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

Abstract

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 fetal bovine serum as a source of enrichment in synthetic culture medium for the isolation of Mycobacterium bovis

“…A utilização do Middlebrook 7H11 modificado em camada delgada para visualização precoce de micobactérias já foi realizada em outros estudos (Dib et al, 2006;Marcondes et al, 2006;Mejia et al, 1999;Robledo et al, 2006;Rosário et al, 2014;Silva et al, 2007). O tempo médio para detecção macroscópica das micobactérias em camada delgada, variou entre 12 e 25 dias pós-semeadura, resultado inferior ao normalmente obtido nos meios tradicionais à base de ovos, que podem demorar entre dez a doze semanas (OIE, 2022).…”

“…O meio de Middlebrook 7H11 enriquecido com suplemento OADC (ácido oléico, albumina, dextrose e catalase), permite o isolamento precoce reduzindo o tempo de incubação para três semanas ou menos (Issa et al, 2017). Além disso, permite a visualização das microcolônias (colônias microscópicas) em estágios iniciais e a identificação preliminar baseada nas características morfológicas, quando utilizado em camada delgada (Dib et al, 2006;Marcondes et al, 2006;Mejia et al, 1999;Robledo et al, 2006;Rosário et al, 2014;Silva et al, 2007). No entanto, a baixa qualidade dos suplementos OADC comerciais disponíveis podem inibir o crescimento ideal dos bacilos (Issa et al, 2017), além das dificuldades inerentes a variação das taxas de dólar, e longos períodos necessários para importação, que reduzem as taxas de validade para sua utilização (Sabagh et al, 2012).…”

Influence of the incubation conditions on culture media to optimize primary isolation of Mycobacterium bovis

“…Slopes with loosened screw caps were incubated in air (IC-1) and in air containing 10% CO 2 (IC-2), and slopes closed with hydrophobic cotton were incubated in air. After 24 hours, the lids were tightened, and the cotton was burned and subsequently closed with a cork to create a higher CO 2 tension (IC-3) (ROSÁRIO et al, 2014). The incubation continued up to 90 days.…”

Tuberculosis diagnostic methods in buffaloes

“…Researchers have shown that the use of a Middlebrook 7H11 thin layer agar plate produces a marked difference in the speed of observation of the colonies of M. bovis and M. tuberculosis (MARCONDES et al, 2006). Apparently, in a relatively short period (between 12 to 25 days), it is possible to observe the growth of mycobacteria, which is an advantage over commonly used media, especially when it is associated with the use of polymerase chain reaction (PCR) for detection of microcolonies (ROSÁRIO et al, 2014).…”

“…Tuberculosis in cattle is a public health concern and a potential threat to human health (DAVIES, 2006). Some authors believe that zoonotic tuberculosis is one of the many consequences of the adaptability of the Mycobacterium species to different hosts (ANAELOM et al, 2010), although this is controversial (ROTHSCHILD et al, 2001). …”