Isolamento e detecção molecular de Mycobacterium avium subespécie paratuberculosis (MAP) em rebanhos bovinos leiteiros na região de Viçosa - MG

• Autor(a) principal: Carvalho, Isabel Azevedo
• Data de Publicação: 2008
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/4968
• topic: Paratuberculose; PCR; Leite; Paratuberculosis; PCR; Milk; CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::MEDICINA VETERINARIA PREVENTIVA::DOENCAS INFECCIOSAS DE ANIMAIS

Paratuberculosis is a chronic infectious enteric disease caused by Mycobacterium avium ssp. paratuberculosis that affects all ruminants, characterized by malabsorption, diarrhea, and weigh loss. The disease has a great economic importance due to the milk production decrease, culling of infected cows, and the increase of sanitary costs. The disease has also an importance in Public Health, considering that the etiological agent can be associated with Crohn s Disease in humans and the main transmitting vehicle would be the milk. In Brazil, there are few research groups about the subject and the disease s economic impact was not measured yet. This study aimed at initiating a survey of the disease in dairy herds in the region of Viçosa and evaluating the detection limit of the PCR for the MAP detection in raw milk samples. 204 fecal samples and 220 pooled quarter and bulk tank milk samples were analyzed. It was observed contamination in 334 (41%) fecal samples and in 132 (15%) milk samples. After the faeces samples processing, colonies with morphological and chemical features similar to MAP were not observed, and it was found colonies only in HEYM with Mycobactin J from milk samples of an animal with clinic signals similar to paratuberculosis. A smear was prepared from one of these colonies and, in optic microscope, BAAR was detected. The 220 milk samples went through the PCR, with two pairs of primers: IS900BN1/IS900BN2 e ISMav2F/ISMav2B2. Using the first one, 8 samples (3.6%), of which two were of animals with clinical signals similar to the disease, amplified fragments of expected size (626bp), and using the second pair, 5 samples (2.2%) amplified fragments of expected size (494bp). Serum and milk samples of 8 animals that amplified fragments of expected size using the pair of primers based on the insertion sequence IS900, were submitted to ELISA. In the serum ELISA, two samples (25%) were considered positive and one (12,5%), proceeding from the animal with clinical signals similar to the disease, was considered suspect; while in the milk ELISA, all the samples were considered negative. The 8 samples were cloned and sequenced. Of these, 3 (37,5%) were sequenced successfully and the genetic analysis revealed 99% of identities among the sequences obtained in this study and the IS900 sequence deposited in the Genbank. Through sequential dilution of the plasmid DNA extracted from the positive control clone, it was evaluated the detection limit of the PCR for the MAP detection in pooled quarter milk samples. It was found that minimum detection limit ranged approximately from 140 to 180CFU/ml of milk. This study reported the first MAP detection in milk samples in Brazil. Using the IS900 PCR as a diagnosis tool, the estimated rate of paratuberculosis in dairy herds evaluated in the region of Viçosa - MG was of 3,6%, providing a first survey about the disease in the region of Viçosa and in Minas Gerais State.