MOLECULAR CHARACTERIZATION SOME BACTERIAL PATHOGENS CAUSING BOVINE MASTITIS WITH SPECIAL REFERENCE TO Mycoplasma bovis
DOI:
https://doi.org/10.26873/SVR-1581-2022Keywords:
mastitis, multiplex RT- PCR, M. bovis, sequence, molecular diagnosticsAbstract
In dairy industry, bovine mastitis is the most prevalent disease, which reduces milk production and causes economic losses. This study was conducted to estimate the prevalence of Mycoplasma bovis and some bacteria causing mastitis in dairy farms and partial sequencing of 16SrRNA target genes and Quinolones Resistance Determining Regions (QRDRs) (gyrA and parC) in M. bovis isolates. 370 milk samples were obtained from farms located in villages in Fayoum governorate, Egypt. The examined milk samples (8,91%) were positive for the California mastitis test (CMT). Multiplex RT-PCR was used for the recognition of microorganisms causing mastitis (Staphylococcus (S.) aureus, Streptococcus species (spp.), Escherichia (E.) coli, and Mycoplasma (M.) bovis) from mastitic milk. The results revealed that E. coli was the most predominant (84.8%) followed by S. aureus (81.8%) while M. bovis was the lowest one (51.5%). Mixed infection with two or more mastitic bacterial agents was also identified. All 33 examined mastitic milk samples were diagnosed with mixed infection with E. coli, S. aureus, Streptococcus spp. and M. bovis (36.36%), E. coli and S. aureus (21.21%), and rephrase E. coli, M. bovis, and Streptococcus spp. (6.06%). The sequence analysis of M. bovis 16SrRNA genes illustrated a high similarity of examined isolates to strains previously deposited in the GenBank recovered from the same locality. The gyrA amino acids showed no substitution but showed 100% similarity with M. bovis isolates worldwide. However, the amino acid sequence of parC, showed substitution at positions 2 (Gln to Arg) (CAG >>CGT), 75 (Ile to Ser) (ATT>>AGC), and 79 (Asn to Asp) (AAC>>GAT). Sequence results can lead to the creation of appropriate treatment and control measures for M. bovis, while multiplex RT-PCR, can be exploited as a standard diagnostic method for major mastitis pathogens.
References
● 1. Wenjuan HE, Ma S, Lei L, et al. Prevalence, etiology, and economic impact of clinical mastitis on large dairy farms in China. Veterinary microbiology, 2020; 242: 108570.
● 2. Dalanezi FM, Joaquim SF, Guimarães FF, et al. Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows. Journal of Dairy Science, 2020; 103 (4): 3648–55.
● 3. Ayvazoğlu Demir P, EŞKİ F. Estimate by Quantitative Methods of the Effect on Some Milk Yield Traits with CMT Score of Subclinical Mastitis in Cows: Pilot Study. Van Vet J 2019; 30 (3) 177–82.
● 4. Krishnamoorthy P, Suresh K P, Jayamma, K S, et al. An understanding of the global status of major bacterial pathogens of milk concerning bovine mastitis: a systematic review and meta-analysis (Scientometrics). Pathogens 2021; 10 (5): 545.
● 5. Almaw GA, Zerihun A, and Asfaw Y. Bovine mastitis and its association with selected risk factors in smallholder dairy farms in and around Bahir Dar, Ethiopia. Tropical animal health and production 2008; 40 (6): 427–2.
● 6. Bradley AJ. Bovine mastitis: an evolving disease. The veterinary journal 2002; 164 (2): 116–28.
● 7. Capurro A, Aspan A, Unnerstad H E , et al. Identification of potential sources of Staphylococcus aureus in herds with mastitis problems. Journal of dairy science 2010; 93(1): 180–91.
● 8. Hata E, Katsuda K, Kobayashi H, et al. Characteristics and epidemiologic genotyping of Staphylococcus aureus isolates from bovine mastitic milk in Hokkaido, Japan. Journal of veterinary medical science 2006; 68 (2): 165-–70.
● 9. Reinoso EB, Lasagno MC, Dieser SA, et al. Distribution of virulence-associated genes in Streptococcus uberis isolated from bovine mastitis. FEMS microbiology letters 2011; 318 (2): 183–8.
● 10. Green MJ, Green LE, Bradley AJ, et al. Prevalence and associations between bacterial isolates from dry mammary glands of dairy cows. Veterinary record 2005; 156 (3): 71–7.
● 11. Calcutt M.J, Lysnyansky I, Sachse K, et al. Gap analysis of Mycoplasma bovis disease, diagnosis and control: an aid to identify future development requirements. Transboundary and emerging diseases 2018; 65: 91–109.
● 12. Abd El Tawab AA, El-Hofy FI, Hassan NI, et al. Prevalence of Mycoplasma bovis in bovine clinical mastitis milk in Egypt. Benha Veterinary Medical Journal 2019; 36 (2): 57–5.
● 13. Fox LK. Mycoplasma mastitis: causes, transmission, and control. Veterinary Clinics: Food Animal Practice 2012; 28 (2): 225–37.
● 14. Sindhu N, Sharma A and Jain V K. Molecular detection of Staphylococcus aureus mastitis in crossbred cows based on genus specific gap gene and species specific aroA gene PCR assay. Indian Journal of Animal Sciences 2010; 80: 275–78.
● 15. Mweu MM, Toft N, Katholm J, et al. Evaluation of two herd-level diagnostic tests for Streptococcus agalactiae using a latent class approach. Veterinary microbiology 2012; 159 (1-2): 181–6.
● 16. Charaya G, Sharma A, Kumar A, et al. Detection of major mastitis pathogens by multiplex polymerase chain reaction assay in buffalo milk. Indian J Anim Sci 2015; 85 (3): 122–5.
● 17. Baştan A, Kaçar C, Acar DB, et al. Investigation of the incidence and diagnosis of subclinical mastitis in early lactation period cows. Turkish J Vet Anim Sci 2008; 32: 119–21.
● 18. Naikare H, Bruno D, Mahapatra D, et al. Development and evaluation of a novel Taqman real-time PCR assay for rapid detection of mycoplasma bovis: comparison of assay performance with a conventional PCR assay and another Taqman real-time PCR assay Veterinary Sciences 2015; 2: 32–42. (Multidisciplinary Digital Publishing Institute)
● 19. El-Demerdash A, Bakry N, Aggour M, et al. Bovine mastitis in Egypt: bacterial etiology and evaluation of diagnostic biomarkers. Int J Vet Sci 2023; 12(1): 60–9. https://doi.org/10.47278/journal.ijvs/2022.161
● 20. Goto M, Takahashi H, Segawa Y, et al. Real-time PCR method for quantification of Staphylococcus aureus in milk Journal of food protection 2007; 70: 90–6. (Allen Press)
● 21. Hogan S, González N, Harmon J, et al. Laboratory Handbook on Bovine Mastitis. 1999; Revised Edition, National Mastitis Council, Madison, WI, USA.
● 22. Alberti A, Addis M, Chessa B, et al. Molecular and Antigenic Characterization of a Mycoplasma Bovis Strain Causing an Outbreak of Infectious Keratoconjunctivitis. Journal of Veterinary Diagnostic Investigation 2006; 18 (1): 41–51. https://doi.org/10.1177/104063870601800106
● 23. Yleana R, Chave Gonzalez C, Goran C, et al. In vitro amplification of the 16S rRNA genes from Mycoplasma agalactiae by PCR. Vet Microbiol 1995; 47: 183–90.
● 24. Hata E, Harada T, Itoh M. Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin. Applied and Environmental Microbiology 2019; 85 (13), May 3, https://doi.org/10.1128/AEM.00575-19
● 25. Altschul S, Gish W, Miller W, et al. Basic Local Alignment Search Tool. J Mol Biol 1990; 215: 403–10.
● 26. Sudhir K, Glen S, Michael L, et al. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 2018; 35: 1547–9.
● 27. Nicholas RA, Fox LK, Lysnyansky I. Mycoplasma mastitis in cattle: to cull or not to cull. Vet J 2016; 216: 142–7.
● 28. Khan J, Rasool M, Arshad M, et al. Comparative Evaluation of Leukotoxic Activities of Indigenous Staphylococcus aureus Isolates from Subclinical and Clinical Mastitic Milk Samples of Buffalo and Cattle. Open Vet J 2013; 7: 24–7.
● 29. Koskinen MT, Holopainen J, Pyörälä S, et al. Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens. J Dairy Sci 2009; 92 (3): 952–9. doi: PMID: 19233788
● 30. OUDA S E, El-Shafii S S, and Hefny EG. Studies on bacterial infection of cow's milk with special reference to Mycopasma Bovis Recoverd from marketing and mastitic milk. Egyptian Journal of Chemistry and Environmental Health 2016; 2 (2): 516–33.
● 31. Katholm J, Bennedsgaard T, Koskinen M, et al. Quality of bulk tank milk samples from Danish dairy herds based on real-time polymerase chain reaction identification of mastitis pathogens. J Dairy Sci. 2012; 95(10):5702–8. doi: PMID: 22921631.
● 32. Algammal A, Enany M, El-Tarabili R , et al. Prevalence, antimicrobial resistance profiles, virulence and enterotoxins-determinant genes of MRSA isolated from subclinical bovine mastitis in Egypt. Pathogens 2020; 9 (5): 362.
● 33. Awad A, Ramadan H, Nasr S, et al. Genetic Characterization, Antimicrobial Resistance Patterns and Virulence Determinants of Staphylococcus aureus Isolated form Bovine Mastitis. Pakistan Journal of Biological Sciences PJBS 2017; 20: 298–305. https://10.3923/pjbs.2017.298.305
● 34. Hande G, Arzu F, Nilgün G, et al. Investigation on the etiology of subclinical mastitis In Jersey And Hybrid Jersey dairy cows. Acta Veterinaria 2015; 65: 358–70. https://doi.org/10.1515/acve-2015-0030
● 35. Liapi M , Botsaris G, Arsenoglou C, et al. Rapid Detection of Mycoplasma bovis, Staphylococcus aureus and Streptococcus agalactiae in Cattle Bulk Tank Milk in Cyprus and Relations with Somatic Cell Counts. Pathogens 2021; 10 (7): 841.
● 36. Abd El-Tawab A, and Mohsen A. Bacteriological and molecular studies on Shiga-Toxin producing Escherichia coli ,causing cattle clinical mastitis. Benha veterinary medical journal 2017; 33.2: 17–26.
● 37. Dudek K, and Szacawa E. Mycoplasma bovis Infections, Occurrence, Pathogenesis, Diagnosis and Control, Including Prevention and Therapy. Pathogens 2020; 9, 994. https://doi.org/10.3390/pathogens9120994
● 38. Van Kuppeveld F, Johansson K, Galama J, et al. Detection of mycoplasma contamination in cell cultures by a Mycoplasma group-specific PCR. Appl Environ Microbiol 1994; 60:149–52.
● 39. Vega-Orellana O, Poveda J, Rosales R, et al. Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes. BMC Vet Res 2017; 13, 195. https://doi.org/10.1186/s12917-017-1116-2
● 40. Bokma J, Vereecke N, Nauwynck H, et al. Genome-wide association study reveals genetic markers for antimicrobial resistance in Mycoplasma bovis . Microbiol Spectr 2021 Oct 31; 9 (2): e0026221. doi: 10.1128/Spectrum.00262-21.
● 41. Ammar A, Abd El-Hamid M, Mohamed Y, et al. Prevalence and Antimicrobial Susceptibility of Bovine Mycoplasma Species in Egypt. Biology 2022; 11, 1083. https://doi.org/10.3390/biology11071083
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