J Vet Sci.  2007 Jun;8(2):151-154. 10.4142/jvs.2007.8.2.151.

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

• Kalorey DR
• Shanmugam
• Kurkure NV
• Chousalkar KK
• Barbuddhe

• KMID: 1089666
• DOI: http://doi.org/10.4142/jvs.2007.8.2.151

Abstract

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds.

Keyword

genotyping; India; Staphylococcus aureus; subclinical mastitis

MeSH Terms

Animals
Bacterial Proteins/chemistry/genetics
Cattle
Coagulase/chemistry/genetics
DNA, Bacterial/chemistry/genetics
Endonucleases/chemistry/genetics
Female
Mastitis, Bovine/*microbiology
Micrococcal Nuclease/chemistry/genetics
Milk/microbiology
Polymerase Chain Reaction/veterinary
Staphylococcal Infections/microbiology/*veterinary
Staphylococcus aureus/*genetics/pathogenicity
Virulence Factors/chemistry/*genetics

Figure

• 

  Fig. 1 Amplicons of the genes encoding Staphylococcal coagulase (coa), clumping factor (clfA), thermonuclease (nuc), spa gene X-region, and IgG-binding regions. Lane M: DNA molecular weight marker MBD 13 (Bangalore Genei, India); Lane 1-3: coa; Lane 4: clfA; Lane 5: nuc; Lane 6-8: spa X-region; Lane 9-11: spa IgG-binding region.

Reference

1. Akineden Ö, Annemüller C, Hassan AA, Lämmler C, Wolter W, Zschöck M. Toxin genes and other characteristics of Staphylococcus aureus isolates from milk of cows with mastitis. Clin Diagn Lab Immunol. 2001. 8:959–964.

2. Annemüller C, Lämmler C, Zschöck M. Genotyping of Staphylococcus aureus isolated from bovine mastitis. Vet Microbiol. 1999. 69:217–224.

3. Cowan ST, Steel KJ. Manual for the Identification of Medical Bacteria. 1970. London: Cambridge University Press;52–57.

4. Cruickshank R, Duguid JP, Harmoin BP, Swain RHA, editors. The practice of medical microbiology. Medical Microbilogy. 1975. Vol. 2:12th ed. Edinburgh: Churchill Livingstone;356–366.

5. El-Sayed A, Alber J, Lämmler C, Bonner B, Huhn A, Kaleta EF, Zschöck M. PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from birds. J Vet Med B Infect Dis Vet Public Health. 2005. 52:38–44.

6. Fitzgerald JR, Hartigan PJ, Meaney WJ, Smyth CJ. Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection. J Appl Microbiol. 2000. 88:1028–1037.

7. Fitzgerald JR, Meaney WJ, Hartigan PJ, Smyth CJ, Kapur V. Fine-structure molecular epidemiological analysis of Staphylococcus aureus recovered from cows. Epidemiol Infect. 1997. 119:261–269.

8. Foster G, Ross HM, Hutson RA, Collins MD. Staphylococcus lutrae sp. nov., a new coagulase-positive species isolated from otters. Int J Syst Bacteriol. 1997. 47:724–726.

9. Foster SJ. Molecular characterization and functional analysis of the major autolysin of Staphylococcus aureus 8325/4. J Bacteriol. 1995. 177:5723–5725.

10. Jarraud S, Cozon G, Vandenesch F, Bes M, Etienne J, Lina G. Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J Clin Microbiol. 1999. 37:2446–2449.

11. Kapur V, Sischo WM, Greer RS, Whittam TS, Musser JM. Molecular population genetic analysis of Staphylococcus aureus recovered from cows. J Clin Microbiol. 1995. 33:376–380.

12. Lachica RVF, Genigeorgis C, Hoeprich PD. Metachromatic agar-diffusion methods for detecting Staphylococcal nuclease activity. Appl Microbiol. 1971. 21:585–587.

13. Miles H, Lesser W, Sears P. The economic implications of bioengineered mastitis control. J Dairy Sci. 1992. 75:596–605.

14. Radostits OM, Gay CC, Blood DC, Hinchcliff KW. Veterinary Medicine. A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses. 2003. 9th ed. Philadelphia: Saunders;615.

15. Salasia SIO, Khusnan Z, Lämmler C, Zschöck M. Comparative studies on pheno- and genotypic properties of Staphylococcus aureus isolated from bovine subclinical mastitis in central Java in Indonesia and Hesse in Germany. J Vet Sci. 2004. 5:103–109.

16. Smeltzer MS, Gillaspy AF, Pratt FL Jr, Thames MD, Iandolo JJ. Prevalence and chromosomal map location of Staphylococcus aureus adhesin genes. Gene. 1997. 196:249–259.

17. Stephan R, Annemüller C, Hassan AA, Lämmler C. Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland. Vet Microbiol. 2001. 78:373–382.

18. Straub JA, Hertel C, Hammes WP. A 23S rRNA-targeted polymerase chain reaction-based system for detection of Staphylococcus aureus in meat starter cultures and dairy products. J Food Prot. 1999. 62:1150–1156.

19. Wilson K. Ausuhel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, editors. Preparation of genomic DNA from bacteria. Current Protocols in Molecular Biology. 1987. Vol. 1. New York: John Wiley & Sons;2.4.1–2.4.2.

20. Zhang S, Iandolo JJ, Stewart GC. The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiol Lett. 1998. 168:227–233.