1. Rapid identification of Bovine Mastitis pathogens by High Resolution Melt Analysis of 16S rDNA sequences Praseeda Ajitkumar Jeroen De Buck Herman Barkema Department of Production Animal Health Praseeda Ajitkumar Jeroen De Buck Herman Barkema Department of Production Animal Health CAHLN 2010

2. Background  Mastitis: persistent problem and the most expensive disease of dairy cows  Coagulase-negative staphylococci (CNS) are a frequent cause of bovine mastitis in many countries.  CNS are not identified further by species but are treated as a uniform group

3. Identification of mastitis pathogens  Bacteriological culture- gold standard  PCR based assays- to complement or replace conventional identification methods  DNA sequencing

4. High Resolution Melt (HRM)  Rapid molecular technique introduced in 2002  Generation of melting curves after PCR amplification  Based on differences in the thermal stability of DNA  Genotyping of several organisms (Chlamydia psittaci, Mycoplasma pneumoniae, Mycobacterium tuberculosis, M. avium subsp.paratuberculosis (Castellanos et al.,2010a and 2010b), Influenza A virus)

5. HRM versus Melt curve HRM is an extended analysis of melt curve  Requires additional analysis software - Normalize melt curves - Apply an optional temperature shift - Plot curves in a difference graph for easy visualization - Clusters curves into groups representing different genotypes/sequences

6. HRM versus Melt curve  “Saturation” dyes are less inhibitory to PCR than SYBR ( Evagreen, LC green dyes )  Observed melting behaviour is characteristic of a particular DNA sample Target - 16S rRNA gene Gold standard for broad-range microbial identification Feasibility of using high-precision melting for bacterial speciation (Cheng et al., 2006) Highly specific species identification of clinically relevant biothreat bacterial agents (Yang et al., 2009)

7. Hypothesis  High resolution analysis of melting curves generated after PCR amplification can lead to rapid speciation of mastitis pathogens

8. Objective  Development of novel and rapid assays to speciate major and minor mastitis pathogens based on real-time PCR and HRM

9. Pathogens in Clinical Mastitis n-3,024 Olde Riekerink et al., 2008

10. Serial No. Bacterial speciesCBMRN 1Staphylococcus aureus ATCC 33862 2Streptococcus agalactiae ATCC 12386 3Streptococcus uberis ATCC 9927 4Fusobacterium necrophorum ATCC 27852 5Bacterioides fragilis ATCC 25285 6Prevotella melaninogenica ATCC 43982 7Klebsiella pneumoniae ATCC 43816 8Escherichia coli CM090903 9Corynebacterium bovis CM090710 10Arcanobacterium pyogenes CM090701 11Streptococcus dysgalactiae CM091011 12Mycoplasma bovis CM091001 Bacterial species included in HRM

11. Coagulase-negative staphylococci Serial No. Bacterial speciesCBMRN 1Staphylococcus chromogenes22705099 2Staphylococcus hyicus 32902167 3Staphylococcus epidermidis11211860 4Staphylococcus simulans11110774 5Staphylococcus capitis20309206 6Staphylococcus warneri32107630 7Staphylococcus xylosus10613450 8Staphylococcus haemolyticus11501077 9Staphylococcus sciuri11501046 10Staphylococcus auricularis41808610 11Staphylococcus cohnii41813355 12Staphylococcus hominis32411508 13Staphylococcus saprophyticus31915182 14Staphylococcus intermedius11007210

12. Materials & Methods Extraction of bacterial genomic DNA  7 bacterial strains from ATCC and 6 isolates from mastitis milk samples subjected to DNA extraction  14 coagulase-negative staphylococci isolates from CBMRN  Genomic DNA extracted with the DNeasy Blood and Tissue Kit (Qiagen)

13. Materials & methods (contd…)  Amplification of 16S rRNA gene using real-time PCR  Real-time PCR amplification of 16S rRNA gene using BioRad CFX thermal cycler  Cycling conditions 1: 98.0°C for 2:00 min 2: 98.0°C for 0:05 3: 55.0°C for 0:10 Plate Read 4: GOTO 2, 39 more times 5: 95.0°C for 1:00 6: 70.0°C for 1:00 7: Melt Curve 70°C to 95°C : Increment 0.2°C for 0:10 Clustering

14. Result 1 2 3 5 4 6,7 8 9 10,11 12 13 HRM-common mastitis pathogens 1. A. pyogenes 2. C. bovis 3. S. agalactiae 4. S. dysgalactiae 5. E. coli 6. K. pneumoniae 7. S. uberis 8. P. melaninogenica 9. F. necrophorum 10. S. aureus 11. B. fragilis 12. M. bovis

15. Result 1. S. auricularis 2. S. chromogenes 3. S. intermedius 4. S. hyicus 5. S. aureus 6. S. capitis 7. S. epidermidis 8. S. sciuri 9. S. simulans 10. S. warneri 11. S. saprophyticus 12. S. cohnii 13. S. xylosus 14. S. haemolyticus 15. S. hominis HRM- coagulase-negative staphylococci

16. Advantages of HRM  Inexpensive  High sensitivity & specificity  Rapid-completed in about 1 h 30 min

17. Conclusions  High resolution melt analysis is a rapid molecular tool for the identification of mastitis pathogens  Validation of the technique is necessary  Applicability of the technique in speciation of pathogens in mastitis milk samples needs to be evaluated

18. Future Directions  Test and validate HRM assays on DNA extracts of subclinical and clinical mastitis cases (CNS and other mastitis pathogens)  Culture-negative mastitis samples

19. Acknowledgements  Supervisors Herman Barkema & Jeroen De Buck  John Middleton, Faculty of Vet. Med, Missouri  Lab mates Elena Castellanos Amanda Reith Nick Mackenzie Vineet Saini Rienske Mortier Joel David  Faculty of Veterinary Medicine, University of Calgary for the UCVM scholarship  National Mastitis Research Foundation

20. Thanks