Clonality Test by PCR - PARR in Real Time of Canine Lymphomas

Background: Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin.Materials, Methods & Results: The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the Collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco UFRPE. Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major (LB), IgH Minor (LB), and TCRγ (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative control. After amplification, the melting curve gradually increased the temperature by 1C/5 s to 95C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T.Discussion: Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysismay vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3C when compared to that in the control sample for LB and LT, 83.5C and 80C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.Keywords: immunophenotyping, lymphoproliferative disease, real-time PCR, TCRγ, IgH

Gastroesophageal Intussusception in Canine

Background: Gastroesophageal intussusception is characterized by the invagination of the stomach into the esophagus, with or without the involvementof adjacent organs such as the spleen, pancreas, and omentum. In dogs, this condition has no breed or sex predisposition. As it is an infrequent disease in routine veterinary medical practice, this study reports a case of gastroesophageal intussusception in a dog necropsied at the Veterinary Hospital of the Federal Rural University of Pernambuco (HOVET - UFRPE), Recife, Brazil.Case: The body of a 12-year-old black mixed breed male dog was sent to the Pathology Department (Necropsy Sector of the Federal Rural University of Pernambuco - UFRPE) for a necropsy. The animal had a previous 4-year history of recurrent emesis and limb weakness, primarily in the anterior limbs, that worsened in the previous months and progressed to death. No previous treatments were reported by the owner. On external examination, the animal had a low body score (cachectic), forelimb joints with great flexibility, congested oral and ocular mucous membranes, enophthalmos, and increased volume in the perianal region. At the opening of the thoracic cavity, the final third of the esophagus was dilated and gastroesophageal intussusception, edema, and pulmonary congestion were noted. In the abdominal cavity, there was hepatic and renal congestion and large intestine and rectal ampoule dilation, with a large amount of solid and retained feces (fecaloma), perianal hernia, and testicular neoformation. These findings were consistent with those observed in death caused by cardiorespiratory failure secondary to gastroesophageal intussusception.Discussion: The pathophysiology of gastroesophageal intussusception is still not elucidated and is probably multifactorial. This condition causes reverse gastric peristalsis associated with a sudden and sustained increase in abdominal pressure. Some probable predisposing factors for this pathological condition are esophageal motility disorders, lower esophageal sphincter dysfunction, and hiatal enlargement. In dogs, gastroesophageal intussusception is associated with increased intra-abdominal pressure owing to emesis or blunt trauma, negative intrathoracic pressure caused by respiratory, and previous esophageal diseases, especially megaesophagus. Partial or total obstruction caused by intussusception leads to circulatory disorders in the organs, especially decreased venous return. Persistence of this condition can lead to gastric necrosis and rupture followed by endotoxic (or septic) shock and release of inflammatory mediators that can cause cardiovascular and respiratory dysfunction and rapid death. Impaired circulation is macroscopically evident in several organs, characterized by mucosal, lung, liver, and kidney congestion, in addition to cardiac dilation and mitral valve endocardiosis. The occurrence of stomach invagination into the esophagus dilated in the final portion is characteristic of gastroesophageal intussusception. Moreover, death owing to cardiorespiratory failure is related to cardiac (dilatation and endocardiosis) and pulmonary (edema and congestion) involvement secondary to gastroesophageal intussusception. Since thispotentially fatal condition has a low incidence in small animals and often goes unnoticed by professionals, early and correct diagnosis along with surgical treatment are essential for a good prognosis and favorable progression

Anti-Leptospira spp. Antibodies in Pigs Slaughtered in the Agreste Region of Pernambuco, Brazil

Background:  Swine production and productivity rates can be influenced by several factors, such as genetics, environmental conditions, nutritional factors, previous infections and others. Among infectious diseases, leptospirosis is a well-known cause of reproductive disorders in pigs. These animals are considered carriers of the disease when they are in the terminal stage of the infection and the Veterinary Inspection Service has not been notified when they are slaughtered. Considering the lack of epidemiological information on Leptospira infection in pigs in the state of Pernambuco, the aim of this study was to investigate anti-Leptospira antibodies in pigs slaughtered in the Agreste region of the state of Pernambuco, Brazil.Materials, Methods & Results: Blood samples were collected from 305 pigs in 11 municipalities in the Agreste region of Pernambuco. The animals had no history of vaccination, and were raised on subsistence-oriented family farms. The sera were subjected to the Microscopic Agglutination Test (MAT) for the detection of antibodies. The serovars used in the MAT were: Icterohaemorrhagiae, Copenhageni, Javanica, Canicola, Castellonis, Pyrogenenes, Cynopteri, Autumnalis, Sentot, Djasiman, Australis, Pomona, Grippotyphosa, Hebdomadis, Wolffi, Sejroe, Saxkoebing, Bataviae, Tarassovi, Panama, Patoc, Andamana, Celledoni, Shermani, Brastilava and Hardjo. Sera showing titers of ≥100 were considered positive. The MAT results indicated that 78/305 (25.57%) of the samples were positive, and were distributed in the 11 municipalities. The most frequent serovars were Icterohaemorrhagiae, Copenhageni, and Djasiman, with frequencies of 55.13%, 17.95% and 6.41%, respectively.Discussion: Swine infected with leptospirosis showed few or no signs of the disease. However, the bacteria can be carried for long periods in convoluted tubules of the kidney and their urinary excretion may last for years. A study of Leptospira spp. risk factors on pig farms in the state of Alagoas revealed an absence of rodent control, suggesting that this was the probable cause of infection by this serovar, Icterohaemorrhagiae. Farms that did not control their rodent populations showed a 7.8-fold higher risk of infection among their swine. This exposure poses a problem, since these animals can contaminate the environment, as well as food and water, making them an important source of infection of other animals. The findings of this study indicate that the pigs were exposed to Leptospira spp. The reason that some atypical serovars were identified  in this study may be have been incidental infection, since  these animals came  from a non-technified system, putting them at greater risk of contact with other animal species. This may also be a reason for the identification of the other serovars in this research. Although the serovar Copenhagen is part of the Icterohaemorrhagiae serogroup, few studies have described its importance. The main reservoirs of Copenhageni are also synanthropic rodents, which underscores the importance of these animals as possible sources of contamination on farms that supply pigs to the slaughterhouses of the region under study. The data obtained here indicate the need for animal health surveillance programs in the region. Moreover, they may also be a source of infection of the professionals directly involved in handling and slaughtering pigs. This indicates the need to implement surveillance programs in the region, such as high vaccination coverage at pig farms, as well as integrated pest management against rodents

Systemic Cryptococcosis in a Miniature Schnauzer Dog

Background: Cryptococcosis is an important zoonotic disease that occurs worldwide. The disease is caused by a soilborne opportunistic fungus of the genus Cryptococcus, which can also be found in the feces of birds, especially pigeons. In Brazil, the geographical distribution of the agent is fairly defined, with the species C. gattii predominantly found in thenorth-eastern states. Diagnosis is based on the clinical history, physical examination findings, and results of complementary testing, such as cytopathological and histopathological examinations. This report aims to describe the clinical and anatomopathological findings in a case of systemic cryptococcosis in a dog.Case: A 4-year-old female Miniature Schnauzer was referred for necropsy. For 3 months prior to death, the dog had displayed generalized alopecia, pruritus, and severe weight loss, Initially, a private veterinarian had suspected leishmaniasis. Gross examination was performed and samples for cytopathology, histopathology, and fungal culture were obtained. Themacroscopic examination revealed generalized alopecia, congested mucosa, cachexia, hyperemia of the brain, and multiple white nodules measuring 0.5 cm to 4 cm in the lungs and the kidneys. None of the other organs showed significant lesions. Cytopathological examination of the nodules revealed a highly cellular sample, with a severe chronic inflammatory response, characterized by multinucleated giant cells and round-to-elliptical, yeast-shaped structures (5-10 μm), suggestive ofCryptococcus organisms. Histopathological examination of the lungs, kidneys, and brain revealed a severe diffuse chronic inflammatory process, with lymphocytic infiltration and multinucleated giant cells; countless yeast-shaped, round-to-ovoid structures (similar in appearance to “soap bubbles”) that were negative in hematoxylin-eosin stain were also present.Grocott’s methenamine silver stain was then applied, which positively stained the organism capsules black, confirming Cryptococcus. For mycological diagnosis, samples from the lungs nodules were cultured on Sabouraud’s dextrose agar supplemented with chloramphenicol 0.4% and incubated for 7 days at 25-30ºC. Abundant small, smooth, irregular-sized,cream-colored colonies were obtained, and a mycological smear, stained by India ink, was performed.Discussion: Considered an uncommon disease, canine cryptococcosis can be misdiagnosed. Clinical findings, such as alopecia, lymphadenopathy, and cutaneous nodules that may be ulcerated, are commonly seen in these patients due to the hematogenous spread of the organism. In the case described, the dog’s owner was contacted and asked to provide epidemiological information. The owner reported that the dog had lived in an apartment, with little access to the street; however, pigeons were frequently observed on the balcony, along with pigeon feces in the dog feeder. The presence of pigeon feces is a risk factor for the development and spread of the fungus, which may be problematic to public health. Cytopathological examination is a simple, quick diagnostic tool with good sensitivity for some infectious agents. Mycological culture can provide results due to the characteristic of the Cryptococcus sp., since the encapsulated yeast produces a mucous-like colony. Cryptococcosis has a relevant role in public health, so a precise diagnosis is imperative. Its diagnosis is based upon culture, cytology and serological tools. When the animal does not survive to the ilness, the association between necropsyfindings and histopathological examinations is essencial.Keywords: canine, diagnostic, yeast, anatomopathology

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Detecção do Mycobacterium avium subespécie paratuberculosis em amostras de leite de bovinos na microrregião de Garanhuns, Pernambuco

Objetivou-se com este estudo detectar a região IS900 do MAP em amostras de leite bovino utilizando-se as técnicas de Reação em Cadeia da Polimerase em Tempo Real (qPCR) e a PCR convencional, e realizar um estudo de concordância entre os testes utilizados. Foram coletadas 121 amostras de leite bovino de propriedades consideradas foco para o MAP pertencentes à microrregião de Garanhuns, Pernambuco, Brasil. Das 121 amostras de leite analisadas foi possível detectar o DNA do MAP em 20 (16,52%) amostras na PCR convencional e em 34 (28,09%) na qPCR. O DNA do agente foi detectado em todas as seis propriedades estudadas, variando de 10,00% a 23,81% na PCR convencional e de 10,00% a 36,84% na qPCR. A concordância entre a qPCR e PCR foi moderada (Kappa =0,53; χ2 = 38,08; p<0,000) e a sensibilidade e especificidade da PCR convencional com relação a qPCR foi de 50% e 96,6%, respectivamente. Conclui-se que o DNA da região IS900 do MAP está presente no leite bovino na região estudada e que a qPCR é uma técnica sensível e rápida para detecção desse agente em amostras de leite.The aim of the present study was to detect the IS900 region of MAP in bovine milk samples using the Real Time Polymerase Chain Reaction (qPCR) and conventional PCR, as well as to study the agreement between the tests used. In total, 121 bovine milk samples were collected from properties considered as a focus of MAP in the micro-region of Garanhuns, Pernambuco, Brazil. Of the 121 milk samples analyzed, it was possible to detect the DNA of MAP in 20 samples (16.52%) using conventional PCR and in 34 samples (28.09%) using qPCR. The DNA of the agent was detected on all of the six properties studied, ranging from 10.00% to 23.81% in the conventional PCR and from 10.00% to 36.84% in the qPCR. The agreement between qPCR and conventional PCR was moderate (Kappa =0.53; χ2 = 38.08; p<0.000). The sensitivity and specificity of conventional PCR in relation to qPCR was 50% and 96.6%, respectively. Based on these results, it was possible to conclude that the IS900 region of MAP is present in bovine milk in the micro-region of Garanhuns, Pernambuco. It was also possible to conclude that the qPCR technique used in the present study was more sensitive than conventional PCR in terms of detecting these bacteria in milk samples.Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqCoordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPE

Low prevalence of infection by Sarcocystis neurona in horses from the State of Alagoas, Brazil

Abstract The aim of this study was to determine the prevalence of infection by Sarcocystis neurona in horses and identify potential risk factors. Were analyzed 427 samples from 36 farms in 21 municipalities in the Alagoas State, Brazil. Presence of anti-S. neurona antibodies was diagnosed by indirect immunofluorescence antibody test (IFAT) and was confirmed using the immunoblot test. Risk factors were assessed through investigative questionnaires on animal management on the farms. The prevalence of anti-S.neurona antibodies was 2.8% (confidence interval, CI: 1.5-4.9%) from IFAT and 1.6% (CI:0.8-3.34%) from immunoblot, and there were positive horses on 16.6% of the studied farms. None of the variables studied presented associations with serological status for S. neurona. This is the first report on infection by S. neurona in horses reared in Alagoas, Brazil showing a low exposure to S. neurona in this region, but with significant numbers of foci