Detection methods for Mycobacterium avium paratuberculosis in goats

摘要

We describe molecular, acid fast staining, histopathology and cultivation methods for diagnosis of Mycobacterium avium paratuberculosis in goats. Ileum, cecum, colon and mesenteric lymph nodes were excised from 30 goats suspected of paratuberculosis.Hematoxylin and eosin and acid fast stainings were performed for histopathological and histochemical examinations. For MAP isolation, Middlebrook 7H9 broth-based associated with OADC (oleic acid, albumin, dextrose, and catalase) supplement with/without mycobactin J were used as a medium. In order to cultivation, the double incubation method was used for tissue decontamination. IS900 specific DNA probe detection by the polymerase chain reaction was performed from broth based media and DNA extractions oftissue speciemens. MAP was cultured after 3-10 days in Middlebrooke 7H9 medium with and without mycobactin J from tissue samples of 15 suspected goats. Bacterial culture revealed all of 15 goats were infected with MAP. Acid-fast staining of broth based cultures were positive in 47% of ileums, 60% of cecums, 33% of colons and 40% of mesenteric lymph nodes with mycobactin J and in 60% of ileums, 53% of cecums, 40% of colons and 33% of mesenteric lymph nodes without mycobactin J. MAP specific IS900 gene with 413bp was demonstrated in 70% of intestinal tissue specimens and in all of the samples in both of with and without mycobactin J cultures. Histopathological examination revealed diffuse granulomatous enteritis and lymphadenitis. Acid fast staining of these lesions showed two different paucibacillary or multibacillary lesions. Positive acid fast staining was observed in 47% and 43% of intestinal tissue specimens as paucibacillary and multibacillary, respectively. Multifocal granulomatous lymphadenitisassociated with caseous necrosis and calcification were observed in the mesenteric lymph nodes. The results of this study showed culture of tissue samples with or without mycobactin J associated with IS 900 PCR can detect MAP in almost all of affected goats to paratuberculosis.