Comparison of decontamination methods for primary isolation of Mycobacterium bovis in paucibacillary bovine tissues

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Abstract
Aims: To compare three decontamination methods applied to paucibacillary samples for primary isolation of Mycobacterium bovis from suspect lesions. Tuberculosis caused by Myco. bovis is an important infectious disease of cattle in Brazil and also has zoonotic potential. Although a national campaign based on testing and slaughtering cattle has achieved good results, there is a strong need to develop better diagnostic methods to identify cattle with recent infections harbouring few bacilli. Methods and Results: A dairy herd (274 adult crossbred cows) located in the state of Rio de Janeiro was tested for tuberculosis with both single intradermal tuberculin test and comparative intradermal tuberculin test. Reactive cows (n = 27, 9·8%) were slaughtered and suspect lesions were collected (one sample per cow). Samples considered paucibacillary (based on microscopy) were decontaminated with 0·75% hexadecylpyridinium chloride (HPC), 4% sodium hydroxide (Petroff) or 6% sulphuric acid. Using these methods, 10, five and six, respectively, of the 27 samples yielded positive cultures. Overall, Myco. bovis was isolated from 14 of 24 cows. Although the HPC method resulted in isolation of more Myco. bovis strains than either Petroff or sulphuric acid methods (P = 0·015), it did not result in the recovery of Myco. bovis from all samples. However, using both HPC and 6% sulphuric acid methods for decontamination was possible to identify 13 of 14 (92·9%) of infected cows. Conclusions: At least two methods should be used concurrently for primary isolation of Myco. bovis from bovine tissues, particularly for paucibacillary samples. Significance and Impact of the Study: Detection of low numbers of Myco. bovis in tissue is an important goal in optimizing the detection of bovine tuberculosis and should assist in identification of infected cattle, in particular, those with few Myco. bovis bacilli. This was apparently the first study comparing three decontamination methods for the detection of Myco. bovis in paucibacillary samples from naturally infected cattle.