Background
Survey of human and bovine tuberculosis infection on dairy farms in southwestern China
Chuang Chena, Zhengyuan Raoa, AiguoYang b, LiGuo b, WeiHou b, LiangZhang c, XiulanYang c, YanLiu d, YumeiWu d, Shu Zhang a*
a Sichuan Center for Disease Control and Prevention, Chengdu, Sichuan, China.
bSichuan Animal Epidemic Prevention and Control Center, Chengdu, Sichuan, China.
c Hongya Center for Disease Control and Prevention, Hongya County, Meishan, Sichuan, China.
d Hongya Center for Animal Epidemic Prevention and Control, Hongya County, Meishan, Sichuan, China.
Abstract
Background: Tuberculosis is a zoonotic chronic infectious disease that can be transmitted through respiratory secretions, causing simultaneous infection in both people and cattle. The aim of our study was to identify the co-infections of Mycobacterium tuberculosis between human and cattle in dairy farms in Sichuan Province, southwest China.
Results: We selected 6 large-scale dairy farms and 5 small-scale dairy farms in Sichuan Province, southwest China, as research sites. A total of 378 dairy farm workers (except one pregnant woman)were screened for tuberculosis symptoms and examined by X-ray radiographs. One worker was diagnosed with tuberculosis, but the sputum culture result was negative, and no strain was obtained. The purified protein derivative (PPD) skin tests of 10224 dairy cows showed that 99 cows were positive, with a positive rate of 9.7‰. The Oesophageal-Pharyngeal (OP) secretions of PPD-positive cows were cultured, and 21 strains were obtained. After amplifying and sequencing the 16SrDNA, hsp65 and rpoB genes and the ITS region, sequence alignment in BLAST showed that these strains were nontuberculous mycobacteria (NTM)(18 Mycobacterium nonchromogenicum, 1 Mycobacterium hiberniae, 1 Mycobacterium arupense and 1 Mycobacterium chitae)..
Conclusion: This study indicated that PPD-positive cows on dairy farms were infected with NTM rather than Mycobacterium bovis. Cross-infection of tuberculosis between humans and cows on dairy farms has been controlled.
Keywords: Zoonotic disease; Nontuberculous Mycobacteria; Susceptibility; Bovine
Background
Tuberculosisis a chronic zoonotic disease caused by the Mycobacterium tuberculosis complex(MTBC), which mainly includes M. tuberculosis(human-like), Mycobacterium bovis, Mycobacterium africanum and Mycobacterium microti. Humans and cattle share similar susceptibility to M. tuberculosis(human strain) and M.bovis[1–4].
As stated in previous reports, the infection status of bovine tuberculosis is grim. Livestock, cattle, especially dairy cows, are the most susceptible bovine to tuberculosis[5].Dairy cows can transmit tuberculosis to workers through oral secretions and feces. Milk secreted by cows with tuberculosis and dairy products that do not undergo pasteurization can also transmit tuberculosis to humans[6–7]. Approximately 3% of tuberculosis patients are infected with M.bovis by drinking milk or dairy products that are not sterilized[8–9]. In Asian and African countries and regions, 4.7% to 30.8% of tuberculosis-positive cattle are infected with the human strain[10]. The more serious the prevalence of human tuberculosis on a farm is, the higher the proportion of cattle infected with human tuberculosis bacteria[11]. According to reports from 2010, the prevalence of bovine tuberculosis in China was 0–15%[12]. This indicates that there were simultaneous infections of tuberculosis in humans and cattle[13].Therefore, we conducted a tuberculosis survey of dairy cows and workers in large and small dairy farms in Sichuan Province, southwestern China, to identify simultaneous infections of tuberculosis in cows and humans.
Results
Population survey
A total of 378 workers in 5 large-scale dairy farms and 6 small-scale dairy farms were screened for symptoms; 377(a pregnant woman was not included)were examined by chest X-ray (DR).The symptom screening results showed that all the workers were asymptomatic; 1 person had an abnormal chest X-ray result, with suspected tuberculosis lesions; all 5 worker samples that were subjected to sputum smear and culture were negative. One patient with active pulmonary tuberculosis was clinically diagnosed and was referred for tuberculosis management and treatment.
Investigation of dairy cows
A total of 10224 dairy cows were examined by PPD skin tests. Ninety-nine cows were PPD positive, but none of them had suspicious symptoms. The OP secretions of 99 PPD-positive cows were cultured on acid LJ medium, and 21 samples were cultured for bacterial growth. The OP secretions from 88 cows that were adjacent to PPD-positive cows and 20 environmental samples were cultured, with negative results.
Identification of species
The sequences of the 16SrDNA, hsp65, and rpoB genes and the ITS region were used 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). The MTBC was not detected.
Drug sensitivity testing results
The antimicrobial susceptibility testing results for the 20 isolates(Mycobacterium arupense did not grow)from the PPD-positive cows are shown in Table 2.
Discussion
The tuberculosis epidemic in China is serious; however, it is better than those in India and Indonesia. Sichuan Province is a populous province in southwestern China, and the epidemic of tuberculosis is very serious in this province. There are also many dairy cows raised in Sichuan Province. Tuberculosis is a zoonotic infectious disease caused by M. tuberculosis. Previous reports have shown evidence of tuberculosis cross-infection between humans and cattle[4,9]. We screened dairy farm workers for tuberculosis symptoms and performed X-ray chest radiographs, and all the cows were screened by PPD skin tests. OP secretions were obtained from PPD-positive cattle; 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 of the PPD-positive cattle were negative. Culture was also used to verify that the samples from OP secretions were not contaminated by environmental tuberculosis.
According to previous reports in Chinese journals, the tuberculosis infection rate of cows on dairy farms in China is 10‰. In our study, the PPD-positive rate was approximately 10‰, which is consistent with previous reports. Through the culture of OP secretions from the PPD-positive cows, 21 isolates were obtained; however, they were recognized as NTM after sequencing and alignment the 16SrDNA, hsp65, rpoB gene and ITS region. This finding indicated that there may be an error in the 10‰ tuberculosis infection rate reported in the previous paper in dairy cows in China, as these cows are most likely infected with NTM. Currently, tuberculosis in dairy cows has been controlled, and NTM is the most likely infection source in PPD-positive cows.
In this study, 21 NTM strains(18 Mycobacterium nonchromogenicum, 1 Mycobacterium hiberniae, 1 M.arupense, and 1 Mycobacterium chitae) were obtained. Research in Northern Ireland also cultured M. nonchromogenicum (a member of the Mycobacterium terrae complex, a nonpigmented, slow-growing species) from cattle, indicating that M. nonchromogenicum is a dominant Mycobacterium of cattle strains[17]. In previous reports, M. nonchromogenicum, M. hiberniae, and M. arupensewere also responsible for human infection and disease[18–22].The minimum inhibitory concentrations (MICs) of 21 kinds of drugs against M. nonchromogenicum, M. hiberniae and M. arupense were relatively high in the drug sensitivity test; in contrast, the MIC of M. chitae was low. Therefore, it is difficult to select appropriate drugs for the treatment of these NTMs. Thus, these MICs of NTMs will provid some suggestions for the further research. We believe that our study makes a significant contribution to the literature because it is the first time to identify that PPD-positive dairy cows were infected with NTM but not Mycobacterium tuberculosis in southwestern China.
Conclusions
Our study indicated that the PPD-positive rate of cows on dairy farms in Sichuan Province which located in southwest China was approximately 10‰, but these cows were infected with NTM rather than Mycobacterium bovis and Mycobacterium tuberculosis. Cross-infection of tuberculosis between humans and cows on dairy farms has been controlled.
Methods
Research sites
The study sites were five large-scale dairy farms and six small-scale dairy farms in Hongya County, Sichuan Province, southwest China.
Investigation methods
All the staff at the investigation sites were screened for clinical symptoms and examined by chest X-ray (DR). Then, sputum smears (night sputum, morning sputum and spot sputum)and sputum cultures were performed for those with suspected tuberculosis symptoms and suspected tuberculosis on chest X-ray. All dairy cows at the survey sites were screened by purified protein derivative (PPD) skin tests (purchased from Zhongmu CO., LTD., Chengdu, China). Then, Oesophageal-Pharyngeal (OP) secretions obtained from dairy cows (PPD-positive cows, cows with suspected tuberculosis symptoms, and adjacent cows of PPD-positive cows) that were fasted (water was provided) for 12 hours were subjected to culture; environmental samples of PPD-positive cows were collected for culture as well.
Culture and DNA extraction
After treatment with 4% NaOH, acid Löwenstein-Jensen (LJ) medium (purchased from Celnovte, CO., LTD., Zhengzhou, China) was used to culture the samples. Each sample was cultured in two tubes at 37°C for 1–2 months.
DNA was extracted by the heating method using the culture strains. The culture strains were suspended in 100 µl of sterile distilled water and then heated at 99°C in a heating block for 20 min. The DNA was stored at –20°C.
Amplification and sequencing of the 16SrDNA, hsp65, and rpoB genes and the ITS region
According to previous reports[14–16], the 16SrDNA, hsp65, and rpoB genes and the internal transcribed spacer (ITS)region were used to design the primers for DNA amplification, which were used to accurately identify the Mycobacterium strains.
16S rDNA PCR
To amplify the 16S rDNA of Mycobacterium, the following primers were used: 16S-F(5’-AGAGTTTGATCCTGGCTCAG–3’) and 16S-R(5’-AAGGAGGTGATCCAGCCGCA–3’). Two microliters of culture-suspended DNA was used as the DNA template in a 50 μl PCR mixture containing 25 μl of 2*Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l) and 21 μl of double distilled water. The PCR cycling parameters were as follows: 95°C for 5 min for initial denaturation; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 58°C, and elongation for 1 min at 72°C; and final extension at 72°C for 10 min.
hsp65 PCR
The primers hsp65-F(5’-ATCGCCAAGGAGATCGAGCT–3’) and hsp65-R(5’-AAGGTGCCGCGGATCTTGTT–3’) were used to amplify the hsp65 gene. The 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2*Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μl of DNA template. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 63°C, and elongation for 1 min at 72°C; and a final extension at 72°C for 10 min.
rpoB PCR
rpob-F(5’-GGCAAGGTCACCCCGAAGGG–3’) and rpob-R(5’-AGCGGCTGCTGGGTGATCATC–3’) were used for the amplification of the rpoB gene. The 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2*Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μlof DNA template of the culture suspensions. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 60°C, and elongation for 1 min at 72°C; and a final extension at 72°C for 10 min.
16SrDNA–23SrDNA internal transcribed spacer(ITS) PCR
ITS -F(5’- AAGTCGTAACAAGGTARCCG –3’) and ITS -R(5’-TCGCCAAGGCATCCACC–3’) were used for the amplification of the ITS gene fragment. A 50 μl PCR mixture contained 21 μl of double distilled water, 25 μl of 2*Taq Master Mix (purchased from Cwbiotech, CO., LTD., China), 1 μl of each forward and reverse primer (10 pmol/l), and 2 μl of DNA of the culture suspensions. The PCR cycling parameters were as follows: initial denaturation at 95°Cfor 5 min; followed by 35 cycles of denaturation for 1 min at 95°C,annealing for 1 min at 60°C, and elongation for 1 min at 72°C; and a final extension at 72°Cfor 10 min.
Sequencing of amplification products of the 16SrDNA, hsp65, and rpoB genes and the ITS region
Gene amplification products of the 16SrDNA, hsp65, and rpoB genes and the ITS region were sequenced at Tsingke(Beijing,China).
Alignment of sequences and identification of species
The sequences of the amplified products(16SrDNA, hsp65, rpoB and ITS) were analyzed on the NCBI BLAST platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by MegaBLAST for species identification.
Drug sensitivity tests
The in vitro drug susceptibility of these strains was evaluated by the broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).
List of abbreviations
MTBC: Mycobacterium tuberculosis complex; PPD: purified protein derivative; OP: Oesophageal-Pharyngeal; NTM: nontuberculous mycobacteria; hsp65: heat shock protein 65; rpoB: β subunit of bacterial RNA polymerase; ITS: 16SrDNA–23SrDNA internal transcribed spacer
Declarations
Ethics approval and consent to participate
This study was authorized by Sichuan Center for Disease Control and Prevention, and the Sichuan Medical Association. It’s ethical approval was obtained from the Research Ethics Committee of Sichuan Center for Disease Control and Prevention. These animals in this study were taken for routine diagnostics and not used for experimental studies, and the material consisted of Oesophageal-Pharyngeal Secretions(OP Secretions) used in this study were collected by local vets after verbal approvals from the owners for further researching. All the staff involved in this study gave the informed consent in oral. According to the review of biomedical research ethics (SC/DL-GZ023) of Sichuan Centers for Disease Control and Prevention, collecting animal sputum culture isolates does not require ethical review. We drafted the written informed consents for the participants, but the owners of the dairy farms believed that oral consent was faster and more convenient, and supported oral consent. The ethical review committee agreed to this practice (Sichuan CDC ethical committee ethical review 4.7: Oral informed consent is allowed when signature would bring trouble to someone), so we used oral informed consent.
Consent for publication
Not applicable.
Availability of data and materials
All data support the results of this paper are included in this published article.
Competing interests
The authors declare that they have no competing interests.
Funding
This study was funded by the Sichuan Medical Association(Grant No. S16066).
Authors’ contributions
Chuang Chen supervised the project. Zhengyuan Rao, Aiguo Yang, Li Guo, Wei Hou, Liang Zhang, XiulanYang, YanLiu, YumeiWu and Shu Zhang were participated in the field investigation. Chuang Chen and Shu Zhang finished the lab work. Shu Zhang drafted and revised the manuscript. All authors read and approved the final manuscript.
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