Thin layer microcolony culture associated with PCR for early identification of Mycobacterium bovis

The initial growth of mycobacteria from 49 samples of cattle and buffalo organs collected in commercial slaughterhouses was compared between modified Middlebrook 7H11 thin layer microcolony culture and Stonebrink medium used in the isolation of Mycobacterium bovis. Aliquots were decontaminated by Petroff's method, processed and cultured in both media. The identity of the acid-fast bacilli stained by Ziehl-Neelsen was confirmed by PCR. Optical microscopy showed that results of the early observation of Mycobacterium bovis colonies in thin layer culture were similar to those obtained in macroscopic observation of the colonies in Stonebrink medium. However, early observation of the colonies enabled early confirmation by PCR, given the shorter time to the visualization of colonies when thin layer culture was used (between the 12nd and 25th day of culture).

Evaluation of polymerase chain reaction (PCR) for identifying Mycobacterium bovis isolates from the modified Middlebrook 7H11 agar thin layer technique

The associated use of the modified Middlebrook 7H11 agar thin layer technique and the Polymerase Chain Reaction (PCR) assay enabled to perform the early identification of microcolonies of Mycobacterium bovis from 12th to 25th day of culture. In order to reduce the time for performing the Mycobacterium bovis identification, the combined use of these two techniques was evaluated by analyzing the microcolonies of mycobacteria at the 8th day after culturing. Until the last day of analysis, all of the PCR-positive samples already showed the microcolonies. Therefore, the early diagnosis of bovine tuberculosis is feasible, without an apparent macroscopic colonies growth.

Mycobacterium bovis infection in goats from the Northeast region of Brazil

A total of 8,058 male and female mixed-breed goats and 1-4 years of age were slaughtered over a period of 7 months at the public slaughterhouse of Patos city, Paraíba state, in the Northeast region of Brazil; 822 animals were inspected for gross lesions of tuberculosis, and 12 (1.46 percent) had lesions suggestive of tuberculosis in the mammary gland, lungs, liver and mediastinal, mesenteric, submandibular, parotid and prescapular lymph nodes. Presence of granulomatous lesions was confirmed in the submandibular lymph node of one (8.3 percent) goat at the histopathological examination and at the mycobacterium culture the same sample was confirmed positive. Isolate was confirmed as belonging to the M. tuberculosis complex by PCR restriction enzyme analysis (PRA). Spoligotyping identified the isolate into spoligotype SB0295 on the M. bovis Spoligotype Database website (www.mbovis.org), and it was classified as M. bovis. The occurrence of M. bovis in goats in this study suggests that this species may be a potential source of infection for humans and should be regarded as a possible problem in the advancement of control and eradication program for bovine tuberculosis in Brazil.