Comparative study of Mycobacterium bovis primary isolation methods

Abstract

For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants – 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media – 7H11 Middlebrook with additives and OADC supplement “A” (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement “B”.

Microorganisms and heavy metals associated with atmospheric deposition in a congested urban environment of a developing country: Sri Lanka

“…Appearance of Mycobacteria spp. in Lowenstain-Jensen medium and in Middlebrook 7H11 require more than 4 weeks) (de Azevedo Issa et al, 2016;Palange et al, 2016). The ability to get bacteria into culture gradually decreases with aerosolisation and with increased HM concentrations (Kaushik and Balasubramanian, 2012).…”

Mycobacterium bovis Tuberculosis in Two Goat Farms in Multi-Host Ecosystems in Sicily (Italy): Epidemiological, Diagnostic, and Regulatory Considerations

“…This limitation is mainly related to analytical issues (homogenization, decontamination, culture medium, etc. ), to the sample characteristics (e.g., extent of necrosis and calcification), and to the bacterial load [23,24]. Culture sensitivity appears to be around 80%, and the parallel use of solid and liquid culture media can improve it [25,26].…”

Multispacer Sequence Typing for Mycobacterium bovis Genotyping

“…These isolates were obtained from cattle herds located in six Brazilian states [Minas Gerais ( n = 48), Goiás ( n = 3), Mato Grosso-MT ( n = 1), Rio Grande do Sul-RS ( n = 1), Paraná-PR ( n = 1) and São Paulo ( n = 4)] between the years 2006 and 2010. First isolation of these M. bovis from clinical samples were performed in Lanagro/MG according to previous publications ( 34 ). Isolated strains were identified as M. bovis by using a PCR assay described by Sales et al ( 35 ).…”