Rapid identification of Bovine Mastitis
pathogens by High Resolution Melt Analysis of
16S rDNA sequences
CAHLN 2010
Praseeda Ajitkumar
Jeroen De Buck
Herman Barkema
Department of Production Animal Health
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
Bacterial species included in HRM
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
Coagulase-negative staphylococci
Serial 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
1: 98.0°C for 2:00 min
2: 98.0°C for 0:05
 Cycling conditions
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
Result
HRM-common mastitis pathogens
1
4
5 6,7
8
9
12 10,11
13
3
2
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
Result
HRM- coagulase-negative staphylococci
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
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