Gene Content and Diversity of the Loci Encoding Biosynthesis ofCapsular Polysaccharides of the 15 Serovar Reference Strains ofHaemophilus parasuis

Kate J. Howell,a Lucy A. Weinert,a Shi-Lu Luan,a Sarah E. Peters,a Roy R. Chaudhuri,a* David Harris,b Øystein Angen,c

Virginia Aragon,d Julian Parkhill,b Paul R. Langford,e Andrew N. Rycroft,f Brendan W. Wren,g Alexander W. Tucker,a

Duncan J. Maskell,a on behalf of the BRaDP1T Consortium

Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdoma; The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus,Hinxton, Cambridge, United Kingdomb; Norwegian Veterinary Institute, Oslo, Norwayc; Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la UniversitatAutònoma de Barcelona, Bellaterra, and Institut de Recerca i Tecnologia Agroalimentàries, Barcelona, Spaind; Section of Paediatrics, Department of Medicine, ImperialCollege London, St. Mary’s Campus, London, United Kingdome; The Royal Veterinary College, Hawkshead Campus, Hatfield, Hertfordshire, United Kingdomf; Faculty ofInfectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdomg

Haemophilus parasuis is the causative agent of Glässer’s disease, a systemic disease of pigs, and is also associated with pneumo-nia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNAsequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulencefactors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysac-charide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the exportof polysaccharide capsules in the current capsule classification system. A conserved region at the 3= end of the locus, containingthe wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A po-tential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci forsynteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which areidentical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step inunderstanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogenand opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis andcontrol of Glässer’s disease.

Haemophilus parasuis is a Gram-negative bacterium and amember of the family Pasteurellaceae. It colonizes the upper

respiratory tracts of pigs as a commensal but is also often foundassociated with pneumonia and is the etiological agent of Glässer’sdisease (1, 2). Glässer’s disease depends on invasion of the bacteriainto the systemic compartment and is characterized by fibrinouspolyserositis, polyarthritis, and meningitis (3–5). It imposes a sig-nificant economic and welfare burden on the global pig industry,resulting in a high demand for the use of antimicrobials (1, 6–8).The vaccines available against this bacterium are whole-cell bac-terins, which are protective only against strains of the same sero-var (9–11). Current vaccines are protective only against serovars 1,4, 5, and 6 (12–14); therefore, knowing which serovar is causinginfection in a herd is central to the management of Glässer’s dis-ease (15–17).

The current serotyping scheme is based on reactions betweenantisera and surface antigens that classify the bacteria into 15 se-rovars, with a considerable number of nontypeable isolates alsobeing observed (18, 19). The original Kielstein-Rapp-Gabrielsonserotyping scheme was designed in 1992 and used a gel immuno-diffusion (GID) assay, but this has been superseded by an indirecthemagglutination assay (IHA) that has increased the proportionof typeable strains from 60% to 80% (12, 19–22). The two meth-ods use different antigen preparation techniques, with GID usingautoclaved antigens that are presumed to consist of thermostablepolysaccharide components (19), while IHA uses boiled or salineextracts, which are thought to be composed primarily of lipopoly-

saccharide (LPS) components (20–22). The extracts used in theIHA protocol may vary from the original GID assay, as the pro-duction method differs between testing laboratories, and so addi-tional bacterial components may remain in the extracts that aretested. This adds a source of variation to the serotyping process(21, 22). While there are differences between the two serotypingmethods, there is strong evidence that the important antigens inboth protocols are polysaccharides. The study of these polysaccha-ride components in relation to serovar has only just started withthe publication of structures of the capsular polysaccharide andLPS for two reference strains, and their expression has been mon-itored in only a selection of the reference strains (23–26). Thereare some problems with the serotyping assay, including the diffi-culty of consistently producing specific antisera against severalreference strains, variation in growth conditions, cross-reactions

Received 24 April 2013 Accepted 14 July 2013

Published ahead of print 19 July 2013

Address correspondence to Kate J. Howell, kjh52@cam.ac.uk.

* Present address: Roy R. Chaudhuri, Centre for Genomic Research, University ofLiverpool, Biosciences Building, Liverpool, United Kingdom.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00471-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00471-13

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between serovars, and the very small number of laboratories thatcurrently perform this test (8, 18, 20, 26). Molecular serotypingsystems have been developed for other bacteria based on the genesinvolved in biosynthesis of extracellular polysaccharide structuressuch as LPS O antigens or capsular polysaccharides (27–30).

The study of capsule genetics in other bacteria (predominantlyEscherichia coli) has led to a classification system that separatescapsule loci into four groups (31) based on the genetics and bio-chemical properties of the polysaccharides (32). Capsular polysac-charides in groups 1 and 4 can be present on the cell surface as acapsule or as short oligosaccharides linked to lipid A core in LPS(33). Capsule loci for groups 1 and 4 contain genes encoding prod-ucts involved in sugar biosynthesis, polymerization of the sugarsinto larger structures and translocation of these structures to thecell surface. These genes include, among others, glycosyltrans-ferase genes, wzy (encoding a polymerase), wza (encoding anouter membrane lipoprotein), wzb (encoding a protein-tyrosinephosphatase), and wzc (encoding a tyrosine-protein kinase) (32).These genes are usually grouped by function within the locus,representing export regions (that are common across a number ofserovars) and serovar-specific regions (32, 34, 35). wzx and wzyare typically required for both the LPS translocation pathway andcapsular polysaccharide export, while wza, wzb, and wzc are spe-cific to capsule biosynthesis (27, 33).

Analysis of the first complete H. parasuis genome sequence(strain SH0165) (36) identified a 14-kb polysaccharide biosynthe-sis region that was thought to encode an O antigen, with 12 pre-dicted coding sequences (CDSs) in the same transcriptional direc-tion. This locus was also found in the genome sequence of H.parasuis strain 29755 (37). The assignation of putative function tothe predicted products of these genes was based on their similarityto other glycosyltransferases and polysaccharide processing/ex-port proteins. In fact, the presence of the wza gene and homo-logues of wzb and wzc (ptp and wzs, respectively) strongly indicatesthat the locus is required for the biosynthesis of a polysaccharidecapsule, rather than an O antigen (33, 38, 39). Furthermore, ex-perimental evidence does not support the production of an Oantigen in H. parasuis (23–25, 40). We therefore propose that thisis a capsular polysaccharide biosynthesis locus.

Here we describe the complete DNA sequence and a detailedanalysis of this locus from the 15 serovar reference strains. Thelocus contains considerable serovar-specific variation, which mayhelp to form the basis for a molecular serotyping method andcould facilitate the development of glycoconjugate vaccines.

MATERIALS AND METHODSBacteria. Details of the 15 reference strains that are currently used forthe production of antisera for the H. parasuis serotyping scheme are inTable 1 (18).

Genome sequencing and draft genome sequence assembly. A singlecolony of each strain was picked and passaged on chocolate agar plates(Colombia agar base and 7% defibrinated horse blood supplemented with25 �g/ml NAD). After a minimum of four passages on chocolate agar, thestrains were scraped from the plates for genomic DNA preparation.Genomic DNA was prepared using a blood and tissue DNeasy kit (Qia-gen) as per the manufacturer’s instructions. For library preparation 500ng of genomic DNA was used, and modified Illumina protocols werefollowed (41, 42). Paired-end sequencing was performed at the WellcomeTrust Sanger Institute, Hinxton, Cambridge, United Kingdom. All strainswere sequenced on an Illumina HiSeq 2000 analyzer for 75 cycles, with

repeat sequencing being performed on the Illumina MiSeq for 150 cycleswhen required.

Draft genome sequences were assembled using a custom-made bioin-formatics pipeline, by removing undetermined bases, using Cutadapt(43), Sickle (44), and custom Perl scripts to match paired-end reads. Draftgenomes were finally assembled using Velvet (version 1.2.08) and VelvetOptimizer 2.2.0 (45, 46).

Comparison of the capsular polysaccharide biosynthesis loci fromthe reference strains. The first and last genes from the published poly-saccharide biosynthesis locus (36) were used to test for its presence inthe 15 reference strains. A BLAST database was created using the ge-nome sequencing data from the reference strains and queried using theHAPS0039 (starting at 49,282 bp on the SH0165 chromosome) andHAPS0052 (starting at 64,696 bp on the SH0165 chromosome) genesin a BLASTn search.

The potential capsule loci thus identified were visualized using theArtemis comparison tool (ACT) (47) to look for any variation in the locusbetween the reference strains. These comparisons were performed using acustom Perl script and NUCmer to order, concatenate, and align the draftcontigs of the reference strains using exact matches (100% identity) ofblocks of sequence (65 bp at a time) to the published genome of SH0165(36, 48, 49). Default NUCmer settings were used (48, 49). For each refer-ence strain, all open reading frames (ORFs) of more than 100 bp withinthe locus were examined for predicted CDSs and translated into aminoacid sequences using the SIB ExPASy Bioinformatics Resources Portal(50). These sequences were then queried against the NCBI database (usingBLASTp) to search for the possible functions of these proteins. The pre-dicted functions were recorded based on the highest amino acid identitymatch, with an E value threshold of 1 � 10�6. If the predicted CDS wasone previously identified in SH0165, then the highest non-H. parasuismatch was also recorded. Annotation of this locus was performed usingthis information. Predicted gene functions between the reference strainsof the 15 serovars were compared for similarity in composition to see if thesame gene was found in multiple serovars. Each capsule locus was analyzedfor the presence of promoters using BPROM (http://linux1.softberry.com/berry.phtml?topic�bprom&group�programs&subgroup�gfindb), wherethe top scores were found and mapped to within 150 bp preceding the startcodon of each CDS of the 15 capsule loci.

The nucleotide sequences of all CDSs from the 15 capsule loci werealigned using MUSCLE and then viewed by eye using SeaView (17, 21) togroup homologous genes present in the different serovars. A predicted

TABLE 1 Background information on the serovar reference strains ofH. parasuis that have been sequenced (18, 22, 63, 83)a

SerovarReferencestrain

Country oforigin

Isolationsite Diagnosis

1 No. 4 Japan Nose Healthy2 SW140 Japan Nose Healthy3 SW114 Japan Nose Healthy4 SW124 Japan Nose Healthy5 Nagasaki Japan Meninges Septicemia6 131 Switzerland Nose Healthy7 174 Switzerland Nose Healthy8 C5 Sweden Unknown Unknown9 D74 Sweden Unknown Unknown10 H555 Germany Nose Healthy11 H465 Germany Trachea Pneumonia12 H425 Germany Lung Polyserositis13 IA-84-17975 United States Lung Unknown14 IA-84-22113 United States Joint Unknown15 SD-84-15995 United States Lung Pneumoniaa These strains were supplied by National Veterinary Institute, Technical University ofDenmark, and Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma deBarcelona.

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function and gene name were given to these groups based on the BLASTpannotations (see Table S2 in the supplemental material). In a few caseswhere there were different gene function assignations for individual geneswithin a homology group, the most commonly assigned function andgene name were used.

To cluster the reference strains based on shared genes, the presenceand absence of all predicted CDSs across the capsule loci of the 15 refer-ence strains were recorded. This did not take into account gene order inthe locus. The presence or absence of genes was recorded as 0 or 1, respec-tively. To be able to use the pairwise distance matrix function from the Rpackage APE (51), this binary code was converted into a pseudo-nucle-otide-based code (A or T, respectively), which could be treated as a nu-cleotide alignment. Using R, the pairwise distance matrix was generatedbased on the coded presence/absence alignment, and a neighbor-joining(NJ) tree was then generated based on these data to show the relationshipbetween the gene compositions of the different loci using the APE, Phang-orn, and SeqinR software packages (51–53).

Further analysis of the similarities between the individual genes pres-ent in the capsule biosynthesis loci of serovars 5 and 12 was performed byaligning the amino acid sequences derived from the CDSs therein usingMUSCLE and viewing them using SeaView (54, 55). Amino acid differ-ences that might influence capsule structure, which determines the differ-ence between these two serovars, were recorded.

Comparison to polysaccharide loci of other bacteria. In order tovalidate our assignation of the capsule as being from group 1, a compar-ative analysis between H. parasuis and the characterized capsule groupsfrom different bacteria was carried out. A BLAST database was createdusing the 15 H. parasuis reference strains and queried using amino acidsequences (via tBLASTn) of known lipooligosaccharide (LOS), LPS, andcapsule genes from better-studied bacteria (Haemophilus influenzae, E.coli, and Klebsiella pneumoniae). Proteins involved in LOS and capsulebiosynthesis were selected from H. influenzae, as this bacterium is a some-what closer relative of H. parasuis than the other two and has knownpolysaccharide structures and protein sequences (56–58). Using the cap-sule proteins from H. influenzae allowed us to look for any homology to agroup 2 capsule within the H. parasuis genomes. E. coli was selected as amodel organism for both LPS and the different capsule groups (groups 1to 4), while K. pneumoniae is another example of a bacterium with a group1 capsule locus (59–62). The full list of genes used in this series of BLASTqueries can be found in Table S3 in the supplemental material.

Comparison of the capsule loci to multilocus sequence type (MLST)profiles. The relationship between the capsule loci was examined based onthe presence or absence of genes within the loci. We sought to comparethis to the relationship with the rest of the genome by looking at the MLSTprofiles of the reference strains. The MLST housekeeping genes from theSH0165 strain were used to query the BLAST database of the referencestrains (BLASTn). These genes were then selected from the genomes usingthe BLAST hit coordinates and a custom Perl script. The MLST house-keeping genes were concatenated in the order established by the MLSTscheme (63) and aligned using MUSCLE, and the phylogenetic tree wasbuilt using RAxML (54, 64).

Nucleotide sequence accession numbers. GenBank accession num-bers for the individual CDSs from the capsule loci of the 15 referencestrains are KC795279-KC795293, KC795295-KC795314, KC795316-KC795331, KC795333-KC795335, KC795351-KC795360, KC795362-KC795364 KC795336-KC795346, KC795348-KC795350, KC795365,KC795367-KC795379, KC795381-KC795384, KC795386-KC795400,KC795402-KC795417, KC853023, KC795418, KC795420-KC795423,KC795425-KC795436, KC795438-KC795441, KC795443-KC795455,KC795457-KC795476, KC795478-KC795491, KC795493-KC795508,KC795510-KC795528, KC795530-KC795533, KC795535-KC795546,and KC795548-KC795550 in order of serovar and gene position withinthe capsule locus.

RESULTS AND DISCUSSIONIdentification of capsular polysaccharide biosynthesis loci. Wereport the DNA sequences of the capsular polysaccharide biosyn-thesis loci in the reference strains of all 15 serovars of H. parasuis.The ACT comparisons of the 15 reference strains have been com-bined in Fig. 1. Strains are ordered by similarity, as determined bythe phylogeny based on gene presence and absence for the locus.This is just one representation of the similarities (and differences)between the capsule loci, ordered here to maximize similarity ofthe loci based on gene composition.

The genes marking the start (HAPS0039) and end (HAPS0052)of the locus were found in all strains, with the genes being foundon a single contig for all strains except the reference strain forserovar 2. We were able to align the capsule locus for the serovar 2reference strain to another serovar 2 genome, which we also se-quenced (isolate 9904791), in which the locus assembled onto oneclosed contig. All the predicted CDSs that were found were thesame between the two different serovar 2 isolates.

The location of the capsule loci was the same for all 15 referencestrains, based on the available contigs. The genes found at the 5=end of the locus include genes for four potential antibiotic resis-tance proteins, a helix-turn-helix (HTH) transcriptional regula-tor, an outer membrane protein, and several hypothetical pro-teins. The capsule loci were all followed at the 3= end by genesencoding an iron-sulfur cluster and several more hypotheticalproteins.

Identification of conserved and variable regions in the cap-sular biosynthesis loci. The capsule loci have been classified intotwo regions based on gene content (Fig. 2). Homologues of thewza-wzc genes are found at the 3= end of the locus, and they areconserved in all of the references strains. These genes are involvedin capsular polysaccharide surface expression and export, whichmatches the definition of region 1 from a model group 1 or 4capsule locus (32). This is the main line of genetic evidence for thepresence of a group 1 in H. parasuis (32). This region is followedby HAPS0052, which is predicted to be a HTH transcriptionalregulator that may influence transcription of the capsule loci andis similar to iscR (65). This is in addition to the HTH transcrip-tional regulator found preceding the locus. In comparison, the 5=end of the locus is variable, and so this has not been classified as aseparate region. Only HAPS0039 is present at the 5= end of thelocus in all strains. HAPS0039 was the locus tag given for theSH0165 gene found at the beginning of the locus. We have re-named this gene funA as the first gene in the locus with functionunknown. Region 2 has been designated between funA and wza,with variation in gene composition seen between most of the ref-erence strains (Fig. 2), with the notable exception of the loci fromserovars 5 and 12, which are identical in gene composition. Thisdivergent region may correspond to potential serovar-specific re-gions usually found in capsule loci, including a variety of genesinvolved in synthesis of oligosaccharide repeat units. These maybe distinctive for particular serovars and result in the productionof different serovar-specific polysaccharide structures (32, 33, 66).

The presence of the capsule-specific genes wza, wzb (ptp) andwzc (wzs), indicates that this locus does not in fact encode an Oantigen. Rather, we propose that the locus is required for the bio-synthesis of a group 1 polysaccharide capsule (33). With the reas-signment of the function of this locus, we contend that H. parasuisexpresses a LPS molecule lacking O antigen, sometimes called

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LOS. This fits with newly reported LOS structures for serovars 5and 15, which were found to be identical, both consisting of lipidA, a single phosphorylated 3-deoxy-D-manno-octulosonic acid(Kdo), and a globotetraose terminal sequence, with no O-antigen-like polysaccharide chains identified (23).

Gene compositions and homology groups of the 15 referencestrains’ capsule loci. In total 268 CDSs were found within thecapsule loci of the 15 reference strains (an average of 17.9 CDSsper locus), with 86 individually definable genes being annotated.Full details of the predicted gene functions for this locus orderedby reference strain can be found in Table S1 in the supplementalmaterial. A schematic diagram of the polysaccharide biosynthesisloci of the 15 reference strains, which compares the compositionsof the variable regions and the diverse range of gene functions, isshown in Fig. 3, with predicted genes colored by function and withthe addition of possible promoter locations. One gene was foundin the opposite transcriptional direction and appears to encode atransposase (KC795330) in the serovar 3 reference strain. Sixgenes were present in all 15 reference strains, while many CDSswere unique to a single reference strain based on the amino acidsequences. These genes are candidates for the development of amolecular serotyping assay and represent the huge diversity incapsule structures that might be produced by this bacterium. Tenof the CDSs identified had no significant BLASTp matches and so

have been labeled as function unknown (FUN) genes (67). CDSspredicted to encode hypothetical proteins have also been labeledas FUN genes. Of the top hits of these 268 CDSs, 44% have topBLAST hits from the family Pasteurellaceae. After the Pasteurel-laceae, the top hits were from the Enterobacteriaceae and theMoraxellaceae, with approximately 10% of the genes being fromeach family. These three bacterial families are all gammaproteo-bacteria, but the diversity in capsule genes found within thesegroups suggests that some of these genes were acquired throughhorizontal gene transfer.

An example of the diversity of genes found within a singlecapsule locus is found in the serovar 14 reference strain, whichcontains genes encoding a predicted acetyltransferase, a dehydro-genase, an oxidoreductase, two aminotransferases, two polysac-charide biosynthesis proteins, two epimerases, two glycosyltrans-ferases, and three hypothetical proteins within region 2.

The transcriptional organization of the loci can be predicted fromanalysis of promoter-like sequences. A �35 box promoter consensussequence precedes the funA gene in all the capsule loci apart from thatfrom serovar 13. At least three additional promoters have been pre-dicted for the majority of the loci. Eleven of the capsule loci havepredicted promoter sequences preceding the wza, wzb, and wzc genes,suggesting that they are transcribed separately from the 5= end of thelocus (Fig. 3). Additional promoter sequences were predicted within

FIG 1 Comparison of the reference strains for all 15 serovars of H. parasuis for the capsule loci. Strains are ordered to show the greatest homology between theloci, based on the neighbor-joining tree of gene presence and absence among the capsule loci, as seen on the left of the ACT comparisons. Red areas represent100% sequence identity, while white areas show variation (areas of nonidentical sequence).

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the capsule loci, which suggests that transcription is organized differ-ently in the different loci (Fig. 3).

While the first attempt at annotation of the capsule loci usedBLASTp to assess protein similarity, we also used nucleotide align-ment tools to determine similar/homologous genes with high se-quence identity among the reference strains. The main function/homology groups that we identified were genes for polysaccharidebiosynthesis, epimerases, and transferases. The remaining geneswere considered “not classified,” as they were similar to differentgenes in the public databases covering a wide range of possiblefunctions. A summary of these functions is presented in Table S2in the supplemental material, which gives the number of groupsfound and a tally of unique or orphan genes. Most of the geneswere grouped with genes of similar function (214 CDSs), with 48unique CDSs that did not align well with any other genes. Only 10of these 48 genes shared sequence identity with genes from thePasteurellaceae.

Serovar-specific genes. With the exception of serovars 5 and12, serovar-specific genes were identified in 12 other serovars. TheCDSs in serovar 1 have all been identified in at least one othercapsule locus, and so it contains no serovar-specific CDSs. Acrossthe remaining serovars, the majority of serovar-specific CDSs ei-ther have no known function or encode glycosyltransferases,based on the unique CDSs from the homology group analysis.This wide range of CDSs, particularly the glycosyltransferases,suggests that the structures of the capsules between the serovarscould vary greatly. This is in addition to the 26 glycosyltransferaseCDSs that are found among all the serovars, which could create awide range of polysaccharide structures. Verification of the actualserovar specificity of these CDSs is required to be certain that theycould be candidates for a molecular serotyping assay by analyzingfurther capsule loci from other serotyped field isolates.

Similarities between the capsule loci. Region 2 of the capsulelocus is identical in gene composition in serovars 5 and 12, theonly two regions that are identical among the reference strains(Fig. 3). This was not expected, as serovars 5 and 12 can be distin-guished from one another in the serotyping scheme. However,vaccine-induced cross-protection between serovars 5 and 12 hasbeen reported on several occasions (11, 22, 68), and it is also acommon cross-reaction in the serotyping results of field strains(20, 21). Alignments of the CDSs revealed a total of 48 amino acidsubstitutions among the 12 of the 14 CDSs that are found in bothserovars. Of these differences, 40 amino acid changes are likely toinfluence the structure of the proteins produced. These changescould be sufficient to alter the polysaccharide structures pro-duced, leading to definition as separate serovars. These amino acidchanges have been found in all CDSs except iscR and wzb, with thehighest number being in wbgY (encoding a glycosyltransferase)and wzx (encoding flippase). In addition to these amino acid sub-stitutions, promoter analysis has predicted an additional pro-moter in the serovar 5 strain. At the whole-genome level, the ref-erence strains for serovars 5 and 12 are not identical, with theserovar 12 strain sharing 81.7% nucleotide identity with the com-plete SH0165 strain (serovar 5) (our unpublished data). Furtherdifferences between these serovars may be due to a modification ofthe capsule encoded by a gene outside the main capsule locus ordifferential expression of capsule genes (29, 69, 70).

There are other serovars that are very similar to each other atthis locus, with serovars 1, 2, 7, and 11 forming a group and sero-vars 8 and 10 forming another, whose capsule loci differ by onlythree CDSs. For these serovar groupings, we would expect thecapsular polysaccharide structures to be quite similar based on thehigh identity in gene composition. This similarity in gene compo-sition may also indicate that they have diversified from a single

FIG 2 Separation of the serovar 5 capsule locus into potential capsule regions with details of the predicted functions. This is based on the published strain SH0165(36).

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locus within the serovar group. Other serovars share some CDSsand intergenic regions but to a lesser extent than the examplesgiven above, and so we are less certain of their relationships. Thesesimilarities in gene composition (as seen in Fig. 1) are summarizedin Fig. 4, which, irrespective of gene order, shows the presence andabsence of all the genes in the 15 reference strains.

Genes that can be found in capsule and LPS biosynthesis locifrom other bacteria. The wzx and wzy genes are involved in theassembly of both O antigens and group 1 capsular polysaccha-rides, and so there are similarities between the biosynthetic path-ways of these polysaccharide structures (32). We have found thatthese genes are not present in all capsule loci for H. parasuis basedon the BLASTp annotation (see Table S1 in the supplemental ma-terial). The wzx allele is not present in three serovars (1, 3, and 11),while there is experimental evidence that at least one of thesestrains produces a capsule (26), and so the wzx gene may havebeen replaced by another gene that plays a similar role within thelocus. Alternatively, the presence of the wza export protein may besufficient for the production of the capsule in these serovars. Con-versely, the wzy gene is absent from the majority of capsule loci,but again there are many other hypothetical genes within the locithat might substitute for this gene.

So far, we have discussed the content of the capsule loci with

respect to homologues specific to a group 1 capsule locus. Group 2and 3 capsules are assembled in a processive way, using glycosyl-transferases instead of the wzy product and an ABC transporter inplace of the wzx product for export of the polysaccharide (32, 39).Within the capsule loci we identified one putative ABC trans-porter gene with low sequence identity in the reference strain forserovar 8 (funM8 KC795416) based on initial BLASTp annota-tion, but it aligned well with two hypothetical genes in serovars 6and 10 (funM6 and funM10, respectively), and so these genes havebeen assigned the name funM, with no known function at thistime (Fig. 3). One O-antigen ligase was predicted within the cap-sule locus of serovar 13 (waaL13 KC795502) (Fig. 3), but with only27% sequence identity, this may be an inaccurate assignation (31,39). Further study of the individual genes within the loci and theirfunctional roles in the expression and structure of the capsule isrequired to understand the mechanisms of capsular polysaccha-ride biosynthesis in H. parasuis and the potential impact on sero-typing and virulence (26).

To reinforce the assignation as a capsule locus, we lookedfor homologues to genes involved in capsule assembly for allgroups, in LOS biosynthesis, and in O-antigen biosynthesisusing tBLASTn analyses of the reference strains. The tBLASTnresults can be found in Table S3 in the supplemental material.

FIG 3 Schematic of each capsule locus for all 15 reference strains of H. parasuis. Serovars are ordered based on highest identity of gene composition. Directionof CDS is represented by the direction of the arrow; the gene name assigned is inside the arrow. Coloring represents the predicted function of the gene. Genes aregrouped to show similarity between serovars where possible. Arrows above the CDSs represent predicted promoters from BPROM output.

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We have found homologues of LOS core proteins and group 1capsule loci with high sequence identity throughout the refer-ence strains. In comparison, proteins involved in the produc-tion of the O antigen were not found to have homologues withhigh sequence identity in this bacterium, with the exception ofthose that may also have a role in capsule production (e.g.,Wzx, glycosyltransferases, and WbaP). Similarly, the onlygroup 2 and 3 capsule loci homologues found are ABC trans-porters with low sequence identity (�25%); this is in fact alarge protein family that plays many roles within the cell asidefrom capsule biosynthesis and so may not be involved in cap-sule production (71). While group 4 capsule loci are producedin a similar fashion to a group 1 capsule loci, only two homo-logues of group 4 capsule genes were identified in the H. para-suis reference strains out of 12 known genes (see Table S3) (32).The lack of further proteins involved in O antigen and group 2,3, or 4 capsule biosynthesis in the genomes of the H. parasuisreference strains supports our assignation of the capsule in thisbacterium to group 1.

Relationship between capsule structure and genetics. Thecapsule structures of serovars 5 and 15 were recently published,both of which contained the same main chain with a disacchariderepeating unit of �-glucose-6P and 2,4-diacetamido-2,4,6-tride-oxy-D-galactopyranose substituted with �-Neu5R-3-�-GalNAc-1-P (serovar 15 strain) or �-Neu5R-3-�-Gal-1-P (serovar 5strain) side chains, where R is an N-acetyl or N-glycolyl group(72). The relationship between the capsule genes identified in thisstudy and the capsule structures show some similarities for bothserovars. This includes the presence of the neuA gene in both loci,which is likely to be involved in the generation of the Neu5Rgroups in both capsule structures. For the serovar 5 structure,

wcwK could be involved in phosphorylation of the UDP-Galgroup, and wbgX could be involved in the synthesis of the reduc-ing end sugar. For the serovar 15 structure, the ugeA product islikely to convert glucose to galactose, with the links between theNeu5 group and the main chain being uncertain at this time. Therole of capD in the loci is also uncertain; with its product havingfive predicted transmembrane domains, it may encode an UndPPtransferase (36). The wide gene variation within the capsule loci ofthe 15 reference strains shows that there is the possibility for a widevariety of capsule structures to be produced by this bacterial spe-cies. While the genes in the serovar 5 and 15 loci differ signifi-cantly, the capsule structures contain the same main chain withvarious side chains. This could suggest that while the genes differat the nucleotide level, they could play similar roles, particularlyfor the glycosyltransferases. These genes differ between the loci,but the majority of the sugars are found in both structures. Alter-natively, the additional genes found within the loci could be ex-pressed under different conditions, such as in different growthphases or in response to the host environment, something that ishighly likely given the presence of multiple promoters and homo-logues of the icsR transcriptional regulator.

The capsular polysaccharide acts as the barrier between thebacterium and the environment, including resistance to desicca-tion, adherence, antiphagocytic activity, and interaction withcomplement, which all contribute to its common association withvirulence and reputation as a virulence factor (73–75). Severalpolysaccharide modifications can be found in the structuresthat aid in this role, including sialyl groups and phosphoryl-choline (25, 76–78). The presence of sialyl groups in the newlypublished structures may be an important factor for virulenceof this bacterium, as has been suggested for H. parasuis and

FIG 4 Summary of genes present in the capsule loci of the 15 reference strains. Strains are ordered by identity, based on the neighbor-joining tree of gene presenceand absence among the capsule loci, as seen on the left of the diagram. The genes found in the capsule locus of the 15 reference strains are separated into sixcategories, colored by function. Unique genes have been excluded. Genes are ordered by decreasing frequency of presence across the 15 reference strains from leftto right.

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shown in H. influenzae (25, 76, 77). Homologues of lsgB andneuA have been predicted for the capsule loci of serovars 5and 15, which would allow the modification and transfer ofNeu5 in the capsule structures. We can see that these genes arealso present in other capsule loci (Fig. 3), with an additionalgene, astA, also being predicted as a sialyltransferase. Fromthis, we could predict that sialylation will be present in 11 of thecapsule structures, with the exceptions of serovars 1, 2, 3, and11. The link between genes involved in sialylation and clinicaldisease has previously been suggested for nine of the serovars,while the link between serovar and virulence is less clear (25,79, 80).

Capsule origin and diversity. This is the first group 1 capsulelocus to be found within the genus Haemophilus, and no othermembers of the Pasteurellaceae have yet been recorded as contain-ing a group 1 capsule (based on literature searches of 26 otherPasteurellaceae species). Group 2 or group 3 capsules have beenrecorded for other Pasteurellaceae, including H. influenzae and A.pleuropneumoniae as well as four other Pasteurellaceae species, butthese are quite distantly related species, based on the molecularphylogenies that have been produced to date (58, 66, 81, 82).While many of the predicted CDSs show homology to closely re-lated Pasteurellaceae species, a greater number match a diverserange of bacterial species.

We also investigated the relationship between the capsule lociin the context of the relationships between the strains derivedfrom their MLST profiles (see Fig. S1 in the supplemental mate-rial) and showed that these two data sets indicate different inter-strain relationships. For example, the most distantly related sero-vars are 8 and 10 according to the MLST tree, but they differ byonly three CDSs in the capsule locus. The main similarity that canbe seen is that serovars 5 and 12 are quite closely related, althoughnot identical, in the MLST tree. This information combined withthe wide diversity of BLAST hits used for the capsule annotationsuggests that these capsule loci may have arisen through horizon-tal gene transfer and since diversified.

Concluding remarks. Capsule gene sequences have been usedto develop successful molecular serotyping assays for classifying E.coli (28), H. influenzae (30, 70), and Actinobacillus pleuropneumo-niae (69). We suggest that the capsular polysaccharides are thedominant components in the determination of serovar in H. para-suis, and therefore that the genetic loci encoding proteins requiredfor biosynthesis and assembly of capsule structures might be use-ful for differentiating serovars at the DNA level even though ad-ditional bacterial components may also be involved.

While the locus has not been experimentally validated at thistime, we have attempted to show via extensive computationalanalyses that the genes in this locus show the greatest homology tothose known to be involved in the group 1 capsular polysaccharidebiosynthesis in better-studied organisms (32, 60). This fits withthe current experimental data that show the presence of both LOSand capsule structures in this bacterium (23). Understanding thislocus will greatly enhance our understanding of the basis for sero-type in H. parasuis and will contribute to the field of H. parasuismicrobiology.

In conclusion, we report the gene compositions and sequencesof the putative capsule loci for all 15 reference strains of H. para-suis. These sequences are a great resource for understanding cap-sule biosynthesis in this bacterium and, with the large number of

“orphan” genes found, may be useful for the development of amolecular serotyping assay for H. parasuis.

ACKNOWLEDGMENTS

This work was supported by a BPEX Ph.D. studentship and a Longer andLarger (LoLa) grant from the Biotechnology and Biological Sciences Re-search Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1, and BB/G003203/1), the UK Department for Environment,Food and Rural Affairs and Zoetis, awarded to the Bacterial RespiratoryDiseases of Pigs-1 Technology (BRaDP1T) consortium. The funders hadno role in study design, data collection or analysis, decision to publish, orpreparation of the manuscript.

Consortium members are as follows: Duncan J. Maskell, Alexander W.(Dan) Tucker, Sarah E. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang,Shi-Lu Luan, and Roy R. Chaudhuri (present address: Centre forGenomic Research, University of Liverpool, Liverpool, United Kingdom)(University of Cambridge); Andrew N. Rycroft, Gareth A. Maglennon,and Dominic Matthews (Royal Veterinary College); Paul R. Langford,Janine T. Bossé, and Yanwen Li (Imperial College London); and BrendanW. Wren, Jon Cuccui, and Vanessa Terra (London School of Hygiene andTropical Medicine).

REFERENCES1. White M. 2010. NADIS pig health—March 2010 Glässers disease. BPEX

Knowledge Transfer, p 1–3.2. Kilian M, Frederiksen W. 1981. Ecology of Haemophilus, Pasteurella and

Actinobacillus, p 11–38. In Kilian M, Frederiksen W, Biberstein E. (ed),Haemophilus, Pasteurella and Actinobacillus. Academic Press, London,United Kingdom.

3. Kilian M. 1976. A taxonomic study of the genus Haemophilus, with theproposal of a new species. J. Gen. Microbiol. 93:9 – 62.

4. Oliveira S, Pijoan C. 2004. Computer-based analysis of Haemophilusparasuis protein fingerprints. Can. J. Vet. Res. 68:71–75.

5. Rapp-Gabrielson V, Oliveira S, Pijoan C. 2006. Haemophilus parasuis, p681– 690. In Straw BE, D’Allaire S, Zimmerman JJ, Taylor DJ (ed), Dis-eases of swine, 9th ed. Wiley-Blackwell, Ames, IA.

6. De la Fuente AJM, Tucker AW, Navas J, Blanco M, Morris SJ, Gutiér-rez-Martín CB. 2007. Antimicrobial susceptibility patterns of Haemophi-lus parasuis from pigs in the United Kingdom and Spain. Vet. Microbiol.120:184 –191.

7. Zhou X, Xu X, Zhao Y, Chen P, Zhang X, Chen H, Cai X. 2010.Distribution of antimicrobial resistance among different serovars of Hae-mophilus parasuis isolates. Vet. Microbiol. 141:168 –173.

8. Rafiee M, Blackall PJ. 2000. Establishment, validation and use of theKielstein-Rapp-Gabrielson serotyping scheme for Haemophilus parasuis.Aust. Vet. J. 78:172–174.

9. Takahashi K, Naga S, Yagihashi T, Ikehata T, Nakano Y, Senna K,Maruyama T, Murofushi J. 2001. A cross-protection experiment in pigsvaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, andevaluation of a bivalent vaccine under laboratory and field conditions. J.Vet. Med. Sci. 63:487– 491.

10. Smart NL, Miniats OP. 1989. Preliminary assessment of a Haemophilusparasuis bacterin for use in specific pathogen free swine. Can. J. Vet. Res.53:390 –393.

11. Bak H, Riising HJ. 2002. Protection of vaccinated pigs against experimen-tal infections with homologous and heterologous Haemophilus parasuis.Vet. Rec. 151:502–505.

12. Angen O, Svensmark B, Mittal KR. 2004. Serological characterization ofDanish Haemophilus parasuis isolates. Vet. Microbiol. 103:255–258.

13. Dijkman R, Wellenberg GJ, van der Heijden HM, Peerboom R, OlveraA, Rothkamp A, Peperkamp K, Van Esch EJ. 2012. Analyses of DutchHaemophilus parasuis isolates by serotyping, genotyping by ERIC-PCRand Hsp60 sequences and the presence of the virulence associated trimericautotransporters marker. Res. Vet. Sci. 93:589 –595.

14. Gallant Custom Labs. 2010. Current advances: analysis of Haemophilusparasuis serotype prevalence 2008 –2009. Gallant Custom Labs, Cam-bridge, Ontario, Canada.

15. Miniats OP, Smart NL, Rosendal S. 1991. Cross protection amongHaemophilus parasuis strains in immunized gnotobiotic pigs. Can. J. Vet.Res. 55:37– 41.

Haemophilus parasuis Capsule Loci

September 2013 Volume 195 Number 18 jb.asm.org 4271

on March 6, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

16. Pfizer Animal Health. 2011. A guide to Pfizer animal health products forthe treatment and prevention of swine diseases. Pfizer, New York, NY.

17. Hipra. 2012. Hiprasuis® Glässer. Hipra, Girona, Spain.18. Kielstein P, Rapp-Gabrielson VJ. 1992. Designation of 15 serovars of

Haemophilus parasuis on the basis of immunodiffusion using heat-stableantigen extracts. J. Clin. Microbiol. 30:862– 865.

19. Morozumi T, Nicolet J. 1986. Some antigenic properties of Haemophilusparasuis and a proposal for serological classification. J. Clin. Microbiol.23:1022–1025.

20. Del Río ML, Gutiérrez CB, Rodríguez Ferri EF. 2003. Value of indirecthemagglutination and coagglutination tests for serotyping Haemophilusparasuis. J. Clin. Microbiol. 41:880 – 882.

21. Tadjine M, Mittal KR, Bourdon S, Gottschalk M. 2004. Development ofa new serological test for serotyping Haemophilus parasuis isolates anddetermination of their prevalence in North America. J. Clin. Microbiol.24:839 – 840.

22. Turni C, Blackall PJ. 2005. Comparison of the indirect haemagglutina-tion and gel diffusion test for serotyping Haemophilus parasuis. Vet. Mi-crobiol. 106:145–151.

23. Perry MB, Maclean LL, Gottschalk M, Aragon V, Vinogradov E. 2013.Structure of the capsular polysaccharides and lipopolysaccharides fromHaemophilus parasuis strains ER-6P (serovar 15) and Nagasaki (serovar5). Carbohydrate Res. doi:10.1016/j.carres.2013.04.023. [Epub ahead ofprint.]

24. Xu C, Zhang L, Zhang B, Feng S, Zhou S, Li J, Zou Y, Liao M. 2013.Involvement of lipooligosaccharide heptose residues of Haemophilusparasuis SC096 strain in serum resistance, adhesion and invasion. Vet. J.195:200 –204.

25. Martínez-Moliner V, Soler-Llorens P, Moleres J, Garmendia J, AragonV. 2012. Distribution of genes involved in sialic acid utilization in strainsof Haemophilus parasuis. Microbiology 158:2117–2124.

26. Morozumi T, Nicolet J. 1986. Morphological variations of Haemophilusparasuis strains. J. Clin. Microbiol. 23:138 –142.

27. Kong F, Wang W, Tao J, Wang L, Wang Q, Sabananthan A, Gilbert GL.2005. A molecular-capsular-type prediction system for 90 Streptococcuspneumoniae serotypes using partial cpsA-cpsB sequencing and wzy- orwzx-specific PCR. J. Med. Microbiol. 54:351–356.

28. Durso LM, Bono JL, Keen JE. 2005. Molecular serotyping of Escherichiacoli O26:H11. Appl. Environ. Microbiol. 71:4941– 4944.

29. Poly F, Serichatalergs O, Schulman M, Ju J, Cates CN, Kanipes M,Mason C, Guerry P. 2011. Discrimination of major capsular types ofCampylobacter jejuni by multiplex PCR. J. Clin. Microbiol. 49:1750 –1757.

30. Falla TJ, Crook DW, Brophy LN, Maskell D, Kroll JS, Moxon ER. 1994.PCR for capsular typing of Haemophilus influenzae. J. Clin. Microbiol.32:2382–2386.

31. Whitfield C, Roberts IS. 1999. Structure, assembly and regulation ofexpression of capsules in Escherichia coli. Mol. Microbiol. 31:1307–1319.

32. Whitfield C. 2006. Biosynthesis and assembly of capsular polysaccharidesin Escherichia coli. Annu. Rev. Biochem. 75:39 – 68.

33. Drummelsmith J, Whitfield C. 1999. Gene products required for surfaceexpression of the capsular form of the group 1 K antigen in Escherichia coli(O9a:K30). Mol. Microbiol. 31:1321–1332.

34. Boulnois GJ, Roberts IS, Hodge R, Hardy KR, Jann KB, Timmis KN.1987. Definition of three functional regions for capsule production. Mol.Genet. Genomics 208:242–246.

35. Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, Coplin D,Kido N, Klena J, Maskell D, Raetz CRH, Rick PD. 1996. Bacterialpolysaccharide synthesis and gene nomenclature. Trends Microbiol.4:495–503.

36. Xu Z, Yue M, Zhou R, Jin Q, Fan Y, Bei W, Chen H. 2011. Genomiccharacterization of Haemophilus parasuis SH0165, a highly virulent strainof serovar 5 prevalent in China. PLoS One 6:e19631. doi:10.1371/journal.pone.0019631.

37. Mullins MA, Register KB, Bayles DO, Dyer DW, Kuehn JS, Phillips GJ.2011. Genome sequence of Haemophilus parasuis strain 29755. Stand.Genomic Sci. 5:61– 68.

38. Whitfield C. 1995. Biosynthesis of lipopolysaccharide O antigens. TrendsMicrobiol. 3:178 –185.

39. Whitfield C, Paiment A. 2003. Biosynthesis and assembly of group 1capsular polysaccharides in Escherichia coli and related extracellular poly-saccharides in other bacteria. Carbohydr. Res. 338:2491–2502.

40. Zucker B, Krüger M, Rehak E, Horsch F. 1994. The lipopolysaccharide

structure of Haemophilus parasuis strains in SDS-PAGE. Berl. Munch.Tierarztl. Wochenschr. 107:78 – 81.

41. Quail MA, Swerdlow H, Turner DJ. 2009. Improved protocols for theIllumina genome analyzer sequencing system. Curr. Protoc. Hum. Genet.18:1–27.

42. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R,Swerdlow H, Turner DJ. 2008. A large genome centre’s improvements tothe Illumina sequencing system. Nat. Methods 5:1005–1010.

43. Martin M. 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17.

44. Joshi N. 2012. Sickle—windowed adaptive trimming for fastq filesusing quality. UC Davis Bioinformatics Core, University of California,Davis, CA. https://github.com/najoshi/sickle.

45. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short readassembly using de Bruijn graphs. Genome Res. 18:821– 829.

46. Gladman S. 2009. VelvetOptimiser, version 2.2.5. http://www.vicbioinformatics.com/velvetoptimiser.manual.txt.

47. Carver T, Berriman M, Tivey A, Patel C, Böhme U, Barrell BG, ParkhillJ, Rajandream M-A. 2008. Artemis and ACT: viewing, annotating andcomparing sequences stored in a relational database. Bioinformatics 24:2672–2676.

48. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C,Salzberg SL. 2004. Versatile and open software for comparing large ge-nomes. Genome Biol. 5:R12. doi:10.1186/gb-2004-5-2-r12.

49. Delcher AL, Kasif S, Fleischmann RD, Peterson J, White O, Salzberg SL.1999. Alignment of whole genomes. Nucleic Acids Res. 27:2369 –2376.

50. Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, De CastroE, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, HernandezC, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K,Redaschi N, Rossier G, Xenarios I, Stockinger H. 2012. ExPASy: SIBbioinformatics resource portal. Nucleic Acids Res. 40(W1):W597–W603.

51. Paradis E, Claude J, Strimmer K. 2004. APE: analyses of phylogeneticsand evolution in R language. Bioinformatics 20:289 –290.

52. Schliep KP. 2011. phangorn: phylogenetic analysis in R. Bioinformatics27:592–593.

53. Charif D, Lobry JR. 2007. Seqin® 1.0 –2: a contributed package to the Rproject for statistical computing devoted to biological sequences retrievaland analysis, p 207–232. In Bastolla U, Porto M, Roman HE, VendruscoloM (ed), Structural approaches to sequence evolution: molecules, net-works, populations. Springer Verlag, New York, NY.

54. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accu-racy and high throughput. Nucleic Acids Res. 32:1792–1797.

55. Gouy M, Guindon S, Gascuel O. 2010. SeaView version 4: a multiplat-form graphical user interface for sequence alignment and phylogenetictree building. Mol. Biol. Evol. 27:221–224.

56. Hood DW, Cox a, Wakarchuk DWW, Schur M, Schweda EK, Walsh SL,Deadman ME, Martin a, Moxon ER, Richards JC. 2001. Genetic basis forexpression of the major globotetraose-containing lipopolysaccharidefrom Haemophilus influenzae strain Rd (RM118). Glycobiology 11:957–967.

57. Schweda EKH, Richards JC, Hood DW, Moxon ER. 2007. Expressionand structural diversity of the lipopolysaccharide of Haemophilus influen-zae: implication in virulence. Int. J. Med. Microbiol. 297:297–306.

58. Kroll J, Booy R. 1996. Haemophilus influenzae: capsule vaccine and cap-sulation genetics. Mol. Med. Today 2:160 –165.

59. Regué M, Climent N, Abitiu N, Coderch N, Merino S, Izquierdo L,Altarriba M, Juan M, Toma JM. 2001. Genetic characterization of theKlebsiella pneumoniae waa gene cluster, involved in core lipopolysaccha-ride biosynthesis. J. Bacteriol. 183:3564 –3573.

60. Rahn A, Drummelsmith J, Whitfield C. 1999. Conserved organization inthe cps gene clusters for expression of Escherichia coli group 1 K antigens:relationship to the colanic acid biosynthesis locus and the cps genes fromKlebsiella pneumoniae. J. Bacteriol. 181:2307–2313.

61. Samuel G, Reeves P. 2003. Biosynthesis of O-antigens: genes and path-ways involved in nucleotide sugar precursor synthesis and O-antigen as-sembly. Carbohydr. Res. 338:2503–2519.

62. Orskov I, Orskov F, Jann B, Jann K. 1977. Serology, chemistry, andgenetics of O and K antigens of Escherichia coli. Bacteriol. Rev. 41:667–710.

63. Olvera A, Cerdà-Cuéllar M, Aragon V. 2006. Study of the populationstructure of Haemophilus parasuis by multilocus sequence typing. Micro-biology 152:3683–3690.

64. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phy-

Howell et al.

4272 jb.asm.org Journal of Bacteriology

on March 6, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

logenetic analyses with thousands of taxa and mixed models. Bioinformat-ics 22:2688 –2690.

65. Kiley PJ, Beinert H. 2003. The role of Fe-S proteins in sensing andregulation in bacteria. Curr. Opin. Microbiol. 6:181–185.

66. Jessing SG, Ahrens P, Inzana TJ, Angen Ø. 2008. The genetic organisa-tion of the capsule biosynthesis region of Actinobacillus pleuropneumoniaeserotypes 1, 6, 7, and 12. Vet. Microbiol. 129:350 –359.

67. Hinton JCD. 1997. The Escherichia coli genome sequence: the end of anera or the start of the FUN? Mol. Microbiol. 26:417– 422.

68. Nedbalcova K, Kucerova Z, Krejci J, Tesarik R, Gopfert E, Kummer V,Leva L, Kudlackova H, Ondriasova R, Faldyna M. 2011. Passive im-munisation of post-weaned piglets using hyperimmune serum against ex-perimental Haemophilus parasuis infection. Res. Vet. Sci. 91:225–229.

69. Angen O, Ahrens P, Jessing SG. 2008. Development of a multiplex PCRtest for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and12. Vet. Microbiol. 132:312–318.

70. Billal DS, Hotomi M, Suzumoto M, Yamauchi K, Kobayashi I, FujiharaK, Yamanaka N. 2007. Rapid identification of nontypeable and serotypeb Haemophilus influenzae from nasopharyngeal secretions by the multi-plex PCR. Int. J. Pediatr. Otorhinolaryngol. 71:269 –274.

71. Linton KJ. 2007. Structure and function of ABC transporters. Physiology22:122–130.

72. Perry MB, Maclean LL, Gottschalk M, Aragon V, Vinogradov E. 2013.Structure of the capsular polysaccharides and lipopolysaccharides from Hae-mophilus parasuis strains ER-6P (serovar 15) and Nagasaki (serovar 5). Car-bohydr. Res. doi:10.1016/j.carres.2013.04.023. [Epub ahead of print.]

73. Boyce JD, Chung JY, Adler B. 2000. Genetic organisation of the capsulebiosynthetic locus of Pasteurella multocida M1404 (B:2). Vet. Microbiol.72:121–134.

74. Moxon ER, Kroll JS. 1988. Type b capsular polysaccharide as a virulencefactor of Haemophilus influenzae. Vaccine 6:113–115.

75. Struve C, Krogfelt KA. 2003. Role of capsule in Klebsiella pneumoniaevirulence: lack of correlation between in vitro and in vivo studies. FEMSMicrobiol. Lett. 218:149 –154.

76. Jenkins GA, Figueira M, Kumar GA, Sweetman WA, Makepeace K,Pelton SI, Moxon R, Hood DW. 2010. Sialic acid mediated transcrip-tional modulation of a highly conserved sialometabolism gene cluster inHaemophilus influenzae and its effect on virulence. BMC Microbiol. 10:48.

77. Clark SE, Eichelberger KR, Weiser JN. 2013. Evasion of killing by humanantibody and complement through multiple variations in the surface oli-gosaccharide of Haemophilus influenzae. Mol. Microbiol. 88:603– 618.

78. Shi F, Harada T, Ogawa Y, Ono H, Ohnishi-Kameyama M, MiyamotoT, Eguchi M, Shimoji Y. 2012. Capsular polysaccharide of Erysipelothrixrhusiopathiae, the causative agent of swine erysipelas, and its modificationwith phosphorylcholine. Infect. Immun. 80:3993– 4003.

79. Oliveira S, Pijoan C. 2004. Haemophilus parasuis: new trends on diagno-sis, epidemiology and control. Vet. Microbiol. 99:1–12.

80. Aragon V, Cerdà-Cuéllar M, Fraile L, Mombarg M, Nofrarías M, OlveraA, Sibila M, Solanes D, Segalés J. 2010. Correlation between clinico-pathological outcome and typing of Haemophilus parasuis field strains.Vet. Microbiol. 142:387–393.

81. Christensen H, Kuhnert P, Olsen JE, Bisgaard M. 2004. Comparativephylogenies of the housekeeping genes atpD, infB and rpoB and the 16SrRNA gene within the Pasteurellaceae. Int. J. Syst. Evol. Microbiol. 54:1601–1609.

82. Kielstein P, Wuthe H, Angen O, Mutters R, Ahrens P. 2001. Phenotypicand genetic characterization of NAD-dependent Pasteurellaceae from therespiratory tract of pigs and their possible pathogenetic importance. Vet.Microbiol. 81:243–255.

83. Zehr ES, Lavrov DV, Tabatabai LB. 2012. Comparison of Haemophilusparasuis reference strains and field isolates by using random amplifiedpolymorphic DNA and protein profiles. BMC Microbiol. 12:108.

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