Area of the study
This study was conducted from January 1st to March 31st 2022, confined to five different districts in Sulaimani province, including Tanjaro, Bakrajo, Peramagrun, Chwarta, and Sharazor. These areas include nearly 273 villages and contain 38661 cattle. Around 414 healthy cows were randomly tested, representing about 1% of the target population, according to the Veterinary Directory of Sulaimani (Table 1).
Table 1
The study area, the target population, and the number of samples.
District | Number of villages | Bovine population | Number of samples |
Tanjaro | 60 | 5883 | 68 |
Bakrajo | 65 | 12670 | 130 |
Peramagrun | 58 | 13391 | 134 |
Chwarta | 48 | 2732 | 32 |
Sharazwr | 42 | 3985 | 50 |
Total | 273 | 38661 | 414 |
Sample Collection For Serology And Histopathology
About 10 mL of blood samples were collected from each cow for quantitative interferon-gamma (IF-ℽ) detection using ELISA test special kit (TB-Feron ELISA Plus, BioNote company, South Korea). Then, sera were obtained and frozen at -20° C until use. Conversely, the lymph node (LN) tissues were collected from the cow at the Karagol slaughterhouse in Sulaimani province. LN was removed aseptically and examined for tuberculosis lesions (enlargement and necrosis). Histopathological examination was performed after the tissue fixation in a 10% neutral buffer formalin. Then, LN were embedded in paraffin wax and stained with hematoxylin & eosin.
Human Samples
For human tissues, 12 paraffin-embedded LN tissues that were previously diagnosed as positive for mycobacterium species were obtained from Shorsh General Hospital and Smart Health Towers, Sulaimani province. First, the tissue samples from each paraffin block were obtained by sectioning (10 mm thickness) using a microtome (R. Jung/Heidelberg, Germany), then paraffin was removed [20, 21] and dewaxed with xylene and absolute ethanol and finally preserved at -20º C for extracting genomic DNA.
Pcr Assay
DNA samples were extracted from 12 obtained human deparaffinized LN tissues (25 mg) and three cattle LN using a DNA extraction kit (AddPrep Genomic, Korea). Then two sets of specific primers were selected, encoding CSB2 (168 bp) (F5’-TTCCGAATCCCTTGTGA-3’); (R-5’-GGAGAGCGCCGTTGTA-3’) [22], and oxyR (548-bp) specific primers to distinguishing M. bovis isolates from other members of Mycobacterium (F-5’-GGTGATATATCACACCATA-3’); (R-5’-CTATGCGATCAGGCGTACTTG-3’) [23]. The PCR master mix was prepared by adding 1.0 µL of each forward and reverse primer (CSB2 and oxyR separately), 8.0 µL of the DNA sample, and the volume was completed to 20 µL using distilled water. The PCR cycles included an initial denaturation for 5 min at 94° C, followed by 35 cycles of denaturation (30 sec at 94° C), annealing (1 min at 53.3° C for CSB2 and one min at 54º C for oxyR), pre-extension (1 min at 72° C), and final extension for 5 min at 72° C. Later on, the PCR product (10 µL) from each gene was stained with 7.0 µL Gel stain dye (Safe Gel Stain Dye, Taiwan) and run on a 1.8% agarose gel using a Gel electrophoresis system (Cleaver, UK) at an electrical potential of 90 volts for 60 min. DNA bands were visualized under ultraviolet light using a molecular imager (UV Transilluminator, Ingenious, USA). The size of the amplified DNA strand was estimated by comparing it with a 100 bp DNA ladder (GenDirex, USA).
Sequencing Of Pcr Products And Phylogenetic Construction
The CSB2 genes, including sh1, sh2, sh3, and sh4 and oxyR genes, including Sul-3, Sul-2, and Sul-1, were selected for sequencing at the Macrogene genome centre in South Korea. Then, sequence alignment was performed using the Applied Biosystems 3500 Genetic Analyzer program. The result was published in GenBank under accession numbers of OP716210, OP716211, OP716212, OP716213 for sh1, sh2, and sh3 and OP503571, OP503572, OP503570 for Sul-3, Sul-2, and Sul-1. The sequence of the CSB2 and oxyR genes of the samples was comparatively analyzed, and a phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 10 software (version 10.0.5) [24]. In the bootstrap test (1000 replicates), the percentage of duplicate trees in which the related taxa clustered together was determined [25]. The Kimura 2-parameter method was used to calculate the evolutionary distances [26].
Statistical analysis
All analyses were done using SPSS, version 25. The histograms were used to determine the normal distribution status of the data. The chi-square test was used to compare variables, while frequency and percentage were calculated for categorical data. Mean ± SD estimated for numerical variables. P < 0.05 was considered statistically significant.