Development and use of a multiplex PCR to detect common mastitis pathogens in ewe milk

1. Development and use of a multiplex PCR to detect common mastitis pathogens in ewe milk Emma Monaghan

2. Aim • To develop a multiplex PCR as an accurate and inexpensive research tool to identify six bacterial pathogens associated with mastitis in sheep • The six bacterial pathogens targeted in the multiplex PCR development were S. aureus, E.coli, M. haemolytica, Strep. agalactiae, Strep. dysgalactiae and Strep. uberis

3. Objectives • To develop a technique to isolate bacterial DNA from sheep milk • To identify sensitive and specific primers for the target pathogens

4. Objectives • To develop a multiplex PCR to identify the presence of the target pathogens in a single test • To process approximately 100 sheep milk samples to test the efficacy of the multiplex PCR

5. Mastitis • Mastitis is an inflammation of the mammary gland, usually as a result of infection with pathogenic microorganisms, most often of bacterial origin • The presentation of mastitis can be defined by severity, clinical signs and type of bacterial infection • When severity is used to describe mastitis, subclinical, clinical, acute and chronic are commonly used

6. Consequences of infection • Temporary or permanent loss of milk production • Reduction in milk quality • Reduction in lamb weight • Increased costs from purchase of milk replacements and treatments

7. Multiplex PCR • A multiplex PCR combines multiple primer sets specific for individual DNA sequence targets (unique to a species of bacteria) in a single PCR mixture • This produces amplicons of variable sizes to allow the identification of several targeted bacterial pathogens in a milk sample

8. Relevance to industry • Starting point for progression of knowledge on the microbial community of sheep udders which may aid methods for prevention, detection and treatment of mastitis in sheep, reducing its economic and welfare related impacts • Potentially inexpensive and rapid research tool to allow efficient diagnosis of mastitis and selection of appropriate treatments

9. Results- development of primers • To identify specific PCR assays for the multiplex PCR development, a total of 57 primer sets were tested, of which thirty-two were from published literature and 25 designed in this study • Specific primer sets for each of the six bacterial pathogens were identified and gradually combined in an attempt to produce a single multiplex PCR • However, cross-reactions and incompatibility problems led to the multiplex PCR being split into two reactions with the first targeting Strep. dysgalactiae, E. coli and M . haemolytica and the second targeting S. aureus and Strep. uberis

10. Results- testing of milk • 122 sheep milk samples were processed using the two multiplex PCRs which targeted five of the six intended bacterial pathogen targets • M. haemolytica was detected in 73% of samples, S. aureus was detected in 25% E.coli and Strep. dysgalactiae were both detected in 11.5%, and S. uberis in 2.5%

11. Results- cross contamination • However, cross-contamination as a result of a combination of contamination during bacterial DNA extraction and preparation for processing in the multiplex PCRs detrimentally affected the confidence in the results obtained • Consequently, only 25% of milk samples testing positive in the two multiplex PCRs were validated

12. Conclusions • The two multiplex PCR assays were used to process sheep milk samples • Validity of results limited by contamination issues which are yet to be resolved • The cross-contamination issues highlight the importance of stringent laboratory practices and technique • Identifying more than one specific PCR assay for each bacterial species included in a multiplex PCR is necessary due to the limitations a multiplex PCR assay can place upon individual primer sets and the cross-reactions and incompatibility issues that can arise

13. Future work to resolve contamination • Essential to achieve confidence in the results • Gradient, specificity and multiplex PCR conditions need to be re-assessed in the re-development process to aid achieving specificity

14. Future work to make one multiplex • Combine the amplification of all target bacterial pathogens into a single multiplex PCR • Design more specific PCR assays and test their compatibility with one another and the specific assays already identified

15. Acknowledgements • Professor Laura Green • Dr Kevin Purdy • Dr Edward Smith • Selene Huntley • Office B134 • EBLEX