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      Genotyping of Brucella strains isolated from humans and cattle of different geographical regions of Pakistan using MLVA‐15

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          Abstract

          Background

          Prevalence of brucellosis and MLVA genotyping in animals and humans, isolated from different regions of Pakistan was performed. Animals having history of brucellosis from the field and local farms of Bannu, Mardan, Peshawar, Swat, Lahore and Islamabad were selected for blood collection. Humans that work with them were also selected for sampling in this study. Total of 600 samples were taken from cattle and humans and subjected to Rose Bengal plate Test (RBPT) for the initial screening of positive samples. Designed primers of B.abortus for cattle and B.melitensis for humans were utilised to perform PCR. Culturing and isolation was carried to further to perform MLVA genotyping assay through the selection of two panels of primer markers.

          Results

          RBPT showed more number of cases of brucellosis in animals and humans compared to the PCR findings. Genotyping findings based upon MLVA‐15 set of markers demonstrated that the isolated strains of B.abortus fall in the same clade with strain A1, P8 and A2 from Pakistan and also similar with BCCN#02‐45 strain from India. On the other hand, B.melitensis isolated from different districts of Pakistan shared the same clade with BwIM‐AFG 63, BwIM‐AFG 44 strains from Afghanistan and BwIM IRN 37 strain from Iran. Selected VNTR alleles were sequenced for calibration purposes.

          Conclusion

          It is concluded that B rucella is prevalent in animals and humans in studied districts of Pakistan. Moreover, A1, P8, BwIM‐AFG 63, BwIM‐AFG 44 and A2 were found the common genotypes in Pakistan.

          Abstract

          Biovars; zoonotic; MLVA; Pakistan; Brucella; genotyping

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          Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity.

          P Hunter, M Gaston (1988)
          An index of discrimination for typing methods is described, based on the probability of two unrelated strains being characterized as the same type. This index may be used to compare typing methods and select the most discriminatory system.
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            Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

            Philippe Le Flèche, Isabelle Jacques, Maggy Grayon … (2006)
            Background The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. Results Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). Conclusion The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.
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              Colonization Dynamics of Cefotaxime Resistant Bacteria in Beef Cattle Raised Without Cephalosporin Antibiotics

              Raies A. Mir, Thomas Weppelmann, Lin Teng … (2018)
              The emergence of infections caused by antimicrobial resistant microorganisms (ARMs) is currently one of the most important challenges to public health and medicine. Though speculated to originate at least partially from the overuse of antibiotics during food animal production, we hypothesized that cattle are exposed to ARMs in the environment. In this cohort study, a herd of beef calves with no previous exposure to antibiotics was followed during the first year of life in order to investigate the rate of colonization by bacteria resistant to the third-generation cephalosporin cefotaxime. Fecal samples were collected from the recto anal junction of cattle at the age of ~3, 6, 9, and 12 months and tested for cefotaxime resistant bacteria (CRB) and the presence of extended spectrum β-lactamases (ESBLs). The colonization dynamics of CRB in calves (n = 188) was evaluated with samples collected from four periods using longitudinal statistical analyses. Colonization by CRB was a dynamic process with over 92% of the calves testing positive for CRB at least once during the first year of life. All isolates subjected to antimicrobial susceptibility test were resistant to at least four different antibiotics and carried multiple variants of the blaCTX-M genes. Metagenomic analysis revealed significant differences in microbiota of the calves with and without CRB colonization at different ages. This study provides evidence that colonization of beef calves by ARMs is a dynamic process that can occur in the absence of veterinary or agricultural use of antibiotics.
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                Author and article information

                Contributors
                Muhammad Muddassir Ali:
                ORCID: https://orcid.org/0000-0001-7930-7782
                muddassir.ali@uvas.edu.pk
                Journal
                Journal ID (nlm-ta): Vet Med Sci
                Journal ID (iso-abbrev): Vet Med Sci
                Journal ID (doi): 10.1002/(ISSN)2053-1095
                Journal ID (publisher-id): VMS3
                Title: Veterinary Medicine and Science
                Publisher: John Wiley and Sons Inc. (Hoboken )
                ISSN (Electronic): 2053-1095
                Publication date (Electronic): 10 July 2021
                Publication date Collection: September 2021
                Volume: 7
                Issue: 5 ( doiID: 10.1002/vms3.v7.5 )
                Pages: 1688-1695
                Affiliations
                [ 1 ] Department of Pathology University of Veterinary and Animal Sciences Lahore Pakistan
                [ 2 ] Institute of Biochemistry and Biotechnology University of Veterinary and Animal Sciences Lahore Pakistan
                [ 3 ] Department of Theriogenology University of Veterinary and Animal Sciences Lahore Pakistan
                [ 4 ] Planning and Development Division Pakistan Agriculture Research Council Islamabad Pakistan
                Author notes
                [*] [* ] Correspondence

                Muhammad Muddassir Ali, Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan.

                Email: muddassir.ali@ 123456uvas.edu.pk

                Author information
                https://orcid.org/0000-0001-7930-7782
                Article
                Publisher ID: VMS3550
                DOI: 10.1002/vms3.550
                PMC ID: 8464260
                PubMed ID: 34245235
                SO-VID: 22086638-9b7d-46b7-9a98-7368f95ea3de
                Copyright © © 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.
                License:

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                Page count
                Figures: 5, Tables: 1, Pages: 8, Words: 4274
                Funding
                Funded by: Pakistan Agricultural Research Council Agricultural Linkages Programme: AS 123; Higher Education Commission Pakistan: HEC‐TDF 02‐023
                Categories
                Subject: Original Article
                Subject: Original Articles
                Custom metadata
                source-schema-version-number 2.0
                cover-date September 2021
                details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.8 mode:remove_FC converted:25.09.2021

                Keywords: biovars,brucella,genotyping,mlva,pakistan,zoonotic
                Data availability:
                Keywords: biovars, brucella, genotyping, mlva, pakistan, zoonotic

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