Detecção de DNA de Brucella spp. em amostras de sangue e de suabe vaginal ou prepucial de cães do município de Natal, Rio Grande do Norte, Brasil

The aim of this work was to detect Brucella spp. DNA in samples of blood and vaginal or preputial swabs in 80 seropositive dogs for brucellosis by agar gel immunodiffusion test (AGID) from the county of Natal, Rio Grande do Norte State, Brazil. Whole blood samples were collected with anticoagulant (sodium citrate) and vaginal and preputial swab samples for DNA extraction and polymerase chain reaction (PCR) employing ITS66 and ITS279 primers. Six animals showed amplification of Brucella spp., being one animal in both samples, two dogs only in blood samples, and three only in reproductive tract swabs. It is concluded that infection due to Brucella spp. occurs in dogs from the county of Natal, and the detection of DNA of the agent in reproductive tract swabs may be used as complementary tool in the diagnosis of canine brucellosis.Objetivou-se com este trabalho detectar o DNA de Brucella spp. em amostras de sangue e de suabe vaginal ou prepucial de 80 cães sorologicamente positivos para brucelose pela prova de imunodifusão em gel de ágar (IDGA), no município de Natal, estado do Rio Grande do Norte, Brasil. Amostras de sangue total foram colhidas com anticoagulante (citrato de sódio) juntamente com amostras de suabe vaginal e prepucial, para extração de DNA e posterior realização da reação em cadeia da polimerase (PCR) empregando-se os primers ITS66 e ITS279. O DNA de Brucella spp. foi amplificado em seis animais, sendo um animal em ambas as amostras, dois cães em amostras de sangue e três em amostras de suabe do trato reprodutivo. Concluiu-se que a infecção por Brucella spp. está presente em cães no município de Natal, e que a detecção de DNA do agente em amostras de suabe do trato reprodutivo podem ser utilizadas como ferramenta suplementar no diagnóstico de brucelose canina

Detection of Brucella spp. in artisan cheese commercialized in Parnaíba, Piauí state, Brazil

The aim of this study was to detect Brucella spp. in artisanal cheese commercialized in Parnaíba city, Piauí state, Brazil. For this study, 30 samples of curd cheese (500g) were randomly collected from different points in the commercialization process. In the laboratory, 25g aliquots of the samples were suspended in Brucella broth; after this procedure, 10µL aliquots of this suspension were sown in Thayer Martin agar plates that were supplemented with 10% defibrinated sheep blood and VCNT antimicrobials. After inoculation, the samples were incubated at 37°C in microaerophilic conditions for 14 days. The suspected morphological colonies were identified and confirmed by polymerase chain reaction (PCR). From thirty samples of microbiologically analyzed cheeses, six samples (20%) were confirmed by PCR as bacteria from the Brucella genus. The detection of Brucella spp. was confirmed in cheese commercialized in markets or a public square (3.33%), bakery (3.33%) and small market (13.33%). Brucella spp. was detected in artisanal cheese commercialized in different points from Parnaíba city. Guidelines for the establishment of good practices for the production of artisanal cheese should be determined by the competent authorities. However, for the better control of human brucellosis it is necessary to control or eradicate bovine brucellosis in the herds

Detection of anti-Neospora caninum antibodies in slaughtered sheep

Neospora caninum is a protozoan that causes reproductive disorders in herbivores. Domestic and wild dogs have been considered its definitive hosts, and when these animals are affected, they may suffer from a neuromuscular disease. Recently, this infection has also gained significance in small ruminants. Although neosporosis is already a proven cause of mortality in lambs and congenital infection can occur, this parasite has only recently been considered a cause of abortion in these animals. The aim of this study was to detect anti-N. caninum antibodies in slaughtered sheep. Serum samples (n = 100) collected in a slaughterhouse located in the municipality of Palmeira dos Índios (09°24’26” S and 36°37’39” W), state of Alagoas, northeastern Brazil, were used in this study. Anti-N. caninum antibodies were detected by means of the Immunofluorescence Antibody Test (IFAT). The cut-off point was set at 50 and samples were titrated up to 800. Anti-N. caninum antibodies were detected in 13% (13/100) of the tested samples, 7.69% of which came from male sheep (1/13) and 92.31% (12/13) from females. Titers ranged from 50 to 800, with the majority of animals (46.15%; 6/13) presenting the maximum one (i.e., 800). Data herein reported demonstrated the circulation of N. caninum parasites among sheep in the study area. These findings are pivotal to improve the knowledge about the dynamics of this pathogen in an ovine population. Therefore, it is crucial to adopt sanitary measures to prevent infection by this parasite and thus reduce its economic impact on ovine production

Isolation of Actinobacillus seminis from a goat with clinical epididymo-orchitis in Brazil

The present study reports the first isolation of Actinobacillus seminis from a goat in Brazil. A four-year-old Moxotó breeding goat in a flock of 70 goats and 65 sheep reared together in the county of Patos, semiarid region of Northeastern Brazil, showed clinical signs of unilateral orchitis and epididymitis. Diagnosis of A. seminis infection was confirmed by association of clinical findings, bacterial isolation and 16S rRNA gene sequencing. This result suggests that A. seminis may be an additional cause of infertility in goats, and that sheep may be the source of infection because the mixed farming system allows the contact between sheep and goats in the semiarid region of Northeastern Brazil

Padronização de PCR multiplex para detecção de agentes infecciosos no sêmen ovino

Para otimizar o diagnóstico na detecção de agentes patogênicos no sêmen ovino comercializado, com risco potencial de transmissão venérea, objetivou-se neste estudo padronizar duas reações de PCR Multiplex, utilizando pares iniciadores sensíveis e específicos, uma reação para detectar o DNA dos gêneros Brucella sp. e Leptospira sp. e outra para detectar o DNA de Actinobacillus seminis, Brucella ovis, Toxoplasma gondii e Neospora caninum de forma simultânea em uma única reação. Foram utilizadas concentrações variáveis de DNA de cepas de referência para análise do limite de detecção das reações. Em ambas reações de mPCR, quando utilizados controles positivos em concentrações de DNA genômico igual ou inferior a 100ng/mL para cada espécie observou-se a amplificação simultânea de todos os agentes pesquisados, podendo assim, ser reproduzida na rotina laboratorial para obtenção de um diagnóstico mais rápido, seguro e menos dispendioso quando comparada a outros métodos de diagnóstico.In the present study it was standardized two Multiplex PCR reactions to detect DNA of Brucella sp., Leptospira sp., Actinobacillus seminis, Brucella ovis, Toxoplasma gondii and Neospora caninum. When used positive controls at a concentrations of less than 100ng/ml of genomic DNA it was clearly seen simultaneous amplification of all agents used on both of the reactions. Thereafter these reactions can now be implemented on our laboratory, that way we are going to be able to obtain faster, safer and less expensive diagnosis on those infectious agents

Application of different techniques to detect Toxoplasma gondii in slaughtered sheep for human consumption

Abstract The aim of this study was to investigate occurrence of Toxoplasma gondii in sheep slaughtered in the state of Alagoas, Brazil, by means of different diagnosis techniques. Serum samples and tissues from 100 slaughtered sheep were used. To detect antibodies, the indirect immunofluorescence antibody test (IFAT) was used, and tissues from seropositive animals (cut-off ≥1:64) were submitted to Polymerase Chain Reaction (PCR) and immunohistochemistry (IHC). To assess the concordance between the direct techniques, the kappa test was used. In the IFAT, it was observed that 14% (14/100) of the ovine samples were serum-positive. In the PCR, 21.43% (3/14) of the animals were positive and in IHC, it was observed that 7.14% (1/14) were positively stained for T. gondii in cerebral tissue. Histopathologically, the predominant finding was the presence of mononuclear cell infiltrate in the heart and a perivascular cuff in the cerebrum and cerebellum. The concordance between the direct diagnosis techniques was moderate (k=0.44). Thus, it is important to use different direct techniques in diagnosing toxoplasmosis in naturally infected sheep

Detection of Listeria spp. in food handling areas of retail food stores in the state of Pernambuco, Brazil

Abstract The identification of Listeria spp. in food handling areas is of great concern to health surveillance agencies, and their control is often hampered by the ability of the bacteria to grow and maintain themselves even under adverse conditions. The present study aimed to isolate and identify Listeria spp. in the food handling areas of 10 retail food stores in the state of Pernambuco, Brazil. Eighty-six swab samples were collected from equipment, utensils and surfaces used for processing ready-to-eat meat products. The Dry and Wet Swabbing Methods (3M™ Quick Swabs) and 3M™ Petrifilm™ Plates were used to identify Listeria spp. Contamination by Listeria monocytogenes was confirmed by the Real-time Polymerase Chain Reaction (qPCR). The hygienic and sanitary conditions of the food handling areas of each store were also assessed. Listeria spp. was isolated in eight stores (80%). Of the 86 swab samples analyzed, 27 (31.2%) [confidence interval 21.81% to 42.30%] were positive for Listeria spp. and only one (3.7%) was confirmed as Listeria monocytogenes. The main contamination sites were the floor (50.0%), the plastic cutting board (42.9%) and the knife (40.0%). None of the hygienic and sanitary conditions assessed in the present study were associated with contamination by Listeria spp. (p = 0.700). It was concluded that Listeria spp. was widely distributed in the retail food stores studied, being a possible risk factor for public health