Volume 23, Issue 6 (Feb - Mar 2020)                   2020, 23(6): 526-539 | Back to browse issues page

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Ghaderi H, Haghkhah M, Mosavari N, Tadayon K. Isolation, Molecular Identification and Genomic Pattern of Mycobacterium Bovis Isolates Collected from Tuberculin-positive Cattle in Infected Farms of Shiraz, Iran. Journal of Inflammatory Diseases. 2020; 23 (6) :526-539
URL: http://journal.qums.ac.ir/article-1-2888-en.html
1- Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
2- Reference Laboratory for Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran. , nmosavari@gmail.com
3- Reference Laboratory for Bovine Tuberculosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.
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Extended Abstract
1. Introduction

Bovine tuberculosis has been known for a long time as the most important zoonotic disease and a mater of concern in the dairy industry worldwide [1]. The mycobacterium tuberculosis complex (MTC) consists of a number of species and subspecies that can cause tuberculosis in humans or animals [3, 4]. One of the globally important members of this complex is Mycobacterium bovis (M. bovis) which is the leading cause of tuberculosis in cattle [7]. Tuberculin test is the most commonly used method for detection of M. bovis in cattle [8]. Restriction fragment length polymorphism (RFLP) method using IS6110, direct repeat (DR) and Polymorphic Guanine/cytosine-rich Repetitive Sequences (PGRS) markers have been among the earliest strain typing systems used for genotyping of the M. bovis [12]. The present study aimed to improve the genomic knowledge of the population structure of M. bovis isolates collected from tuberculin-positive cattle in infected cattle farms of Shiraz, Iran.
2. Materials and Methods
In this descriptive cross-sectional study, conducted within 13 months (from January 2016 to February 2017), pathological samples were collected from 50 slaughtered tuberculin-positive cattle belonged to 6 cattle farms located in Shiraz, Iran which had been sent to two abattoirs. The frozen samples were then transferred to Bovine Tuberculosis Reference Laboratoriy of Razi Vaccine and Serum Research Institute in Karaj, Iran. Using sterile scissors, scalpels and forceps, the samples with tuberculosis lesions were dissected. The small cut tissue pieces were then homogenized using sterile sea sand, mortar and pestle. Ten ml of NaOH (4%), according to the Petroff’s decontamination method, was added to the homogenates with continuous but gentle stiring of the pestle content using the mortar for 20 minutes to ensure the completion of decontamination process. After 20 minutes of settling, 5 mL of supernatant was transferred to a Falcon tube and centrifuged at 5,000 x g for  15 minutes. The supernatant was removed and the centrifuge deposit was used to inoculate traditional (glycerinated) and pyruvated (0.2% sodium pyruvate) Lowenstein-Jensen media [19, 20]. The genomic contents of the culture-positive slopes were extracted using the Van Soolingen method [1] and subjected to polymerase chain reaction (PCR)-16SrRNA, PCR-IS6110, and the quad PCR-regions of difference (RD) (RD1, RD4, RD9 and RD12) protocols [21-23]. The detected MTC isolates were further digested by PvuII restriction enzyme and the strain was typed by PGRS-RFLP technique. The obtained patterns were evaluated by the Gel-Pro Analyzer [24].
3. Results
Of 50 study samples, 13 (26%) had mycobacterial growth-positive cuture. The PCR-16SrRNA and PCR-IS6110 results (Figure 1) confirmed their identity as MTC bacteria. No other MTC members execpt M. bovis was detected in PCR-RD typing between the isolates (Figure 2). All isolates were typeable by RFLP strain typing method. The genotype profiling of M. bovis isolates resulted in detection of two patterns among which 10 isolates (76.92%) shared a single profile identical to that of M. bovis Bacillus Calmette-Guerin (BCG) strain (1173 P2; Figure 3A), while the remaining three isolates (23.08%) displayed a different genotype (Figure 3B). Although it has been previously reported in Iran (Urmia City), but this type seems to be new in Shiraz City. 
4. Discussion
Our results showed a relatively high prealence (77%) of the BCG-like M. bovis isolates in cattle farms of Shiraz city which is consisting with previous studies in Iran [9, 17, 30]. Finding homogeneous population of M. bovis isolates in Shiraz and in other cities of Iran at a larger scale indicates the local evolution of new M. bovis strains in the region or the entery of such strains through livestock production, especifically cattle farming.
Ethical Considerations
Compliance with ethical guidelines
The present study used the biological samples and tissues prepared from abattoirs by the inspectors of the Veterinary Department in Shiraz. No any experiment was performed on living animals. 
This study was extracted from the PhD. thesis of the first author approved by the School of Veterinary Medicine at Shiraz University. It received financial support from the Razi Vaccine and Serum Research Institute (Karaj, Iran) (Code: 95GCU4M1304). 
Authors' contributions
Conceptualization: Nader Mosavari, Masoud Haghkhah, Keyvan Tadayon; Field and laboratory experiments: Hossein Ghaderi; Data analysis, draft preparation: Nader Mosavari, Keyvan Tadayon, Hossein Ghaderi; Editing and review: Keyvan Tadayon.
Conflicts of interest
The authors declared no conflict of interest.
The authors would like to thank the staff of Deputy of Tuberculin and Mallein Research and Production Department of Razi Vaccine and Serum Research Institute and the School of Veterinary Medicine at Shiraz University for their cooperation in conducting this study. 
Type of Study: Research | Subject: Bacteriology

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