Longitudinal monitoring of Mycobacterium bovis and non-tuberculous mycobacteria in cow's milk from a rural community

Abstract

Bovine tuberculosis (Bovine TB) is a chronic respiratory disease of cattle caused by Mycobacterium bovis (M. bovis) which also affects other domestic animals, wildlife and humans (zoonotic TB). Raw milk from bovine TB infected animals can serve as a vehicle for the transmission of M. bovis to humans and the disease is therefore a potential threat to human health and livelihoods. The purpose of the study was to 1. Compare the efficacy of decontamination methods and sensitivity of Mycobacteria spp. for the primary isolation of Mycobacterium spp. from milk using M. fortuitum as a model. 2. To investigate the frequency of M. bovis shedding in milk from bovine TB infected cows at regular intervals. For the first objective, a known concentration (109 cells/mL) of M. fortuitum, E. coli and S. aureus were inoculated into pasteurised milk. E. coli and S. aureus were included as contaminants in the experimental samples as these organisms have previously been detected in milk from communal cattle. The spiked milk aliquots were decontaminated with one of the following methods: 1. 5% Oxalic acid (OA) for 15 min 2. 7% NaCl and 4% NaOH (5 mL each) for 15-20 min and 5 mL phosphate buffer 3. 1% CPC for 5 hours 4. 2% HCl for 10 min and distilled sterile water 5. 4% NaOH for 10 min and washed with distilled sterile water. The frequency of M. bovis and NTM shed in milk was investigated by collecting milk samples from skin test positive cattle between March 2017 to March 2018. A total of 41 TB skin test positive cattle were identified. The collection of milk was done 10 times at different time intervals with a total of 131 milk samples targeting preferentially but not exclusively the same cows. This study showed that M. fortuitum survived and grew confluent on culture, incubated at 37°C for 2 months when 2% HCl and 5% OA is used for decontamination and inoculated on LJ pyruvate resulting in a method of isolating the mycobacteria. The frequency of M. bovis shed in milk could not be determined. This is because samples collected in 2017 were found positive for NTM on culture and cPCR, and only 2 samples were positive for M. bovis on cPCR. Samples collected in March 2018 were positive for M. bovis on cPCR yet negative on culture. 14/121 milk DNA samples were positive for M. bovis whereby 12 were collected in 2018. Two slopes were contaminated on culture hence 77/119 slopes were positive for NTM. Isolates were sequenced and resulted in mycobacteria closely related to Mycobacterium brasiliensis, Mycobacterium sp. py137, Mycobacterium sp. strain EPG1. It is speculated that due to poor nutrition, which resulted in reduced milk production caused by drought experienced in the study area, the concentration of M. bovis within the samples was very low to be detected using cPCR. In addition, NTM as a fast growing organism may have inhibited growth of M. bovis on culture.