Detection and Characterization of Bovine Rumen Microorganisms Resistant to Sodium Fluoroacetate

Background: Poisoning of animals due to toxic plants is found in Brazil and other countries. One of the known toxic plants in Brazil, with the active ingredient sodium fluoroacetate (SF), is Palicourea marcgravii. Dehalogenases that inactivate the fluor-carbon bonds are enzymes found in microorganisms and may prevent intoxication. This study evaluated the occurrence of rumen microorganisms naturally resistant to SF.Materials, Methods & Results: Two samples of rumen fluid of cattle from the Experimental Farm of Federal University of Mato Grosso fed with Brachiaria sp. were obtained via fistula in flasks. An aliquot of 2 mL was placed in a microtube and centrifuged at 9000 g for 1 min. Then, the sample was inoculated into 2 tubes, one containing 100 µL of clarified rumen fluid in 2 mL of modified liquid culture medium (0.1% ammonium sulfate, 0.1% potassium phosphate monobasic, 0.05% sodium phosphate dibasic, 0.01% magnesium sulfate, 0.01% yeast extract, pH 7.0) and 0.4% of SF and the other sample containing 2 mL of liquid culture medium and 100 µL of clarified rumen fluid. The 2 samples were incubated at 40°C for 24 h. Dilutions were performed under the same conditions every 24 h until the attainment of microorganisms resistant to SF, and the finaldilution containing 50 µL of each sample was plated in the middle containing SF (0.4%) and incubated at 40°C for 24 h for the isolation of bacteria. The bacterial colonies resistant to SF were identified by morphological methods, stained, and subjected to DNA extraction sequencing using the universal primers 27f and 1492r (16S rDNA) for the identification of the bacterial genus using Blast DNA identity analysis. These bacteria were cultured with and without SF (0.4%), and the presence of fluoride ions was detected by an ion-selective electrode (fluoride) during incubation for 0, 30, 60, 90, and 120 min. Two resistant microorganisms were isolated, one was a Gram-positive coccus and the other was a Gram-positive rod. DNA sequencing identified these organisms as Enterococcus faecalis (98% identity Genbank 1358689) and Bacillus sp. (89% identity Genbank 1358671). Fluoride ions were detected more at 60-min incubation time in both E. faecalis (0.0560 ppm) and Bacillus sp. (0.0488 ppm). Bioassay protection tests were performed in mice ofthe following four groups: negative control (NC) with saline administration, positive control (PC) with administration of plant containing SF, Bacillus group (BG) with administration of plant containing SF plus Bacillus sp., and coccus group (CG) with administration of SF and E. faecalis. Clinical signs were recorded, and statistical analyses were performed to confirm the differences in the groups. Bioassay protection tests showed clinical signs of intoxication in the PC group (83.3%), BG group (100%), and CG group (16.6%) but not in the NC group (0%), with a statistical difference between GC and PC groups (P < 0.05).Discussion: Several environmental bacteria possessing dehalogenase activity have been described, such as Pseudomonas sp., Moraxella sp., and Burkholderia sp. and Pigmentiphaga kullae and Ancylobacter dichloromethanicus isolated from the rumen. No previous study has yet reported an association between dehalogenase activity and E. faecalis, and the protection assay has been observed only in the E. faecalis group. Similar results were observed in experimental intoxication in goats that had previously consumed SF, with the microorganisms identified being Pigmentiphaga kullae and Ancylobacter dichloromethanicus. E. faecalis, isolated from the bovine rumen, exhibited a dehalogenase activity, which could help control animal poisoning by plants containing SF

CHARACTERIZATION OF CANINE LEISHMANIASIS BY PCR-RFLP IN CUIABA, MATO GROSSO, BRAZIL

Leishmaniases are neglected zoonoses that are increasing in Brazil. The dog is considered the main reservoir of the visceral form in urban areas of Brazil and also important in maintaining the cycle of transmission of the cutaneous form in endemic areas. We used PCR-RFLP to identify the species of Leishmania involved in canine infection in Cuiaba City, Mato Grosso. Samples of bone marrow and lymph were collected from 181 dogs, of which 7.2% tested positive with indirect immunofluorescence and 24.9% using PCR-RFLP; a significant difference (p ≤ 0.05), had been possible to characterize the species Leishmania (L.) chagasi. This will aid in developing prevention measures and in the control of disease in Cuiaba and the surrounding area

Pneumonia granulomatosa decorrente de pitiose em quatis sul-americanos (Nasua nasua) de cativeiro

Pythiosis is an emerging infectious disease affecting captive and free-ranging wild animals. We report granulomatous pneumonia due to Pythium insidiosum in two South American coatis (Nasua nasua), who were found dead without any clinical records. Severe granulomatous pneumonia associated with pleural effusion was revealed in the necropsy. Microscopically, variably sized granulomas and pyogranulomas presented negative hyphae profiles at the periphery of their necrotic cores. Grocott methenamine silver stain highlighted these structures, and immunostain (anti- P. insidiosum) was strongly positive. Molecular analysis by polymerase chain reaction amplified P. insidiosum specific DNA. These findings characterized P. insidiosum as a cause of granulomatous pneumonia in coatis and proved that pythiosis needs to be considered in the differential diagnosis of respiratory diseases affecting this species in endemic areas.A pitiose é uma doença infecciosa emergente que afeta animais silvestres de cativeiro e em vida livre. Reportamos dois casos de pneumonia granulomatosa decorrentes da infecção por Pythium Insidiosum em quatis sul-americanos (Nasua nasua), que foram encontrados mortos sem apresentar nenhum quadro clínico prévio. Pneumonia granulomatosa severa associada a efusão pleural foi observada durante a necropsia. Na microscopia, foram observados múltiplos granulomas e piogranulomas de tamanhos variados que continham imagens negativas de hifas na periferia de seus centros necróticos. A coloração de metenamina de prata (Grocott) evidenciou estas estruturas, e a imunomarcação (anti-P. insidiosum) foi fortemente positiva. A análise molecular pela reação de polimerase em cadeia amplificou o DNA específico do P. insidiousum. Estes achados caracterizaram o P. insidiosum como a causa da pneumonia granulomatosa nos quatis e provou que a pitiose deve ser considerada um diagnostico diferencial para outras doenças respiratórias que afetam esta espécie

Pitiose gastrointestinal canina: relato de caso no Pantanal brasileiro a partir de uma abordagem diagnóstica

A pitiose é causada por um organismo aquático semelhante a um fungo (Pythium insidiosum) e considerada um importante problema de saúde pública, pois pode afetar animais e humanos. Este artigo relata um caso de pitiose gastrointestinal em um cão. O paciente ficou internado por quatro dias, período em que o animal recebeu tratamento de suporte e sintomático. No entanto, o tratamento aplicado não teve sucesso e o quadro clínico do cão piorou, culminando com a morte. Exames de imagem complementares, como radiografia e ultrassonografia, bem como exames hematológicos, foram realizados durante o período de internação. O diagnóstico definitivo foi feito na autópsia, pois as características macroscópicas e microscópicas sugeriam a presença de granuloma intestinal e acentuada enterite necrótica multifocal supurativa. Além disso, a avaliação histológica revelou estruturas morfológicas compatíveis com P. insidiosum. Além disso, a nested PCR foi realizada e mostrou amplificação parcial (105 pb) da região ITS1 do gene ribossomal de P. insidiosum.Pythiosis is caused by an aquatic fungus-like organism (Pythium insidiosum). It is considered an important public health issue as it can affect both animals and humans. This paper reports a case of gastrointestinal pythiosis in a dog. The patient was hospitalized for four days, during which the animal received supportive and symptomatic treatment. But the applied treatment was unsuccessful and the dog’s clinical condition worsened, culminating in death. Complementary imaging tests such as radiography and ultrasonography, as well as hematological tests, were performed during the hospitalization period. The definitive diagnosis was reached in the postmortem as macroscopic and microscopic characteristics suggested the presence of intestinal granuloma and accentuated multifocal suppurative necrotic enteritis. Additionally, the histological evaluation revealed morphological structures compatible with P. insidiosum. Also, the results of nested PCR performedshowed partial amplification (105 bp) of the ITS1 region of the ribosomal gene of P. insidiosum

Prevalence and Associated Factors of Canine Visceral Leishmaniasis in an Endemic Area of Mato Grosso, Brazil

Background: Canine visceral leishmaniasis is a chronic and severe disease of great interest to global public health. In Brazil, the main species causing visceral leishmaniasis is Leishmania chagasi [syn. Leishmania infantum], which is transmitted by sandflies of the species Lutzomyia longipalpis and Lu. cruzi. This study aimed to determine the risk factors and prevalence of visceral leishmaniasis in dogs residing in endemic areas of the municipality of Várzea Grande, Mato Grosso.Material, Methods & Results: The study was conducted in the Várzea Grande neighbourhoods Jardim Eldorado, Parque Sabiá and São Mateus, which are located in the Brazilian Cerrado region and are endemic for visceral leishmaniasis. The Várzea Grande is a mostly commercial and industrial municipality and relies on subsistence farming. To describe the general characteristics of the dog population and housing environment, an interview was carried out with the dog owners in each household. This interview addressed local demographics and the identification of dogs to establish epidemiological aspects of canine illness and risk factors for infection. The dogs were examined and physically restrained for blood collection by jugular venipuncture. For serological testing, a commercial enzyme-linked immunosorbent assay (ELISA) was used. A Chi-square test or Fisher’s exact test was used to identify associations between independent variables and the seroprevalence of dogs with anti-Leishmania antibodies. It were collected blood from 521 animals, including 160 belonging to Jardim Eldorado, 129 to Parque Sabiá and 232 to São Mateus. Of these animals, 120 were reactive by ELISA, with a prevalence of 23.0%. There was no statistically significant difference (P > 0.05) according to gender, age, racial definition, origin, time with family, presence of ectoparasites, or the presence of people with skin wounds at home or on other pets. The major risk factor for canine infection was fur length; dogs with short fur were 2.2 times more likely to be infected than dogs with long fur.Discussion: The prevalence of canine leishmaniasis in endemic areas was found to be similar to studies in Cuiaba, a city in Várzea Grande, which also included neighbourhoods endemic for visceral leishmaniasis as study areas. Other studies have shown prevalence rates that differ from those found in this study. This difference may either be because the neighbourhoods in this study had already experienced outbreaks of the disease or because serum samples were cross-reactive with other trypanosomatids. While infection was more readily detected in dogs with clinical signs, asymptomatic dogs with anti-Leishmania antibodies were found in greater numbers, which is consistent with the literature. Hair length was the only factor associated with canine leishmaniasis in this study; short-haired animals were most at risk of contracting the disease because they had more skin exposure to sandflies during blood feeding. In conclusion, endemic districts of Várzea Grande had high prevalence rates of canine visceral leishmaniasis, and a short length coat was associated with infection

Presence of BRCA1 Gene Mutation in Bitches with Malignant Mammary Tumors

Background: Mammary tumors (MTs) in bitches are similar to breast cancers in women. Thus, they can be used as a model for human breast cancer and findings can be extrapolated for use in human medicine. BRCA1 is a tumor suppressor gene. When the gene has a mutation, it cannot repair damaged DNA, which causes genetic instability and tumorigenesis. Therefore, we aimed to study the frequency of single nucleotide polymorphisms (SNPs) in the BRCA1 gene that are associated with distinct histological types of malignant MT in bitches.Materials, Methods & Results: The study population consisted of 91 bitches, including a control group of 6 animals with healthy mammary glands and 85 animals with MTs. All animals underwent a presurgery evaluation consisting of a questionnaire administered to the person responsible for the animal, a physical examination, collection of peripheral blood for hematological and serum biochemistry evaluations, an electrocardiogram, and a preanesthesia evaluation. In addition, distant metastasis was studied via chest radiography and abdominal ultrasound. After evaluations were complete, the animals that could undergo surgery were administered general anesthesia and underwent a mastectomy or mammary gland sample collection. Histopathological examination and molecular analysis were performed to identify mutations in the BRCA1 gene. Histopathological examinations found 10 different types of malignant tumors in 36 sick animals. Tumor samples plus samples from the 6 control animals were subjected to DNA extraction, polymerase chain reaction (PCR) analysis, and genetic sequencing. The tumor with the highest incidence (33.33%) was a complex carcinoma, followed by carcinoma in mixed tumor (13.88), tubular carcinoma (13.88) and carcinosarcoma (13.88). Molecular analysis revealed 3 different SNP points in 5 samples (4006G>A, 3619A>G, and 3761C>T). The allelic variant 4006G>A (1/36) resulted in the alteration of the amino acid valine by isoleucine (V1336 I). The mutation 3619A>G (2/36) inserted the amino acid alanine instead of threonine (T1207 A). The mutation 3761C>T (2/36) led to the alteration of the amino acid serine by phenylalanine (S1254 F), a mutation for which there are no published reports. The histological types that showed BRCA1mutations were complex carcinoma (1/5), carcinoma in mixed tumor (1/5), papillary carcinoma (1/5) and tubular carcinoma (2/5). Software analysis identified the new SNP (nucleotide 3761) in BRCA1 and 2 points mutations in nucleotides 4006 and 3619 and responsible for genetic instability.Discussion: The development of breast cancer is caused by many endogenous and exogenous factors. The results of our study show that these factors have a greater presence in female, mixed breed, uncastrated, and older dogs, confirming the data in the veterinary literature. In the present study, we found different histological types of malignant breast tumors with mutations in the BRCA1 gene, as other authors have reported. However, we also found the mutation 3761C>T, which, to the best of our knowledge, has not been reported in the literature. This shows the need for studies in veterinary medicine that assess mutations in the BRCA1 gene and the most common histological types. In conclusion, SNPs in the BRCA1 gene cause genetic instability, resulting in additional mutations that lead to the development of breast tumors. They are point mutations that affect transcription, resulting in truncated proteins. These proteins may have a loss of function, leading to carcinogenesis.Keywords: polymorphism, BRCA1 gene, cancer, dogs.Descritores:polimorfismo, gene BRCA1, câncer, cães. Título: Presença da mutação do gene BRCA1 em cadelas com tumores mamários malignos.

Levantamento sorológico e molecular de patógenos transmitidos por carrapatos em cães do Norte do Brasil

O estudo teve como objetivo avaliar a ocorrência de anticorpos anti-Rickettsia spp. e anti-Ehrlichia canis, bem como a detecção molecular de DNA de Babesia vogeli, Ehrlichia spp. e Hepatozoon spp. em cães do município de Ji-Paraná, estado de Rondônia, norte do Brasil. Para tanto, foram analisadas 177 amostras de soro e sangue por meio reação de imunofluorescência indireta (RIFI) e reação em cadeia pela polimerase (PCR), respectivamente. Um total de 8,47 (15/177) cães foram sororreativos para pelo menos uma espécie de Rickettsia, com títulos finais variando de 64 a 1.024. Os títulos finais mais elevados foram observados para Rickettsia parkeri e Rickettsia rhipicephali. Além disso, anticorpos anti-E. canis foram detectados em 24,29% (43/177) das amostras de soro testadas, com títulos finais variando de 80 a 2.560. Dentre as amostras de sangue analisadas molecularmente, 5,65% (10/177), 22,03% (39/177) e 48,02% (85/177) estavam infectados por B. vogeli, E. canis e Hepatozoon canis, respectivamente. Neste trabalho, fornecemos evidências sorológicas de Rickettsia spp., e E. canis além disso, confirmamos a ocorrência de DNA de B. vogeli, E. canis e H. canis em cães da área rural do estado de Rondônia. região oeste da Amazônia brasileira