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 CAHLN 2010
Dec 31, 2015
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