Rapid identification of Bovine Mastitis pathogens by High Resolution Melt Analysis of

16S rDNA sequences

Rapid identification of Bovine Mastitis pathogens by High Resolution Melt Analysis of

16S rDNA sequences

Praseeda AjitkumarJeroen De Buck

Herman BarkemaDepartment of Production Animal Health

Praseeda AjitkumarJeroen De Buck

Herman BarkemaDepartment of Production Animal Health

CAHLN 2010

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

Identification of mastitis pathogens

Bacteriological culture- gold standard

PCR based assays- to complement or replace conventional identification methods

DNA sequencing

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)

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

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)

Hypothesis

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

Objective

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

Pathogens in Clinical Mastitis

n-3,024

Olde Riekerink et al., 2008

Serial No. Bacterial species CBMRN

1 Staphylococcus aureus ATCC 33862

2 Streptococcus agalactiae ATCC 12386

3 Streptococcus uberis ATCC 9927

4 Fusobacterium necrophorum ATCC 27852

5 Bacterioides fragilis ATCC 25285

6 Prevotella melaninogenica ATCC 43982

7 Klebsiella pneumoniae ATCC 43816

8 Escherichia coli CM090903

9 Corynebacterium bovis CM090710

10 Arcanobacterium pyogenes CM090701

11 Streptococcus dysgalactiae CM091011

12 Mycoplasma bovis CM091001

Bacterial species included in HRM

Coagulase-negative staphylococciSerial No. Bacterial species CBMRN

1 Staphylococcus chromogenes 22705099

2 Staphylococcus hyicus 32902167

3 Staphylococcus epidermidis 11211860

4 Staphylococcus simulans 11110774

5 Staphylococcus capitis 20309206

6 Staphylococcus warneri 32107630

7 Staphylococcus xylosus 10613450

8 Staphylococcus haemolyticus 11501077

9 Staphylococcus sciuri 11501046

10 Staphylococcus auricularis 41808610

11 Staphylococcus cohnii 41813355

12 Staphylococcus hominis 32411508

13 Staphylococcus saprophyticus 31915182

14 Staphylococcus intermedius 11007210

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)

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 min2: 98.0°C for 0:053: 55.0°C for 0:10 Plate Read4: GOTO 2, 39 more times5: 95.0°C for 1:006: 70.0°C for 1:007: Melt Curve 70°C to 95°C : Increment 0.2°C for 0:10 Clustering

Result

1

235

46,78

910,1112

13

HRM-common mastitis pathogens

1. A. pyogenes2. C. bovis3. S. agalactiae4. S. dysgalactiae5. E. coli6. K. pneumoniae7. S. uberis8. P. melaninogenica9. F. necrophorum10. S. aureus11. B. fragilis12. M. bovis

Result

1. S. auricularis2. S. chromogenes3. S. intermedius4. S. hyicus5. S. aureus6. S. capitis7. S. epidermidis8. S. sciuri9. S. simulans10. S. warneri11. S. saprophyticus12. S. cohnii13. S. xylosus14. S. haemolyticus15. S. hominis

HRM- coagulase-negative staphylococci

Advantages of HRM

Inexpensive

High sensitivity & specificity

Rapid-completed in about 1 h 30 min

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

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

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

Thanks