Effect of age on the viability of pig intestinal explants: a histological and ultrastructural approach

Intestinal explants are a useful model for toxicological and pathological studies, but the maintenance of tissue viability for longer periods of incubation remains challenging. The aim of this study was to evaluate the effect of age on the viability of intestinal explants. Jejunal explants were obtained from 24- and 42-day-old piglets. The explants were incubated for two or four hours and were then processed for histological analysis. The explants from younger pigs showed minor histological changes, as assessed by histological and morphometrical scores. Explants from 24-day-old pigs were incubated for four hours and subjected to ultrastructural analysis to assess cell integrity. The plasma membranes, nuclei and organelles of enterocytes were well preserved, and adsorptive endocytosis was observed. In conclusion, the data indicate that the age of the donor influences explant viability. The intestinal morphology of incubated explants from young donors was better preserved than that of explants from older piglets

Clinical and Pathological Features of Canine Metastatic Adrenocortical Carcinoma

 Background: Adrenocortical carcinomas (ACC) are uncommon in dogs. ACC often invade the posterior vena cava wall and the advential layer of the abdominal aorta; however, metastases to distant organs are rare. Most dogs with ACC show clinical signs of Cushing`s syndrome in a similar way with signs reported in humans. The aim of this study is to report three cases of metastatic ACC in dogs and their clinical and pathological features.Cases: This report describes three cases of metastatic adrenocortical carcinoma (ACC) in dogs diagnosed post mortem through histopathological examination. The animals presented no signs of adrenal hormones overproduction. Case 1. A 13-year-old intact, mixed breed female dog was presented with a history of progressive hepatomegaly during the last four months. The animal was submitted to an exploratory laparotomy, but due to anesthetic complications no tissue specimen was sampled and after one week, the animal died. The necropsy examination showed an increased left adrenal (3 cm in diameter), multiple yellow to whitish nodules measuring 1 to 2 cm of diameter in the lung and severe hepatomegaly. The histopathological diagnosis was established as ACC with pulmonary metastasis. A severe and diffuse accumulation of gly­cogen in hepatocytes was also observed in Periodic acid-Schiff (PAS) staining. Case 2. A 9-year-old female intact, mixed breed dog was presented showing ataxia, left head-tilt and dyspnoea. The clinical signs progressed and the dog died after four days. The gross examination showed a diffuse increase of the left adrenal gland (2 cm of diameter) accompanied by a yellowish colour at cut surface; diffuse bronchopneumonia and cerebral and renal discrete congestion. The microscopi­cal diagnosis was ACC with kidney and CNS metastasis. Case 3. A 16-year-old male, intact, Poodle dog was presented with apathy, anorexia, vomiting and diarrhoea in the last two days. The animal was submitted to treatment, but the clinical signs progressed and the dog died. In the necropsy exam an increased right adrenal gland was observed (5 cm of diameter) showing multiple yellowish nodules when sectioned. The lung showed multiple whitish nodules mainly in pleural region. In the histopathological examination, the definitive diagnosis was ACC with pulmonary metastasis.Discussion: Most of adrenocortical tumours in dogs are functional and secrete excessive amounts of cortisol; therefore commonly animals show clinical signs of Cushing`s syndrome. In the present cases, the animals showed no clinical evidence of hormonal changes. Well-differentiated neoplastic cells were observed in cases 1 and 2, whereas in case 3 tumorous cells showed marked features of malignancy as cellular pleomorphism, binucleation, high mitotic index and atypical mitosis. Despite mild anaplastic features observed on cases 1 and 2, animals showed focus of metastases in lung, kidney and CNS. Apparently features of malignancy in ACC were not associated to the risk of metastasis development, indicating that even well-differentiated ACC may metastasize to distant organs. Metastases of ACC to distant sites are rare in dogs and there was no previous report of metastasis to CNS as observed in case 2. Canine ACC are considered uncommon tumours, how­ever, they should be included in the differential diagnosis of mass in the abdominal cavity since the animals may show no clinical signs of adrenocortical hormones overproduction.Keywords: adrenal tumours, adrenocortical hormones, metastases, dog

Linfoma canino de células T: aspectos epidemiológicos, clínicos e morfológicos de 38 casos

The aim of this study was to evaluate the incident and clinical, morphological and immunological aspects of canine T-cell lymphomas. From 71 canine lymphomas, 38 were classified by immunohistochemistry as T-cell lymphomas. According to the Working Formulation and Kiel classification, 18 were classified as intermediate-grade, 15 as high-grade and 5 as low-grade. The pattern of lymphomatous proliferation was diffuse in all 38 animals. T-cell lymphoma was more common in defined breeds (24/38), such as Boxers, English Cockers and Rottweilers. The occurrence of T-cell lymphoma in males (21/38) was greater than in females and the median age was 5.8 years (1 to 13 years). For the anatomical classification and the clinical staging of T-cell lymphomas, the most common were multicentric form (29/38) and stage IV (15/38) and III (14/38), respectively.O objetivo deste trabalho foi avaliar a ocorrência e aspectos clínicos, morfológicos e imunoistoquímicos de linfomas caninos de células T. Foram avaliados 71 cães com linfoma, dos quais 38 foram classificados imunoistoquimicamente como células T. Conforme a classificação da Working Formulation e Kiel 18 foram classificados como grau intermediário de malignidade, 15 de alta malignidade e 5 de baixa malignidade. O padrão de proliferação linfomatoso foi difuso em todos os animais (38). O linfoma de células T foi mais comum em cães de raça definida (24/38), como Boxer, Cocker Spaniel Inglês e Rottweiler. A ocorrência de linfoma T em machos (21/38) foi maior do que em fêmeas e a idade média foi de 5,8 anos (1-13 anos). Quanto à classificação anatômica e estadiamento clínico do linfoma de células T, a forma multicêntrica (29/38) foi a mais comum e os estádios IV (15/38) e III (14/38), respectivamente

Putative Equine Neorickettsiosis in a Mare from Southern Brazil

Background: This report describes the occurrence of equine neorickettsiosis (EN) in the northern region of Paraná, southern Brazil. EN is a non-contagious infectious disease caused by the Gram-negative bacterium, Neorickettsia risticii. Equine neorickettsiosis was previously known as Potomac horse fever and monocytic ehrlichiosis. The disease occurs predominantly in the USA and Canada; data relative to EN in Brazil is scarce. The aim of this study was to report the first case of putative EN in the state of Paraná due to a combination of IHC and molecular testing.Case: A 2-year-old Quarter Horse was referred to a Veterinary Hospital with episodes of abdominal discomfort, fever, anorexia, tachycardia, and tachypnea. The animal reportedly demonstrated episodes of blackened and fetid diarrhea after the ingestion of hay. A treatment was established upon arrival at the veterinary hospital, but the mare died after 12 hours of monitoring. An autopsy examination performed soon after death revealed severe hyperemia of the mucosa of the cecum and colon, with multifocal cecal erosions and ulcerations. The principal histological lesion observed was necrotizing enterocolitis. Additional significant histopathologic lesions included widespread lymphoid depletion affecting the spleen, tonsils, and lymph nodes. An IHC assay designed to identify the antigens of N. helminthoeca (NH) in formalin fixed paraffin embedded (FFPE) tissues, identified antigens of intralesional neorickettsial organisms within macrophages of the mucosa of the colon. Additionally, a PCR assay designed to amplify the 16S rRNA gene of Neorickettsia, amplified the desired amplicon, but sequencing was frustrating.Discussion: A putative diagnosis of equine neorickettsiosis was established due to the combination of epidemiological evidence, pathologic findings, immunohistochemical identification of intralesional antigens of neorickettsial agents, and amplification of the 16S gene of Neorickettsia spp. by PCR. The gross and microscopic lesions described must be differentiated from lesions observed in equine salmonellosis and Clostridium difficile associated disease of horses. IHC was performed on FFPE intestinal tissue using an anti-NH hyperimmune serum as primary antibody due to high antigenic cross-reactivity between Neorickettsia spp. There was positive intracytoplasmic immunolabeling of macrophages in the mucosa of the large colon indicating the presence of neorickettsial antigens. The PCR amplified the desired amplicon from the colon fragment, but sequencing was frustrating due to poor quality product that resulted in an organism consistent with Methylobacterium spp., probably, a contaminant. EN show a seasonal and endemic tendency in USA, Uruguay and in southern Brazil, occurring in warmer months, as in the case reported. Evidence suggests that an ancient disease named “churrido” known for at least 100 years in southern Brazil and Uruguay is, in fact, equine neorickettsiosis and might have been introduced to North America from South America. Considering the epidemiological evidence and the worldwide distribution of NR, we suggest that EN is probably underdiagnosed in Brazil and that the etiology of equine enterocolitis should be more thoroughly investigated. Therefore, this report broadens the knowledge of EN in our region. In conclusion, considering the epidemiologic, pathologic, immunohistochemical and molecular evidence, and the specie-specificity of Neorickettsia spp., we report a putative case of EN in southern Brazil and the first report in Paraná state. IHC is a useful technique, less technically demanding and time consuming than bacterial culture and isolation, considered the gold standard; and can be used to diagnose EN when compatible pathologic evidence is present concomitantly

Estudo retrospectivo de linfoma canino no período de 1990 - 2004 na região norte do Paraná

Canine malignant lymphoma (CML) is a common lymphoid tumor. Canine lymphomas from 1990 to 2004 (n=186) were analyzed on epidemiological aspects. The occurrence of CML in males ( 58,6%) was greater than in females (41,4%). The middle age of affected dogs was 5,9 years. The most common breeds in this study were mixed breed (40,3%), German Sheepdog (8,6%), Rottweiler (8,1%) and Boxer (7,0%). For the anatomical classification and the clinical staging of CML, the most common were multicentric form (68,8%) and stage III (37,1%) and IV (43,5%), respectively.O linfoma canino é a principal neoplasia de origem hemolinfática. Foram analisados 186 casos de linfoma diagnosticados entre 1990 a 2004. Observou-se que cães machos apresentaram uma ocorrência maior (58,6%) do que em fêmeas (41,4%). A idade média dos animais acometidos foi de 5,9 anos. As maiores ocorrências do linfoma foram observadas em cães sem raça definida (40,3 %), Pastor Alemão (8,6%), Rottweiler (8,1%) e Boxer (7,0%). Quanto à classificação anatômica e estadiamento clínico, a maior ocorrência foi a da forma multicêntrica (68,8%) e os estádios III (37,1%) e IV (43,5%), respectivamente

Spinal Neoplasm in Dogs

Background: Various diseases can cause spinal cord disorders in dogs including neoplasia of the nervous system, which are more common in mature and older animals. Some breeds, as brachycephalics, are more predisposed for certain types of tumors. Spinal neoplasia can be categorized as primary or secondary tumors, and still can be grouped in extradural, intradural/extramedullary or intramedullary. The aim of this study was to investigate the signalment, neurological syndrome, type and source of the mass, presence of metastases, and clinical outcomes in 28 dogs diagnosed with spinal tumors.Materials, Methods & Results: This study included 28 dogs, and in 20 cases, spinal neoplasia was confirmed by necropsy, biopsy, histopathology, and immunohistochemistry. In the other eight animals, the presumptive diagnosis was based on clinical and neurological signs and by observing changes in plain radiographs of the spine, chest radiographs, and myelography. The location of the lesion was established as cervical, cervicothoracic, thoracolumbar, or lumbosacral, and the lesion was determined to be multifocal when clinical signs appeared in more than one location. Spinal cord injury was additionally classified as unilateral, bilateral, symmetrical, or asymmetrical. The following complementary examinations were performed based on the clinical suspicion, indications, and availability of the owner: complete blood count, serum biochemical profile, urinalysis, spinal radiography, cerebrospinal fluid analysis (CSF), myelography, abdominal ultrasound, thoracic X-ray to determine metastases, and Computed Tomography. CSF was collected and analyzed in seven animals, and pleocytosis with a predominance of lymphocytes was found in two cases. Decompression and exploratory surgeries were performed in six dogs to confirm the suspected diagnosis and collect tissues for histopathology. The final diagnosis was made by histopathological examination of the tissue obtained by excisional biopsy or necropsy after the death of the animal or euthanasia. In eight cases, the neoplasia type could not be confirmed as the owner did not provide consent to perform exploratory surgery or necropsy. The dogs most affected were poodles (17.85%) and boxers (17.85%), between the ages of 5 and 10 years, and 67.85% of the cases were female dogs. The thoracolumbar region was the most affected (50%). Metastatic spinal tumors predominated; especially those originating in the mammary gland (38.46%) and on the skin (34.61%), and among the primary neoplasms, meningiomas predominated. The clinical course in relation to the onset of symptoms of neurological dysfunction, until death or euthanasia, ranged from 1-170 days, with an average 42 days of survival.Discussion: The results differ from previous studies, in which the number of male and female dogs with neoplasms in the CNS was the same, as in the present study female dogs were more affected. Unlike other studies, metastatic spinal tumors predominated. After the diagnosis, the survival was short and this may be due to the fact that most dogs in this study already had more than one system affected, and consequently showed significant changes related to systemic metastases during the onset of the neurological symptoms. The surgical treatment presented variable results, and the results observed after surgical resection of neoplasms vary due to the location in relation to the spinal cord, neoplasm type, severity of the clinical signs, and possibility of removal along with margin without causing spinal instability. In dogs with spinal disorders, the veterinary practitioner should suspect cancer, especially if there is a history of excision of neoplasms in other systems

The low intestinal and hepatic toxicity of hydrolyzed fumonisin B1 correlates with its inability to alter the metabolism of sphingolipids

Fumonisins are mycotoxins frequently found as natural contaminants in maize, where they are produced by the plant pathogen Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism.Fumonisin B1 (FB1) is the predominant fumonisins in this family. FB1 is converted to its hydrolyzed analogs HFB1, by alkaline cooking (nixtamalization) or through enzymatic degradation. The toxicity of HFB1 is poorly documented especially at the intestinal level. The objectives of this study were to compare the toxicity of HFB1 and FB1 and to assess the ability of these toxins to disrupt sphingolipids biosynthesis. HFB1 was obtained by a deesterification of FB1, with a carboxylesterase. Piglets, animals highly sensitive to FB1, were exposed by gavage for 2 weeks to 2.8 µmol FB1 or HFB1/kg body weight/day. FB1 induced hepatotoxicity as indicated by the lesion score, the level of several biochemical analytes and the expression of inflammatory cytokines. Similarly, FB1 impaired the morphology of the different section of the small intestine, reduced villi height and modified intestinal cytokine expression. By contrast, HFB1 did not trigger hepatotoxicity, did not impair intestinal morphology and slightly modified the intestinal immune response. This low toxicity of HFB1 correlates with a weak alteration of the sphinganine/sphingosine ratio in the liver and in the plasma. Taken together, these data demonstrate that HFB1 does not cause intestinal or hepatic toxicity in the sensitive pig model, and slightly disrupts sphingolipids metabolism. This finding suggests that conversion to HFB1 could be a good strategy to reduce FB1 exposure

Polycystic Kidney Disease in Rough Toothed Dolphins (Steno bredanensis) founded in the Paraná coast, Southern Brazil

Background: Renal cystic diseases (RCD) are characterized by cystic structures on renal parenchyma associated with obstructive lesions, membranous disruptions, and/or growth disturbances. The polycystic kidney disease (PKD) shows specific pathological characteristics, related to mutations on PKD1 and/or PKD2 chromosome locus on humans. In Persian cats and bull terriers the condition is like the human “adult-onset” PKD, while in Perendale sheep the “childhood”-like is described. In cetaceans, RCD are reported, however the characterization of PKD is scarcely described.  This report aims to describe two cases of PKD and one of RCD in stranded Steno bredanensis, and to discuss the disease associated factors.Cases: Four rough-toothed-dolphins were found stranded in the Paraná coast, southern Brazil between 2016 to 2018, through the Santos Basin Beach Monitoring Project (PMP-BS), one of the systematic monitoring programs required by Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) for the environmental licensing process of oil production and transport by Petrobras in the presalt province. In three animals histological sampling was performed. One of them (animal 3) was founded alive, presenting altered buoyancy with lateralization to the right and signs of pneumonia. The blood analysis showed anemia and leukocytosis. The animal showed poor clinical prognosis, and even with supportive treatment, come to death four days after the rescue. Routine autopsies were performed on all animals. Animals 1 and 2 presented macroscopically enlarged kidneys containing disseminated cystic structures in the parenchyma. On microscopic examination, the cortical region showed diffuse cystic structures delimited by variable thickness of fibrous tissue, usually compressing adjacent glomeruli, without concomitant inflammatory process. In these cases, the death was associated with the end stage renal disease. Animal 3 showed grossly few cystic structures, well delimited and replacing some reniculi. Tracheitis, granulomatous pneumonia, esophagitis, gastritis, enteritis and papilloma on penis and palate were observed. Microscopically, the cysts were lined by a single layer of columnar to cuboidal epithelial cells surrounded by extensive fibrotic tissue. Multifocal tubular necrosis was also noticed. Multifocal moderate nonsuppurative encephalitis with parasitic eggs and bacterial granulomatous hemorrhagic pneumonia were observed. In this case, the death was associated with the lesions in the nervous system. Discussion: Data concerning polycystic kidney disease on cetaceans and wild animals is limited, and no primary genetic pathway was associated. In the present study, the gross and histological aspects observed on two animals (1 and 2) are similar to the characteristics found in the human adult form of PKD, while the characteristics observed on animal 3 are consistent with usual cystic disease. In addition, the animals are aged like humans where the end stage renal disease occurs in patients around 70 years old. The kidney histological aspects observed in all animals are similar, however, animal 3 showed no renomegaly, a characteristic of PKD.  Considering the genetic pathway involved in humans and some animal’s breeds, investigation on gene mutations in S. bredanensis could help to define if this is also a genetic disorder and increase the knowledge about PKD. Keywords: kidney, delphinidae, PKD, diagnostic pathology, cystic disease, rough toothed dolphin

The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression

The gastrointestinal tract represents the first barrier against food contaminants as well as the first target for these toxicants. Deoxynivalenol (DON) is a mycotoxin that commonly contaminates cereals and causes various toxicological effects. Through consumption of contaminated cereals and cereal products, human and pigs are exposed to this mycotoxin. Using in vitro, ex vivo, and in vivo approaches, we investigated the effects of DON on the intestinal epithelium. We demonstrated that, in intestinal epithelial cell lines from porcine (IPEC-1) or human (Caco-2) origin, DON decreases trans-epithelial electric resistance (TEER) and increases in a time and dose-dependent manner the paracellular permeability to 4 kDa dextran and to pathogenic Escherichia Coli across intestinal cell monolayers. In pig explants treated with DON, we also observed an increased permeability of intestinal tissue. These alterations of barrier function were associated with a specific reduction in the expression of claudins, which was also seen in vivo in the jejunum of piglets exposed to DON-contaminated feed. In conclusion, DON alters claudin expression and decreases the barrier function of the intestinal epithelium. Considering that high levels of DON may be present in food or feed, consumption of DON-contaminated food/feed may induce intestinal damage and has consequences for human and animal health

Pathological Findings in the Adrenal Glands of 80 Dogs

Background: The adrenal glands development important endocrine functions and can be affected by primary or secondary diseases. These adrenal gland pathologies may induce clinical syndromes resulting from abnormalities in the production and secretion of hormones. Data about pathological changes in dogs are scarce. Therefore, the aim of the present study was to identify and evaluate the histopathological and epidemiological features of adrenal changes in dogs submitted to necropsy examination from 2005 to 2016 in a Veterinary Teaching Hospital, Londrina, Paraná, Brazil.Material, Methods & Results: During this period, 80 animals presented alterations of adrenal gland, representing 5.5% of all necropsied dogs. The pure breed dogs representing 58.6% and mixed breed 41.4%; 53.4% were female and 46.6% were male. The non-neoplastic adrenal lesions were more frequently (57.5%) compared to the neoplastic changes (42.5%). Most of the adrenal glands lesions occurred in older dogs (60%), following by the middle aged (31.25%) and young dogs (8.75%). The main non-neoplastic lesions observed in the adrenal glands were of hyperplastic (69.5%) and circulatory (26%) origin, among the earlier, 68.8% were diffuse hyperplasia and 31.2% nodular hyperplasia. The nodular hyperplasia was classified as micronodular multifocal in 40% of the glands with nodular hyperplasia, macronodular multifocal in 30%, micronodular diffuse in 20%, and micronodular focal in 10%. The neoplasms observed were adrenocortical adenoma (ACA) in 44.1%, pheochromocytomas in 23.5% and adrenocortical carcinomas (ACC) in 11.7% of the dogs with adrenal tumors. Metastasis from other primary tumors were observed in 20.6%.Discussion: The data in veterinary literature about the frequency of changes in adrenal gland of dogs are scarce and focus features of diagnosis by ultrasound examination. Most of the adrenal changes observed in the present study were incidental findings observed during the necropsy examination and no specific clinical signs were observed. Non-neoplastic adrenal lesions, mainly of hyperplastic origin, were more frequently observed compared to the neoplastic changes. Differing from previous studies, the diffuse hyperplasia was the most common non neoplastic finding in the adrenal glands. Considering the subclassification of nodular hyperplasia, the multifocal micronodular and macronodular multifocal pattern were the most frequent. In veterinary literature, there are no data about frequency of nodular hyperplastic subtypes. However, in humans the subclassification of nodular hyperplasia is associated to development of endocrine disorders. Previous studies reported lower incidence of neoplastic changes in adrenal gland of dogs compared to the present results. The adrenocortical adenoma was the most common primary tumor, followed by the adrenocortical carcinoma, pheochromocytoma and adrenocortical carcinoma. Congestion and hemorrhage were common findings observed in the adrenal gland primary tumors. On the other hand, necrosis and inflammatory infiltrate were observed only in the adrenocortical carcinomas. These histopathological features may be used as tool in the differential diagnosis between well differentiated adrenocortical carcinoma and adenoma. The histopathological examination was fundamental to differential and definitive diagnosis of all canine adrenal gland disorders observed