with MVC, primarily involving skeletal fractures and internal multiorgan damage with extensive bleeding and/or severe central nervous system injury. The most prevalent concurrent histopathological findings, unrelated to the cause of death, were: adrenal gland degeneration, necrosis and loss of fascicular and reticular cells with replacement fibrosis and cortical atrophy (9/15; 60%); interstitial pneumonia (20/34; 59%); glossitis (9/24; 38%); pulmonary anthracosis (12/34; 35%); colitis (9/28; 32%); and cholangitis/pericholangitis (9/35; 26%). The aetiopathogeneses and clinicopathological significance of some of these findings are unclear; however, parasitic infections appear to be common. Our results highlight the importance of wildlife health information obtained through the study of carcasses of roadkills.

Primary Multicentric Pulmonary Low-grade Fibromyxoid Sarcoma and Chelonid Alphaherpesvirus 5 Detection in a Leatherback Sea Turtle (Dermochelys coriacea).

We describe the gross, microscopical, histochemical and immunohistochemical features of a sclerosing pneumopathic disease process resembling primary multicentric pulmonary low-grade fibromyxoid sarcoma in a juvenile female leatherback sea turtle (Dermochelys coriacea). The animal was fresh, presented in good body condition and stranded dead in Aracaju, Sergipe state, Brazil, in September, 2017. Grossly, the lungs were enlarged bilaterally and the parenchyma was replaced by large, coalescing, white, firm masses that extended into the bronchi and bronchioles and to the pleura. Microscopically, these masses consisted of paucicellular populations of well-differentiated, spindle-shaped fibroblasts with low pleomorphism and low mitotic count, but tissue invasion. Abundant collagen in compact areas merged with peripheral fibromyxoid foci and inflamed stroma. Antibodies specific for cytokeratins AE1/AE3 and smooth muscle actin (SMA) labelled pneumocytes lining the remaining distorted alveoli and the hypertrophied and hyperplastic bronchial muscles, respectively. Tumour cells were negative for SMA; neither neoplastic nor normal tissues cross-reacted with antibodies specific for vimentin or Ki67. Chelonid alphaherpesvirus 5 (ChHV5) polymerase chain reaction analysis from formalin-fixed, paraffin wax-embedded lung tissue sections amplified a 450 base pair fragment of DNA-polymerase (UL30 region) that had 100% homology to sequences previously detected in green sea turtles (Chelonia mydas) on the Brazilian coast. Enterocolitis was a concomitant condition that likely caused morbidity in this case. These findings contribute to the body of knowledge on sea turtle health and expand the known geographical range for ChHV5 in the southern hemisphere.

Cerebral and cardiac congenital malformations in neonatal West Indian manatees (Trichechus manatus).

Strandings of live new-born West Indian manatees (WIMs; Trichechus manatus) are one of the main challenges for the conservation of this species in Brazil, particularly in the northeastern states. Congenital malformations (CMs) are rare in sirenians. We identified CMs in two of 19 stranded WIMs that were rescued, rehabilitated and subjected to complete pathological examinations in Ceará and Rio Grande do Norte States between 1992 and 2017. In case 1, dilation of the cerebral lateral and fourth ventricles with abundant cerebrospinal fluid (internal hydrocephalus), was diagnosed. Furthermore, this animal developed necrotizing enterocolitis associated with pneumatosis intestinalis and aspiration pneumonia late during rehabilitation. Cardiac malformations in case 2 included: right ventricle hypoplasia with marked stenosis of the tricuspid outflow, high ventricular septal defect, segmental pulmonary artery aneurysm, mitral valve haemocyst and left ventricular hypertrophy. Herein, we provide the first description of a neural tube defect, specifically a developmental internal hydrocephalus, and multiple cardiac congenital anomalies, together with their respective clinicopathological features in manatees. Although the aetiology of the CMs remains unknown in these cases, a genetic basis is plausible given the low genetic variability in this population. These cases add to the body of knowledge on health and disease aspects of manatees and may provide scientific basis for future medical and conservation efforts on neonatal WIMs.

Histopathological Findings in the Viscera of Malformed Neotropical Viperids.

Congenital malformations (CMs) or developmental abnormalities are pathological processes defined as morphological defects that originate during embryonic stages. A wide variety of CMs is observed in reptiles, most of which are incompatible with normal life. Characterization of CMs and their pathogenesis are of great importance for the conservation of reptiles, including neotropical viperids (NVs). External CMs are often reported in these species, but little is known about concomitant microscopical findings in the viscera of malformed individuals. In order to partially fill in this gap in knowledge, we aimed to investigate and characterize histopathological findings in the viscera of malformed NVs, jararacas (Bothrops jararaca) and South American rattlesnakes (Crotalus durissus), correlating these findings with features of external CMs. From 78 malformed snakes analysed, 100% (19/19) of rattlesnakes and 98.3% (58/59) of jararacas had histopathological changes in the viscera. Liver and/or kidney fatty degeneration was the most common finding in malformed and control animals. Renal glomerulocystic and tubular disease was significantly correlated with brachygnathia (P < 0.05) in jararacas. These results add to the body of knowledge of snake pathology and demonstrate some associations between external CMs and visceral histopathological findings, some of which could have implications for individual survival.

Immunohistochemical investigation of the cross-reactivity of selected cell markers in formalin-fixed, paraffin-embedded lymphoid tissues of Franciscana (Pontoporia blainvillei).

A considerable amount of knowledge on natural and anthropogenic pathologic conditions affecting different cetacean species has been gained over the last decades. Nonetheless, the immunopathological bases for most of these processes have been poorly documented or remain unknown. Comparative immunopathological investigations in these species are precluded by the limited number of specific antibodies, most of which are not commercially available, and the reduced spectrum of validated and/or cross-reactive ones. To partially fill in this gap of knowledge, a set of commercially available primary antibodies were tested for cross-reactivity against leukocytes and cytokines in formalin-fixed, paraffin-embedded (FFPE) lymphoid tissues (lymph nodes, spleen and thymus) of three bycaught, apparently healthy and fresh Franciscanas (Pontoporia blainvillei) using immunohistochemistry. On the basis of similar region specificity within the lymphoid organs, cellular morphology and staining pattern with human control tissues, 13/19 primary antibodies (caspase 3, CD3, CD57, CD68, FoxP3, HLA-DRα, IFNγ, IgG, IL4, IL10, Lysozyme, TGFß and PAX-5) exhibited satisfactory cross-reactivity. Our results expand the spectrum of suitable cross-reactive primary antibodies in FFPE cetacean tissues. Further comparative immunopathological studies focused on infectious diseases and ecotoxicology may benefit from establishment of baseline expression of immunologically relevant molecules in various cetaceans species.

Multicentric cutaneous keratoacanthomas in a free-living marmoset (Callithrix sp.).

Cutaneous neoplasia is common in non-human primates. We describe the gross and microscopic features of multicentric cutaneous keratoacanthomas in a free-living marmoset (Callithrix sp.). Immunohistochemistry for human papillomavirus and herpes simplex virus type I and simplex virus type II was negative. Keratoacanthomas should be included in the differential diagnosis for cutaneous masses in non-human primates.

Parotid Salivary Gland Basal Cell Adenocarcinoma in a Big-eared Opossum (Didelphis aurita).

The opossum (family Didelphidae) is a marsupial endemic to the Americas. Apart from the South American short-tailed opossum (Monodelphis domestica) and the Virginia opossum (Didelphis virginiana), there is considerable lack of knowledge about the health and diseases of most opossum species. Among these, the big-eared opossum (Didelphis aurita) is found in Argentina, Brazil and Paraguay. Natural and experimental studies have shown this species to be susceptible to infectious agents with zoonotic potential and the animals may play a role in transmission of such agents. However, neoplasia appears to be uncommon in this species. We describe the gross, microscopical and immunohistochemical features of a parotid salivary gland basal cell adenocarcinoma in a free-living big-eared opossum. This case represents the first report of salivary gland neoplasia in opossums.

Schistosomes and Microfilarial Parasites in Magellanic Penguins.

The Magellanic Penguin ( Spheniscus magellanicus) is native to Argentina, Chile, and the Falkland/Malvinas Islands, and is a regular winter migrant in Uruguayan and Brazilian coastal waters. The species is known to be susceptible to a variety of gastrointestinal nematodes, cestodes, trematodes, and acanthocephalans, as well as renal trematodes and pulmonary nematodes. Schistosomes (Platyhelminthes, Trematoda, Schistosomatidae) and microfilariae (Nematoda, Secernentea, Onchocercidae) were histologically identified in Magellanic Penguins ( Spheniscus magellanicus) that died while under care at rehabilitation centers in southern Brazil. Phylogenetic analysis of the COI gene, ITS-1 region, 5.8S rRNA gene, ITS-2 region, and 28S rRNA gene sequences of the schistosome revealed that it is closely related to, but distinct from, a schistosome reported from the African Penguin ( Spheniscus demersus). The schistosomes from Magellanic and African Penguins were grouped with Gigantobilharzia huronensis, Gigantobilharzia melanoidis, and Dendritobilharzia pulvurenta; however, the lack of a clearly monophyletic origin precludes determining their genus. The incidental discovery of novel parasites during a study that did not specifically aim to investigate the occurrence of helminths underscores the value of histopathological examination as an exploratory diagnostic approach.

Spontaneous pulmonary adenosquamous carcinoma in a free-living black capuchin monkey (Sapajus nigritus).

Pulmonary neoplasia is rare among wild New World primates. We report the gross, microscopical, and immunohistochemical features of a primary multicentric pulmonary adenosquamous carcinoma in a free-living black capuchin monkey (Sapajus nigritus). Herein, the spectrum of pulmonary neoplasms in non-human primates is widened and briefly reviewed.

Hepatocellular carcinoma in a free-living marmoset (Callithrix sp.) with concomitant biliary trematodiasis.

Hepatocellular carcinoma (HCC) is rare in New World primates. We report the gross, microscopical, and immunocytochemical features of a spontaneous HCC in a free-living marmoset (Callithrix sp.). Hepatitis B and C virus and aflatoxin immunohistochemistry were negative; however, concomitant intra- and extrahepatic biliary trematodiasis could have played a role.

Apoptotic intrinsic pathway proteins predict survival in canine cutaneous mast cell tumours.

Mast cell tumours (MCTs) are the most frequent canine round cell neoplasms and show variable biological behaviours with high metastatic and recurrence rates. The disease is treated surgically and wide margins are recommended. Adjuvant chemotherapy and radiotherapy used in this disease cause DNA damage in neoplastic cells, which is aimed to induce apoptotic cell death. Resisting cell death is a hallmark of cancer, which contributes to the development and progression of tumours. The aim of this study was to investigate the expression of the proteins involved in the apoptotic intrinsic pathway and to evaluate their potential use as prognostic markers for canine cutaneous MCTs. Immunohistochemistry for BAX, BCL2, APAF1, Caspase-9, and Caspase-3 was performed in 50 canine cases of MCTs. High BAX expression was associated with higher mortality rate and shorter survival. BCL2 and APAF1 expressions offered additional prognostic information to the histopathological grading systems. The present results indicate that variations in the expression of apoptotic proteins are related to malignancy of cutaneous MCTs in dogs.

Choroid Plexus Cyst in a Neonatal Burmeister's Porpoise (Phocoena spinipinnis).

Neuroectodermal developmental anomalies are reported rarely in cetaceans and central nervous system cysts are not described. We describe the gross, microscopical, histochemical and immunohistochemical features of a neuraxial myelencephalic cyst in a stranded neonatal Burmeister's porpoise (Phocoena spinipinnis). Grossly, a subdural, extra-axial, well-demarcated, yellow fluid-filled cystic structure (1.9 × 1.6 × 1 cm) expanded the left foramen of Luschka, the left caudolateral cerebellar recess and the left cranioventral myelencephalon. The cyst displaced the ipsilateral ventral paraflocculus and distended the underlying cranial nerves IX, X, XI and XII. Microscopically, the cystic structure was lined by a monolayer of low cuboidal to flattened epithelium supported by a thin fibrovascular matrix. Immunohistochemistry (IHC) revealed strong and diffuse expression of AE1/AE3 and focal positivity for vimentin. IHC for epithelial membrane antigen, glial fibrillary acid protein, synaptophysin and S100 was negative. Based on these findings, an extra-axial cyst of the choroid plexus of the fourth ventricle (CCPFV) was diagnosed. The pathological relevance of the CCPFV in this case is uncertain. The cause of death involved severe perinatal interspecific (shark) trauma. The present case provides the first evidence of a neuroepithelial cyst in cetacean species.

Increased expression of tissue inhibitor of metalloproteinase-1 correlates with improved outcome in canine cutaneous mast cell tumours.

Canine mast cell tumour (MCT) is a biologically heterogeneous disease. The extracellular matrix degradation promoted by matrix metalloproteinases (MMPs) has been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The aim of this study was to characterize the expression of MMP-2 and -9 and tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in canine cutaneous MCTs and to evaluate their prognostic values. Immunohistochemical staining for MMP-2, MMP-9, TIMP-2 and TIMP-1 was performed in 46 canine cases of MCTs. TIMP-1 expression showed an independent prognostic value for post-surgical survival and disease-related mortality. Dogs with MCTs showing less than 22.9% mast cell TIMP-1 positivity were more prone to die because of the disease and had a shorter post-surgical survival. This article suggests the involvement of TIMP-1 in MCT progression, by contributing to a good outcome in patients with MCTs.