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

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

2. I shall be talking about....... • Aim and objectives of the project • Background on mastitis • Clinical definitions of mastitis • Mastitis pathogens in sheep • Multiplex PCR • Designing and testing of primers • Results so far • Summary of progress to date

3. Aim of Project • To develop and assess the use of a multiplex PCR as a tool to identify bacterial species commonly associated with mastitis in sheep.

4. Objectives of Project • To develop an effective technique for the isolation of DNA directly from sheep milk for use in a multiplex PCR. • To develop a multiplex PCR for the most commonly recognised mastitis pathogens (Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Mannheimia haemolytica, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis), using specific primers to amplify products of varying fragment sizes.

5. Objectives of project continued • To use the multiplex PCR technique developed to process approximately 500 sheep milk samples and analyse the occurrence of the previously specified mastitis-associated bacterial species. • To look at the infection status of sheep over time.

6. Mastitis Background • Defined as ‘inflammation of the udder’, usually as a result of infection with pathogenic bacteria. • Can lead to temporary or permanent loss in milk production, reduction in milk quality, low weaning weights, loss of milk as a result of discarding after antibiotic treatment, culling and premature death, increased surveillance and labour costs. • Defined by severity and clinical signs into clinical, subclinical and chronic infection.

7. Subclinical Mastitis • No outward signs of infection. • Identified through changes in milk composition, reduction in milk yield and presence of inflammatory components in the milk, indicating the presence of an infection.

8. Clinical Mastitis • Visible with changes to the udder, milk or ewe behaviour. • Can be mild or acute. • Mild cases- symptoms include watery milk, inflammation of the udder. • Severe cases – lameness, increases in body temperature, swelling of udder, with infected gland(s) becoming hot, swollen, painful and purple rapidly, becoming necrotic and sloughing off with surrounding tissue.

9. Chronic Mastitis • Persistent, long-term infection. • Symptoms include milk becoming clotted and the teat of the affected gland thickened. • Infected udder develops hard lumps from the “walling off” of bacteria causing irreparable tissue damage. • Treatment considered futile, as sheep with one infected gland can only rear one lamb, which is seen as uneconomic in most flocks.

10. http://www.southdownsheepsociety.co.uk/May09NadisSheepBulletin-MastitisinEwes.pdfhttp://www.southdownsheepsociety.co.uk/May09NadisSheepBulletin-MastitisinEwes.pdf

11. Impact of Mastitis Direct effects: • Temporary of permanent reduction in milk production • Milk quality reduction • Animal welfare issue/ quality of life: Mastitis can be very painful. Affected ewes become uninterested in their lambs • Premature culling and death as a result of infection Economic: • Annual loss of £300 million to UK dairy farmers • Prevention costs: antibiotics, disinfectants • Treatment costs: drugs, veterinary costs etc

12. Mastitis pathogens in sheep • Staphylococcus aureus • coagulase-negative staphylococci • Escherichia coli • Mannheimia haemolytica • streptococcal species- Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis.

13. Multiplex PCR • Multiplex PCR works by combining multiple primer sets specific for individual sequence targets in a single PCR mixture. • This produces amplicons of variable sizes, allowing the identification of targeted bacterial pathogens in a milk sample.

14. Primers • Specific primers aiming to combine in a multiplex PCR are targeting: E.coli, Mannheimia haemolytica, Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. • So far, I have optimised and tested the specificity of a mixture of previously published and validated primers and newly designed ones.

15. Specificity results designed primers 1

16. Specificity results designed primers 2

17. Specificity results of designed primers 3 KEY  primer amplified DNA of bacterial species specific amplification of bacterial species X no amplification seen TBC to be confirmed

18. Specificity Results Previously Published Primers 1

19. Specificity Results Previously Published Primers 2

20. Specificity results previously published primers 3 KEY  primer amplified DNA of bacterial species specific amplification of bacterial species X no amplification seen TBC to be confirmed (i.e. the primer set has not been specificity tested against this primer set.

21. Summary of progress • DNA isolation directly from milk technique has been identified, tested and optimised. • Successfully tested the DNA isolation technique with universal and specific primers sets. • Have Streptococcus uberis and Streptococcus dysgalactiae specific primer sets. • Continue to design, optimise and test primer sets, in addition to testing of previously published and validated primers to combine in the multiplex PCR.

22. Thanks for listening! Any questions?