Survey of human and bovine tuberculosis infection on dairy farms in southwestern China

Abstract Background: Tuberculosis is a zoonotic chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC) that can be transmitted between humans and cattle. The aim of our study was to identify cross-infections of MTBC between humans and cattle on dairy farms in Sichuan Province, southwestern China. Results: We selected 6 large-scale and 5 small-scale dairy farms in Sichuan Province as research sites. A total of 378 dairy farm workers (except one pregnant woman) were screened for tuberculosis symptoms and examined by X-ray. One worker was diagnosed as tuberculosis, though the sputum culture was negative. In total, 99 of 10,224 (0.97%) cows showed positive results for the purified protein derivative (PPD) skin tests. Esophageal-pharyngeal (OP) secretions from PPD-positive cows were cultured and 21 isolates were obtained. Sequences of 16s rDNA , hsp65 and rpoB and 16s-23s rRNA spacer region were amplified and sequenced. BLAST analysis classified these isolates as nontuberculous mycobacteria (NTM) (18 M. nonchromogenicum , 1 M. hiberniae , 1 M. arupense, and 1 M. chitae isolates). Conclusion: This study indicates that the PPD-positive cows in these dairy farms were infected with NTM rather than MTBC. Tuberculosis cross-infection between humans and cows on dairy farms has been well controlled in this region.

samples collected from 5 workers (one pregnant woman; one abnormal chest X-ray person; three slight cough persons) were subjected to sputum smear and culture, and all samples showed negative results. One patient with abnormal chest X-ray result was clinically diagnosed and referred for tuberculosis management and treatment.

Investigation of dairy cows
A total of 10,224 cows were examined by PPD skin tests. Ninety-nine cows were PPD positive, but none of them displayed suspicious symptoms. Esophageal-pharyngeal (OP) secretions of the 99 PPD-positive cows were cultured on acid LJ medium, 21 bacteria isolations were obtained from these samples. OP secretions from 88 PPD-negative cows and 20 environmental samples that were adjacent to PPD-positive cows were also cultured, and all samples showed negative results.

Identification of species
The sequences of the 16S rDNA, hsp65, and rpoB genes and ITS region were sequenced to identify species by multilocus sequence analysis. The species of the 21 strains are presented in Table 1. All of the strains were nontuberculous mycobacteria (NTM).

Drug sensitivity testing results
Antimicrobial susceptibility testing results for the 20 isolates(Mycobacterium arupense did not grow)from the PPD-positive cows are shown in Table 2.

Discussion
According to Global Tuberculosis Report released by the WHO in recent years, the tuberculosis epidemic in China is high; however, it is not as severe as those in India and Indonesia. Sichuan Province is a populous province in southwestern China, and the epidemic of tuberculosis in this province is very concerning. In addition, many dairy cows raised in Sichuan Province. Tuberculosis is a zoonotic infectious disease caused by MTBC.
Previous reports have shown evidence of tuberculosis cross infection between humans and cattle[1,2,6,8].
We screened dairy farm workers for tuberculosis symptoms and performed chest X-ray; all cows were screened by PPD skin tests. OP secretions were obtained from PPD-positive cattle, and the cows were fasted for 12 hours (with water accessible) to ensure that the OP secretions were not contaminated by ruminate. The results of the environmental sample cultures for the PPD-positive cattle were negative. Culture was also used to verify that the samples from OP secretions were not contaminated by environmental mycobacteria.
According to previous Chinese reports, the infection rate of MTBC in cows on dairy farms in China is 1% by the PPD methods. In our study, the PPD-positive rate was approximately 1% ,too, which is consistent with previous reports. Through the culture of OP secretions from PPD-positive cows, 21 isolates were obtained; however, these isolates were classified as NTM after 16S rDNA, hsp65, rpoB gene and ITS region sequencing and alignment. This finding indicates that there may be an error in the 1% MTBC infection rate reported in the previous paper mentioned above, as these cows were likely infected with NTM. According to our current data, tuberculosis in dairy cows has been well controlled, and NTM is the most likely infection source in PPD-positive cows. So, it is important to confirm the true MTBC infection rate of dairy cows in China by culture and PCR.
In this study, 21