Background: Staphylococcus aureus is one of the most important contagious bacteria causing subclinical bovine mastitis. This bacterial infection is commonly identified by determine the pathogen in bovine milk samples through conventional technique including coagulase test. However, this test has several disadvantages as low sensitivity, risk of biohazard, cost expensive, and limited preparation especially in local area.

Aim: Aim of this study was to compare and assess the screening method, Mannitol fermentation test (Mannitol salt agar [MSA]), and deoxyribonuclease (DNase) test, for S. aureus identification in milk samples.

Materials and Methods: A total of 224 subclinical bovine mastitis milk samples were collected from four provinces of Thailand and determined S. aureus using conventional method and also subjected to the screening test, MSA and DNase test. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) among both tests were analyzed and compared to the tube coagulase test (TCT), as reference method. Immunological test by latex agglutination and molecular assay by determined spa gene were also used to identify and differentiate S. aureus.

Results: A total of 130 staphylococci were isolated by selective media, Gram-stain, and catalase test. The number of S. aureus which identified using TCT, MSA and DNase test were 32, 102, and 74 isolates, respectively. All TCT results were correlated to results of latex agglutination and spa gene which were 32 S. aureus. MSA showed 100% sensitivity, 28.57% specificity, 31.37% PPV, and 100% NPV, whereas DNase showed 53.13% sensitivity, 41.84% specificity, 22.97% PPV, and 73.21% NPV. DNase test showed higher specificity value than MSA but the test presented 26.79% false negative results whereas no false-negative result from MSA when comparing to TCT.

Conclusion: MSA had a tendency to be a good preference for screening S. aureus because of its high sensitivity and NPV. The result from this study will improve a choice to use a screening test to diagnose S. aureus of veterinary field for prompt disease controlling and effective treatment.

Keywords: bovine mastitis, deoxyribonuclease test, mannitol fermentation test, screening methods, Staphylococcus aureus.

1. Hillerton, J.E. and Berry, E.A. (2005) Treating mastitis in the cow-a tradition or an archaism. J. Appl. Microbiol., 98(6): 1250-1255. [Crossref] [PubMed]

2. Islam, M.R., Ahamed, M.S., Alam, M.S., Rahman, M.M., Sultana, T., Roh, Y.S. and Kim, B. (2011) Identification and antibiotic sensitivity of the causative organisms of sub-clinical mastitis in sheep and goats. Pak. Vet. J., 32(2): 179-182.

3. Yadav, S., Sharma, M., Yadav, A. and Chaudhary, U.A. (2014) Study of biofilm production in Staphylococcus aureus. Int. J. Pharm. Biol. Sci., 3(2): 61-63.

4. National Mastitis Council. (1996) Current Concepts of Bovine Mastitis. 4th ed. National Mastitis Council, Arlington, VA.

5. Plozza, K., Lievaart, J.J., Potts, G. and Barkema, H.W. (2011) Subclinical mastitis and associated risk factors on dairy farms in New South Wales. Aust. Vet. J., 89(1-2): 41-46. [Crossref] [PubMed]

6. Jurgen, S., Barbel, K., Wilfried, W., Michael, Z., Axel, S. and Klaus, F. (2003) The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme. Vet. Microbiol., 96(1): 91-102. [Crossref]

7. Witaya, S. (2011) Epidemiology of subclinical mastitis and their antibacterial susceptibility in smallholder dairy farms, Chiang Mai province, Thailand. J. Anim. Vet. Adv., 10(3): 316-321. [Crossref]

8. Jaruwan, K., Varaporn, S., Anantachai C., Sarinya, R. and Arunee, P. (2010) Chronic mastitis in small dairy cattle herds in Muang Khon Kaen. Thai J. Vet. Med., 40(3): 265-272.

9. Balows, A., Hausler, W.J., Hermann, K.L., Isenberg, H.D. and Shadomy, H.J. (1991) Manual of Clinical Microbiology. 5th ed. American Society for Microbiology, Washington, DC.

10. McDonald, C.L. and Chapin, K. (1995) Rapid identification of Staphylococcus aureus from blood culture bottles by a classic 2-hour tube coagulase test. J. Clin. Microbiol., 33(1): 50-52. [PubMed] [PMC]

11. Moreillon, P., Entenza, J.M., Francioli, P., McDevitt, D., Foster, T.J. and Francois, P. (1995) Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis. Infect. Immun., 63(12): 4738-4743. [PubMed] [PMC]

12. Bello, C.S.S. and Qahtani, A. (2006) Pitfalls in the routine diagnosis of Staphylococcus aureus. Afr. J. Biotechnol., 4(1): 83-86.

13. Cheng, A.G., McAdow, M., Kim, H.K., Bae, T., Missiakas, D.M. and Schneewind, O. (2010) Contribution of coagulases towards Staphylococcus aureus disease and protective immunity. PLoS Pathog., 6(8): e1001036. [Crossref]

14. Yousef, A.E. and Carolyn, C. (2003) Food Microbiology: A Laboratory Manual. John Wiley & Sons, United States of America.

15. Sperber, W.H. and Tatini, S.R. (1975) Interpretation of the tube coagulase test for identification of Staphylococcus aureus. Appl. Microbiol., 29(4): 502-505. [PubMed] [PMC]

16. Berke, A. and Tilton, R.C. (1986) Evaluation of rapid coagulase methods for the identification of Staphylococcus aureus. J. Clin. Microbiol., 23(5): 916-919. [PubMed] [PMC]

17. Makwana, D.G.E., Gadhavi, D.H. and Sinha, D.M. (2012) Comparision of tube coagulase test with mannitol fermentation test for diagnosis of Staphylococcus aureus. Natl. J. Integr. Res. Med., 3(4): 73-75.

18. Varettas, K., Mukerjee, C. and Taylor, P.C. (2005) Anticoagulant carryover may influence clot formation in direct tube coagulase tests from blood cultures. J. Clin. Microbiol., 43(9): 4613-4615. [Crossref] [PubMed] [PMC]

19. D'Souza, H.A. and Baron, E.J. (2005) BBL CHROMagar Staphylococcus aureus is superior to mannitol salt for detection of Staphylococcus aureus in complex mixed infections. Am. J. Clin. Pathol., 123(6): 806-808. [Crossref]

20. Weckman, B.G. and Catlin, B.W. (1957) Deoxyribonuclease activity of micrococci from clinical sources. J. Bacteriol., 73: 747-753. [PubMed] [PMC]

21. Anyanwu, N.C.J. and John, W.C. (2013) Conventional and rapid methods for identification of Staphylococcus aureus from clinical specimens. Sci. J. Microbiol., 2(9): 174-178. [Crossref]

22. Murray, P.R., Baron, E.J., Jorgensen, J.H., Pfaller, M.A. and Yolken, R.H. (2003) Manual of Clinical Microbiology. 8th ed. American Society for Microbiology, Washington, DC.

23. Remel Europe Ltd. (2011) Staphaurex^TM Plus Latex Agglutination. Remel Europe Ltd., Dartford, UK.

24. Bhati, T., Nathawat, P., Sharma, S.K., Yadav, R., Bishnoi, J. and Kataria, A.K. (2016) Polymorphism in spa gene of Staphylococcus aureus from bovine subclinical mastitis. Vet. World, 9(4): 421-424. [Crossref] [PubMed] [PMC]

25. Akineden, O., Annemuller, C., Hassan, A.A., Lammler, C., Wolter, W. and Zschock, M. (2001) Toxin genes and other characteristics of Staphylococcus aureus isolates from milk of cows with mastitis. Clin. Diagn. Lab. Immunol., 8(5): 959-964. [Crossref]

26. Indrawattana, N., Sungkhachat, O., Sookrung, N., Chongsa-nguan, M., Tungtrongchitr, A. and Voravuthikunchai, S.P., Kong-Ngoen, T., Kurazono, H. and Chaicumpa, W. (2013) Staphylococcus aureus clinical isolates: Antibiotic susceptibility, molecular characteristics, and ability to form biofilm. BioMed. Res. Int., 2013: 314654. [Crossref] [PubMed] [PMC]

27. Kateete, D.P., Kimani, C.N., Katabazi, F.A., Okeng, A., Okee, M.S., Nanteza, A., Joloba, L.M. and Najjuka, C.F. (2010) Identification of Staphylococcus aureus: DNase and mannitol salt agar improve the efficiency of the tube coagulase test. Ann. Clin. Microbiol. Antimicrob., 9: 23. [Crossref] [PubMed] [PMC]

28. Lagace-Wiens, P.R.S., Alfa, M.J., Manickam, K. and Karlowsky, J.A. (2007) Thermostable DNase is superior to tube coagulase for direct detection of Staphylococcus aureus in positive blood cultures. J. Clin. Microbiol., 45(10): 3478-3479. [Crossref] [PubMed] [PMC]