Research sites
The study sites were in Hongya County, Sichuan Province, southwestern China. All dairy farms in this county were in this survey, including five large-scale dairy farms and six small-scale dairy farms.
Investigation methods
All the staff at the investigation sites were screened for clinical symptoms (coughing lasts two or more weeks, coughing up blood, chest pain, unintentional weight loss, Fatigue, Fever, night sweats, chills, loss of appetite) and examined by chest X-ray (DR). 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 single intradermal cervical comparative test (SICCT) using purified protein derivative (PPD) of Mycobacterium bovis tuberculin(purchased from Zhongmu CO., LTD., Chengdu, China). The cows were inoculated with 0.1ml of bovine PPD in the position of anterior neck, the results were computed as the difference in skin fold thickness in millimeters(mm) before and 72(±4) hours after injection of the bovine PPD. The skin fold thickness increased 4 mm or more, or severe local clinical signs(pain, edema, necrosis, exudation, emphysema, swelling, and/or local lymph node hypertrophy) were observed, it was considered the reactor to be positive.
Esophageal-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 cultured; 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 from the cultured strains by the heating method. The culture strains were suspended in 100 µl of sterile distilled water and heated at 99°C in a heating block for 20 min. The DNA obtained was stored at -20°C.
Amplification and sequencing of the16S rDNA, hsp65, and rpoB genes and ITS region
Primers were designed to amplify the 16S rDNA, hsp65, and rpoB genes and internal transcribed spacer (ITS) region to accurately identify Mycobacterium strains,vaccording to previous reports[19-21].
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 a final extension at 72°C for 10 min.
hsp65PCR
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°C for 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.
rpoBPCR
rpob-F(5’-GGCAAGGTCACCCCGAAGGG-3’) and rpob-R(5’-AGCGGCTGCTGGGTGATCATC-3’) were used for 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 μl of DNA template from the culture suspensions. The PCR cycling parameters were as follows: initial denaturation at 95°C for 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.
16S rDNA-23S rDNA internal transcribed spacer(ITS) PCR
ITS-F(5’-AAGTCGTAACAAGGTARCCG-3’) and ITS-R(5’-TCGCCAAGGCATCCACC-3’) were used for amplification of the ITS gene fragment. The50-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°C for 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.
Sequencing of 16S rDNA, hsp65, and rpoB genes and the ITS region amplification products
Gene amplification products of the16S rDNA, hsp65, and rpoB genes and the ITS region were sequenced by Tsingke (Beijing, China).
Alignment of sequences and identification of species
The sequences of the amplified products (16S rDNA, hsp65, rpoB and ITS) were analyzed using the NCBI BLAST platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with MegaBLAST for species identification.
Drug sensitivity tests
The in vitro drug susceptibility of the strains was evaluated by the broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).