Download Using genomic sequences to develop new diagnostic methods for

Using genomic sequences to develop new diagnostic methods for Glässer’s disease
Kate Howell
PhD Student
Supervisors: Dr Tucker & Prof. Maskell
INTRODUCTION
Glässer’s Disease and Haemophilus parasuis (HPS)
Symptoms:
•
Fibrinous polyserositis ‐ Meningitis, Arthritis, Pericarditis, Septicaemia
•
Also able to cause pneumonia
Occurrence:
•
Used to be a sporadic disease in recently weaned pigs. •
Current production practices (multi‐site, high health) have coincided with more frequent larger outbreaks, affecting various ages with increased mortality and morbidity •
Multisourcing of weaners has been a major driver to increased disease prevalence
Diagnostics:
•
Most commonly used technique is serotyping, but no labs in the UK perform this test. Treatment & Disease Control •
Antibiotics (penicillin based) •
Prophylactic antibiotic treatment in feed is common
•
Several vaccines are available but they are serotype specific.
Management of disease •
Optimise maternal antibody delivery
•
Optimise acquired immunity
•
Minimise introduction of new strains of HPS
INTRODUCTION
Scale of Virulence by Serotype
Serotype: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 NT
?
Avirulent
Moderately virulent
Highly virulent
OBJECTIVES
Aims to fill technology gaps in respiratory disease control by designing efficient diagnostics
Identify molecular markers from variation in the genetic sequences of H. parasuis
Two main strategies:
1.Molecular serotyping •
Identify genes or markers characteristic of serotypes
•
Would indicate usefulness of vaccination & useful for stock mixing
2.Virulence Associated Gene Identification •
Compare clinical and non‐clinical strains of H. parasuis
GENOME SEQUENCES + CLINICAL DATA + SEROTYPING DATA INDUSTRY FOCUS
1. For diagnosis of HPS disease
2. For management of disease
3. For surveillance and herd monitoring prior to trade
UK Statistics: 5% of systemic swine disease and 5% respiratory disease attributable to HPS in 2010 AHVLA surveillance report % Diagnosis in Pigs Attributable to HPS
Economic Impact on the UK Industry is ~ £27/pig for an outbreak and £20/pig/year for vaccination (White 2010).
3
2.5
2
1.5
Pneumonia
Glassers
1
0.5
0
2002
2003
2004
2005
2006
Year
2007
2008
2009
MATERIALS & METHODS
1. Strains were collected by the AHVLA which were both disease‐associated and non‐disease associated (carrier strains), with clinical data.
2. Strains were also supplied by collaborators in Denmark & Spain including reference strains used in the current serotyping assays
3. All of these strains were sent for whole genome sequencing
4. Subset of strains sent for serotyping
5. Bioinformatics analysis •
•
•
•
•
Assembly of sequencing data into draft sequences
Core genome built
Capsule loci identified & compared
Phylogenetic trees built for genes of interest
Pangenomes built – core & accessory genes identified – analysis continuing. RESULTS
H. parasuis
Core Genome – Disease association & Serotype Preliminary whole genome sequences
 103 clinical isolates (red)
 47 non‐clinical tonsil isolates (blue)
 12 not defined (black)
Preliminary whole genome sequences
Coloured by serotype
Serotypes 4, 5 & 13 (most common disease causing serotypes) in bottom clade
RESULTS Polysaccharide biosynthesis locus comparisons
Serotype
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
EKB467
77.1
EGC72791.1/EIJ
71310.1
EHC0357
9.1
ABM39
011.1
capD
wza
wzz
ZP_024726
06.1
*
ptp
wzz
HAPS0052
putative HTH‐type transcriptional regulator
HAPS0052
tyrosine kinase
ptp
putative HTH‐type transcriptional regulator
putative HTH‐type transcriptional regulator
Possible variable region ~ 5kb
cytoplasmic tyrosine phosphatase
ZP_02472
606.1
tyrosine kinase
cytoplasmic tyrosine phosphatase
tyrosine kinase
wzz
Hypothetical protein
cap5M
polysaccharide export protein
wza
Capsular polysaccharide biosynthesis protein
ZP_049783
54.1
ptp
Hypothetical protein
wza
cytoplasmic tyrosine phosphatase
polysaccharide export protein
polysaccharide biosynthesis protein CapD
putative glyscosyltransferase/ lipopolysaccharide biosynthesis protein
capD
Capsular polysaccharide biosynthesis protein
ZP_05285
452.1
polysaccharide export protein
putative LPS sugar transferase
wciP
glycosyltransferase
DegT/DnrJ/EryC1/StrS aminotransferase
Predicted glyscosyltransferase
Glyscosyltransferase/ phosphotransferase
wbgY
PilT domain‐containing protein
EFX908
71.1
wbgX
UDP‐glucose 4‐
epimerase
EIJ713
27.1
ZP_0915
9580.1
Lipopolysaccharide biosynthesis protein
EKE579
54.1
wcfQ
Pilin glyscosyltransferase
ACA3157
wcwK
Hypothetical protein
ADO96837.1 8.1
Glyscosyltransferase
EIW18
727.1
Hypothetical protein
HAPS0043
Glyscosyltransferase
wzx
lsgB
Putative membrane protein/ Hypothetical protein
neuA1
glyscosyltransferase
wzx
glyscosyltransferase
putative lipooligosaccharide flippase
0
Glyscosyltransferase
neuA1
putative lipooligosaccharide flippase
CMP‐N‐acetylneuraminic acid synthetase
Hypothetical protein
neuA1
CMP‐N‐acetylneuraminate‐beta‐galactosamide‐
alpha‐2, 3‐sialyltransferase/ lipopolysaccharide biosynthesis protein/ hypothetical protein
HAPS0039
putative lipooligosaccharide flippase
HS228
Serotype 13
HAPS0039
CMP‐N‐acetylneuraminic acid synthetase
HS238
Serotype 4
HAPS0039
CMP‐N‐acetylneuraminic acid synthetase
Hypothetical protein
SH0165 Serotype 5
Hypothetical protein
RESULTS
Predicted Gene Functions between dominant serotypes
15k
Gene Name
Predicted Function
HAPS0052
CONCLUSIONS
Tree of Capsule Gene Cluster
All Strains
Serotyped samples ONLY
HS375-NK-5R
HS353-Y-5
HS311-Y-5
HS094-Y-12-5*
HS070-Y-12-5*
HS061-Y-5
HS056-N-5
HS069-Y-5
HS053-Y-5
HS044-Y-5
HS0006-Y-13.gnm
HS381-NK-12R
HS310-Y-5
HS092-N-5-12*
HS091-N-5
HS088-N-5
HS345-Y-5
HS369-Y-5
HS343-Y-5
HS318-Y-7
HS312-Y-5
HS376-NK-6R
HS366-Y-6
HS348-Y-NT
HS333-Y-NT
HS068-NK-6-7*
HS033-Y-6-9*
HS064-Y-12-4*5*
HS045-N-14-7*
HS357-Y-14
HS383-NK-14R
HS306-NK-14R
HS405
HS034-Y-14-9*
HS370-NK-10R
HS378-NK-8R
HS302-NK-8R
HS077-Y-12-2*4*
HS039-Y-5-7
HS373-NK-3R
HS303-NK-3R
HS0011-N-3.gnm
HS046-Y-7-2*4*
HS062-Y-4
HS099-Y-NT
HS032-Y-NT
HS075-Y-4-8*
HS042-Y-4
HS041-Y-7-2*
HS344-Y-4
HS342-Y-4
HS040-Y-7-4*
HS074-Y-4-8*
HS374-NK-4R
HS101-Y-NT
HS097-Y-4-8*
HS063-Y-7
HS054-Y-4
HS035-Y-7
HS0005-Y-4-8*.gnm
HS058-Y-7-2*4*
HS350-Y-NT
HS060-Y-NT
HS368-Y-NT
HS377-NK-7R
HS305-NK-7R
HS334-Y-NT
HS078-N-NT
HS036-Y-NT
HS304-NK-11R
HS358-Y-1
HS346-Y-2
HS038-Y-2
HS100-Y-2
HS076-Y-2
HS0007-Y-2.gnm
HS352-Y-9
HS379-NK-9R
HS087-N-NT
HS073-Y-7
HS055-Y-9-7*
HS384-NK-15R
HS301-NK-15R
HS367-Y-13
HS356-Y-13
HS355-Y-13
HS349-Y-13
HS254-Y-14
HS250-Y-11
HS065-Y-13-7*
HS359-Y-NT
HS052-Y-13
HS037-Y-13
HS0004-Y-NT.gnm
HS239-N-ND
HS374-NK-4R
HS032-Y-NT
HS001-Y-ND
HS018-Y-ND
HS042-Y-4
HS095-Y-ND
HS211-N-ND
HS227-Y-ND
HS219-N-ND
HS054-Y-4
HS101-Y-NT
HS021-Y-ND
HS217-N-ND
HS004-Y-ND
HS074-Y-4-8*
HS075-Y-4-8*
HS010-Y-ND
HS041-Y-7-2*
HS011-Y-ND
HS205-Y-ND
HS043-Y-ND
HS063-Y-7
HS0005-Y-4-8* .gnm
HS062-Y-4
HS040-Y-7-4*
HS035-Y-7
HS099-Y-NT
HS015-Y-ND
HS019-Y-ND
HS097-Y-4-8*
HS012-Y-ND
HS325-N-ND
HS344-Y-4
HS342-Y-4
HS026-Y-ND
HS303-NK-3R
HS373-NK-3R
HS0011-N-3.gnm
HS301-NK-15R
HS329-N-ND
HS336-Y-ND
HS332-N-ND
HS323-N-ND
HS209-N-ND
HS379-NK-9R
HS352-Y-9
HS330-N-ND
HS055-Y-9-7*
HS073-Y-7
HS201-N-ND
HS233-N-ND
HS087-N-NT
HS203-N-ND
HS003-Y-ND
HS0004-Y-NT.gnm
HS089-N-ND
HS367-Y-13
HS356-Y-13
HS355-Y-13
HS349-Y-13
HS254-Y-14
HS250-Y-11
HS002-Y-ND
HS085-N-ND
HS232-N-ND
HS086-N-ND
HS228-Y-ND
HS017-Y-ND
HS037-Y-13
HS022-Y-ND
HS359-Y-NT
HS052-Y-13
HS016-Y-ND
HS218-N-ND
HS065-Y-13-7*
HS014-Y-ND
HS304-NK-11R
HS358-Y-1
HS080-N-ND
HS337-Y-ND
HS309-Y-ND
HS023-Y-ND
HS090-N-ND
HS083-N-ND
HS081-N-ND
HS050-Y-ND
HS038-Y-2
HS100-Y-2
HS024-Y-ND
HS346-Y-2
HS0010-Y-ND.gnm
HS0007-Y-2.gnm
HS235-N-ND
HS076-Y-2
HS025-Y-ND
HS350-Y-NT
HS027-Y-ND
HS320-N-ND
HS036-Y-NT
HS060-Y-NT
HS028-Y-ND
HS006-Y-ND
HS368-Y-NT
HS377-NK-7R
HS005-Y-ND
HS0003-Y-ND.gnm
HS013-Y-ND
HS305-NK-7R
HS334-Y-NT
HS098-Y-ND
HS030-Y-ND
HS029-Y-ND
HS079-N-ND
HS082-N-ND
HS009-Y-ND
HS078-N-NT
HS370-NK-10R
HS302-NK-8R
HS378-NK-8R
HS221-N-ND
HS045-N-14-7*
HS357-Y-14
HS306-NK-14R
HS220-N-ND
HS034-Y-14-9*
HS376-NK-6R
HS240-N-ND
HS057-Y-ND
HS051-Y-ND
HS366-Y-6
HS348-Y-NT
HS223-N-ND
HS333-Y-NT
HS068-NK-6-7*
HS033-Y-6-9*
HS312-Y-5
HS345-Y-5
HS343-Y-5
HS318-Y-7
HS369-Y-5
HS212-Y-ND
HS0008-Y-ND.gnm
HS091-N-5
HS092-N-5-12*
HS314-Y-ND
HS020-Y-ND
HS088-N-5
HS315-Y-ND
HS381-NK-12R
HS313-Y-ND
Predict serotype 4
5 & 12
6
14Predict serotype 13
3
4 & 7
2
Predict serotype 5 or 13 12
HS310-Y-5
HS094-Y-12-5*
HS307-Y-ND
HS069-Y-5
HS317-Y-ND
HS056-N-5
HS0001-Y-ND.gnm
HS353-Y-5
HS0012-N-ND.gnm
HS070-Y-12-5*
HS375-NK-5R
HS311-Y-5
HS061-Y-5
HS0009-Y-ND.gnm
HS044-Y-5
HS053-Y-5
HS0006-Y-13.gnm
HS335-Y-ND
HS008-Y-ND
HS316-Y-ND
HS238-NK-ND
NEXT STEPS
Virulence Gene Identification ‐ Strategy
Non‐Disease associated strains
Accessory genome
Core genome
Disease associated strains
Accessory genome
Candidate virulence markers
Core genome
Acknowledgements & References
•
•
•
•
Dr. Tucker and Prof. Maskell
Roy Chaudhuri
Shi‐Lu Luan
Lucy Weinert
•
•
•
•
Sarah Peters
Tracy Wang
Dhaarini Raghunathan
BPEX
References
Angen, O., Svensmark, B., & Mittal, K. R. (2004). Serological characterization of Danish Haemophilus parasuis isolates. Veterinary microbiology. Elsevier. Retrieved from http://www.sciencedirect.com/science/article/pii/S0378113504002706
Dijkman, R., Wellenberg, G. J., van der Heijden, H. M. J. F., Peerboom, R., Olvera, A., Rothkamp, A., Peperkamp, K., et al. (2011). Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC‐PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker. Res Vet Sci. Elsevier Ltd. doi:10.1016/j.rvsc.2011.10.013
Olvera, A., J. Segalés, et al. (2007). "Update on the diagnosis of Haemophilus parasuis infection in pigs and novel genotyping methods." The Veterinary Journal 174(3): 522‐529.
White, M. (2010). NADIS Pig Health – Glässers Disease. BPEX NADIS Health Bulletin (pp. 1‐3).
Xu, Z., M. Yue, et al. "Genomic Characterization of Haemophilus parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China." PLoS ONE 6(5): e19631.