Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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.