Highlighting on Mycobacterium Bovis in cattle, focusing on its antimicrobial resistance

Highlighting on Mycobacterium Bovis in cattle, focusing on its

antimicrobial resistance

El-Gedawy, A .A.*, Abeer Elrefaie**,Yousry A. El-Shazly***,  Mohamed A. Elkady**, Mahmoud A. Abdelrahman** and Marwa Shalaby**

*Bacteriology Dept., Animal Health Research Institute, Dokki. **Bacteriology Dep., AHRI, Mansoura.

***Veterinary Hospital, Faculty of Vet. Med., Zagazig university

ABSTRACT

The current state of multidrug resistance among Mycobacterium bovis (M.bovis) isolates originating from cattle in Egypt underscores the need for control measures based on accurate diagnostic approaches. Conducting antibiogram surveillance of M.bovis using advanced molecular techniques can greatly contribute to the prevention of transmission of multidrug-resistant (MDR) strains to humans. In this study, a total of 153 tuberculous lesion samples were collected from  slaughtered cows in the abattoir  and subjected to bacteriological examination. Acid-fast bacilli were detected in 54.2% of samples using ZN staining, while M.bovis was isolated from 65.4% of samples through culture. Out of the 65 M.bovis isolates confirmed by PCR, 25 showed resistance to at least one anti-tuberculous drug. The resistance rates to EMB, STR, INH, and RIF were 3.1%, 20%, 30.8%, and 32.3% respectively. MDR was detected in 21.5% of isolates. DNA sequencing of six rifampicin-resistant isolates revealed three specific mutations (H526Y, S531L, and D516V). These findings highlight the importance of investigating ante-mortem M.bovis infection in cattle to minimize public health risks associated with bovine tuberculosis

Keywords:

bovine tuberculosis- multidrug-resistant tuberculosis-cattie

INTRODUCTION

Bovine tuberculosis (BTB) is a chronic disease in cattle caused by Mycobacterium bovis (M.bovis), a member of the Mycobacterium tuberculosis complex (MTBC). It has significant economic and public health implications due to its impact on animal production and the potential for human infection (Mohamed 2020). In humans, tuberculosis (TB) is a major global health concern (WHO 2017). M.bovis accounts for approximately 10-15% of TB cases in developing countries. The emergence and spread of multidrug-resistant TB (MDR-TB), particularly resistance to rifampicin (RR), is a serious threat to TB control efforts.

Isoniazid (INH) resistance is caused by different mutations affecting different genes (Vilchèze et al. 2014), while on molecular basis rifampicin (RIF) resistance is less difficult, as absolutely all resistant strains exhibit rpoB gene mutations (Telenti et al. 1993). Moreover, many countries use rifampicin resistance detection by molecular techniques as a potential marker of MDR-TB, because more than 90% of cases resistant to rifampicin exhibit resistance to isoniazid (Mboowa et al. 2014).

This study aimed to isolate MTBC from animal tissue samples obtained after post-mortem examination, confirm M. bovis using PCR targeting Mpb70, assess the drug sensitivity of isolates, and investigate rifampicin resistance mutations through DNA sequencing.

MATERIALS AND METHODS

1.1 Collection of Samples:

A total of 153 tissue samples, exhibiting tuberculous lesions, were gathered from slaughtered cows that tested positive for tuberculin in the abattoir after postmortem examination, following the guidelines set forth by the Egyptian authorities for cattle inspection.

2.1 solation and Identification:

The tissue samples were processed and inoculated into modified LJ medium (Oxoid, England) as instructed by the manufacturer (Marks 1972). Additionally, direct smears of the processed samples were stained with Ziehl-Neelsen (ZN) stain according to the manufacturer's instructions (Kubica 1973)

3.1Confirmation of M.bovis by Conventional PCR Targeting Mpb70:

DNA extraction from 65 selected isolates was conducted using the QIAamp® DNA Mini Kit (Qiagen, Germany) following the manufacturer's guidelines. These isolates were then subjected to PCR using primers designed to target the mpb70 gene: forward (5'-ACCCTCAACAGCGGTCAGTAC-3') and reverse (5'-TTACGCCGGAGGCATTAGCAC-3'). This specific PCR amplifies a 314 bp product that is specific to M.bovis, as described by Zhang et al. (2016).

4.1 Antibiogram by the Gold Standard Agar Proportion Method in Middlebrook 7H11 Medium:

The susceptibility of the 65 M.bovis isolates, confirmed by the presence of Mpb70, to certain anti-mycobacterial drugs was determined using the gold standard agar proportion method in Middlebrook 7H11 medium. This method follows the approved standard (M24A) provided by the Clinical and Laboratory Standards Institute (CLSI 2011) for the susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes.

5.1 PCR and Sequencing for rpoB gene of Some Drug-Resistant M.bovis Isolates:

DNA from six selected isolates showing resistance to rifampicin ( Code N0:M-LN-2, M-LN-9, M-LN-12, S-LN-5, S-LN-7, and Q-LN-8) was amplified using PCR with primers targeting the rpoB gene: forward (5'-GGAGCGGATGACCACCC-3') and reverse (5'-GCGGTACGGCGTTTCGATGAAC-3'). This PCR amplifies a 350 bp product specific for rifampicin-resistant isolates, as described by Siddiqi et al. (2002). The PCR products were purified with the QIAquick PCR Product extraction kit (Qiagen Inc. Valencia CA) and then sequenced in both directions using the Big dye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, FC, CA). Purification of the sequencing reaction was performed using Centrisep (spin column), and the sequencing reaction was carried out on an Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA). Sequence comparisons were conducted following the method described by Thompson et al. (1994). Phylogenetic analysis utilizing neighbor joining, maximum likelihood, and maximum parsimony methods in MEGA6 was performed, as outlined by Tamura et al. (2013)