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Centre de Recerca en Sanitat Animal (CReSA), Campus de Bellaterra-Universitat Autònoma de Barcelona, 08193-Bellaterra, Barcelona, Spain
Correspondence
Virginia Aragon
virginia.aragon{at}cresa.uab.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are DQ781411–DQ782327.
A supplementary table giving strain details is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition to Glässer's disease, H. parasuis produces other clinical outcomes, such as pneumonia, and colonizes the upper respiratory tract of healthy animals (Rapp-Gabrielson et al., 2006). Although it is commonly accepted that one strain is responsible for each clinical outbreak, diagnosis of H. parasuis infection is complicated by the fact that it is usual to detect several strains in a farm and even within a single animal. Therefore, it is essential to determine the causative strain by its isolation from organs with the characteristic lesions of the disease.
Differences among strains in phenotypic and genotypic characteristics have been reported, although no clear association with virulence could be determined (Oliveira & Pijoan, 2004; Rapp-Gabrielson et al., 2006). However, several studies have confirmed that different strains of H. parasuis have different pathogenic capacity (Kielstein & Rapp-Gabrielson, 1992; Nielsen, 1993; Rapp-Gabrielson et al., 1992; Vahle et al., 1995). Classically, strains of H. parasuis have been classified by serotyping, and although this method is useful for vaccine implementation, it is not discriminative enough for epidemiology. Moreover, a high percentage of strains are non-typable by serotyping (Oliveira & Pijoan, 2004; Rapp-Gabrielson et al., 2006). Recently, different genotyping methods have been proposed to differentiate H. parasuis strains. The majority of them are fingerprinting methods, and, even though the reported techniques have a higher level of discrimination than serotyping, they present application problems, such as limited resolution (de la Puente-Redondo et al., 2000, 2003; del Rio et al., 2006) or difficulty in comparing results from different laboratories (Rafiee et al., 2000; Smart et al., 1988). To improve the epidemiological study of H. parasuis strains, a single-locus sequence typing method was recently used by our group (Olvera et al., 2006). The heterogeneity of H. parasuis field isolates was confirmed by hsp60 partial sequencing, but although a virulent cluster was detected, the classification of the strains was not satisfactory. Moreover, the results of our study with hsp60 and the 16S rRNA gene indicated a possible lateral gene transfer within H. parasuis strains and between H. parasuis and Actinobacillus spp. Thus, to achieve robustness against the effects of recombination and maintain an adequate resolution, we have developed a multilocus sequence typing (MLST) for H. parasuis.
MLST is based on the sequencing of 450–600 bp fragments of core genes and the assignment of allelic profiles, which leads to sequence types (ST). The advantages of MLST for local and global epidemiology have been extensively discussed elsewhere (Cooper & Feil, 2004; Enright & Spratt, 1999; Maiden et al., 1998; Spratt, 1999). MLST has been successfully used for the determination of clonal complexes (CC) of several human and animal pathogens (Dingle et al., 2001; Enright & Spratt, 1998; Enright et al., 2001; Feavers et al., 1999; Heym et al., 2002; Homan et al., 2002; King et al., 2002; Kriz et al., 2002; Lemee et al., 2004; Nallapareddy et al., 2002; Noller et al., 2003; Shi et al., 1998; van Loo et al., 2002; Wang et al., 2003), including Haemophilus influenzae (Meats et al., 2003). Since the genomic sequence of H. parasuis is not available, we used primers designed for H. influenzae (Meats et al., 2003), universal primers (Christensen et al., 2004), or primers designed to areas of homology of the selected genes in other bacteria, including Pasteurellaceae. On the other hand, a major problem of current MLST databases is the poor representation of non-pathogenic isolates within species of clinical interest. In some cases, non-virulent strains can represent a significant part of the population, which is important in order to define population structures and to estimate population parameters (Perez-Losada et al., 2006). For that reason, an effort was made to have a representative sample of the natural population of H. parasuis by also sampling asymptomatic carriers.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Clinical strains were isolated from characteristic lesions from diseased animals or kindly provided by the Department of Infectious Diseases of the Veterinary School of the Universitat Autònoma de Barcelona (Spain), by Dr E. Rodríguez Ferri (Universidad de León, Spain), by Dr Gustavo C. Zielinski (Instituto Nacional de Tecnología Agropecuaria-INTA, Argentina), Dr Øystein Angen (Danish Institute for Food and Veterinary Research, Denmark) and by Dr T. Blaha (Federal Institute for Health Protection of Consumers and Veterinary Medicine, Germany). All the strains were maintained in 20 % glycerol-Brain Heart Infusion at –80 °C and routinely cultured in chocolate agar plates at 37 °C with 5 % CO2 for 24–48 h.
DNA extraction, primers and PCR conditions.
Genomic DNA from each strain was extracted using a commercial kit (Nucleospin Blood, Macherey-Nagel) following the manufacturer's instructions.
Primers were preliminarily tested with the 11 H. parasuis reference strains. All the primers of the H. influenza MLST (Meats et al., 2003) were tested, but finally only the primers for the malate dehydrogenase gene (mdh) were useful for H. parasuis. Previously published primers for rpoB, atpD and infB were also tested (Christensen et al., 2004). The remaining primers were designed by homology with other bacterial genes (sequences from Pasteurellaceae were used when available). Target genes were those for the 
chain of ATP synthase (atpD), 60 kDa heat-shock protein (hsp60), translation initiation factor IF-2 (infB), ribosomal protein subunit (rpoB), superoxide dismutase A (sodA), phosphoglucomutase (pgm), 6-phosphogluconate dehydrogenase (6pgd), glyceraldehyde-3-phosphate dehydrogenase (g3pd) and fumarate reductase B (frdB). Primers amplifying all reference strains without non-specific bands were selected. The seven selected were atpD, infB, mdh, rpoB, 6pgd, g3pd and frdB (Table 1). All PCR amplifications were carried out in a final volume of 50 µl containing 1.5 U Taq polymerase and 200 µM dNTP. MgCl2 and primer concentration were optimized in order to perform all the PCR reactions under the same cycling conditions (Table 1). Cycling conditions were 5 min at 95 °C, 35 cycles of 1 min at 95 °C, 30 s at 50 °C and 30 s at 72 °C, followed by a final step of 10 min at 72 °C.
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), BigDye terminator v.3.1 kit and the ABI 3100 DNA sequencer (Applied Biosystems).
Fingerprinting II v.3.0 software (Bio-Rad) was used to edit, assemble and align the sequences and to carry out allele assignment. Congruence between loci was calculated by Pearson product-moment correlation coefficient comparing neighbour-joining (NJ) trees for each gene. Mean diversity for each locus was calculated as previously described (Blackall et al., 1997). Afterwards, START (Jolley et al., 2001) was used to perform Burst analysis (one strain was assigned to a CC when it shared five alleles with any other strain in the same group), cluster analysis [unweighted-pair group method with arithmetic mean (UPGMA) dendrogram using the matrix of pairwise differences] and recombination analysis [index of association (IA) and square sum of the condensed fragment length statistic of Sawyer's test]. To examine the association of the clustering with the origin of the strains, the number of clinical isolates within a cluster was compared to the number of other isolates by chi-squared test (significance at P<0.001) using SPSS 12.0 software. Finally, the partial sequences of the seven genes were concatenated using DAMBE (Xia & Xie, 2001), multiple alignments were constructed using BioEdit (Hall, 1998) and NJ trees using 10 000 bootstraps were constructed using MEGA3.1 (Kumar et al., 2004). MEGA3.1 was also used to calculate the overall mean distances and the overall mean difference between non-synonymous and synonymous codon changes (dN–dS) for the seven genes independently.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The overall mean distance and dN–dS indicated differences in the variability of the loci and absence of strong positive or negative selection. In summary, we obtained a mean of 29 alleles per locus and an approximate potential resolution of 1010 STs. The 131 isolates studied were assigned to 109 STs, with a mean diversity per locus of 0.777. The most frequent ST was ST46, which was present in 3.8 % of the dataset.
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When a UPGMA dendrogram was built, six monophyletic clusters of related genotypes were defined (Fig. 1). Notably, cluster A was mainly formed by nasal isolates from asymptomatic carriers (Table 3). Statistical analysis by chi-squared test showed a significantly (P<0.001) higher number of nasal isolates in this cluster. Clusters B, C, D and E did not show any significant percentage of nasal or clinical isolates (Table 3). Finally, cluster F included a high percentage of systemic isolates (Table 3) and showed a significantly (P<0.001) higher number of clinical isolates when compared using the chi-squared test. It is noteworthy that cluster F also included virulent reference strains Nagasaki, 1A-84-22113 and 5D-84-15995 (Rapp-Gabrielson et al., 2006). Indeed, some nasal isolates (e.g. CA38-4 and CC6-7) included in cluster F came from farms affected by Glässer's disease. When the alleles were compared, strains in cluster F had 54 alleles that were not present in other clusters (although 36 of them were only present in a single isolate) and 22 alleles that were shared with other clusters.
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). Strains were divided into two main branches strongly supported by high bootstrap values (>95 %). There was little structure inside the two main branches, although many of the bootstrap values were above 50 %. The first branch (branch 1, Fig. 2) included 65 nasal isolates out of a total of 101 (64 %) and non-virulent reference strains C5, D74, 174 and SW114. On the other hand, it also included virulent reference strain SW124. The second branch (branch 2, Fig. 2) included 83 % clinical isolates and virulent reference strains Nagasaki, 5D-84-15995 and 1A-84-22113 (Rapp-Gabrielson et al., 2006). This branch contained almost all the strains included in cluster F of Fig. 1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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As expected, the mean diversity per locus in the MLST scheme was higher (0.777) than the mean diversity reported for multilocus enzyme electrophoresis (0.405) (Blackall et al., 1997). In agreement with previous reports (Oliveira et al., 2003; Olvera et al., 2006; Rafiee et al., 2000; Smart et al., 1988), our results confirmed that several strains (between one and five) can circulate in a farm. In addition, different strains can be isolated from the same animal [e.g. IQ7N7 (ST56) and IQ7N8 (ST84)] and even from the same systemic lesion [e.g. RU15-4P (ST51) and RU15-5P (ST75)]. Contrary to what is commonly accepted, the latter results indicate that more than one strain can be involved in a clinical outbreak. Nevertheless, some clones seem to have a wider distribution, since some STs could be detected in different farms (e.g. ST44, ST97, ST56 and ST34).
In this study, we also confirmed the high heterogeneity of H. parasuis. Accordingly, MLST analysis did not detect any predominant ST (the highest frequency of a ST is 3.8 %) and singletons were very frequent, even when a relaxed CC definition (five common alleles instead of six) was used in the Burst analysis. Although a certain geographical association of strains was found, our sample could be biased by a more intense sampling in Spain and this association should be studied further.
Taking into account the IA values, the sign of recombination in 6pgd and the lack of congruence between individual gene trees, it seems that recombination events have significant incidence in H. parasuis. Indeed, this bacterium seems to have no clonal framework. Furthermore, the shared alleles among groups indicate a recent exchange of alleles, a phenomenon that has also been reported for H. influenzae (Meats et al., 2003). Suitably, the facts that H. parasuis is naturally transformable (Bigas et al., 2005; Lancashire et al., 2005) and that more than one strain can colonize an individual create suitable conditions for frequent recombination.
In some bacteria, disease is caused by specific clones, which spread out, causing outbreaks. Those genotypes are favoured by selection and expand, thus creating an ‘epidemic’ population structure (Smith et al., 2000). In H. parasuis, few CCs were identified by Burst analysis, and although a link between some of them and clinical (putative virulent) isolates was found, no dominant CC associated with systemic infection could be demonstrated. However, it is possible that, when more clinical isolates are tested, CCs with a worldwide distribution and linked to disease onset could be found.
When a UPGMA dendrogram was constructed, the 131 isolates were divided into six monophyletic clusters. Interestingly, cluster A (Fig. 1) was clearly associated with nasal isolation, and it is probably formed by non-virulent strains. Cluster B showed a tendency to include mainly pulmonary isolates, which were also present, although at a lower percentage, in cluster E. Finally, systemic isolates were primarily found in cluster F, although they were also included in clusters C and D. Our results, together with the clinical background of the strains, suggest that H. parasuis comprises strains with three levels or capacities of virulence: first, non-virulent strains, belonging to the biota of the upper respiratory tract; second, pulmonary strains with the pathogenic capacity to produce bronchopneumonia but not invasive disease; and third, systemic strains with the capacity to produce Glässer's disease. Unfortunately, the putative virulence of some isolates was difficult to establish, since, even when systemic infection is observed, lung tissue is frequently used for diagnosis of H. parasuis infection. Nevertheless, these results should be confirmed by experimental animal infections in order to determine the real virulence of the strains.
Finally, the NJ tree indicated the existence of two divergent branches within H. parasuis. Other algorithms were not used due to excessive computation time. However, there are several studies indicating that all methods tend to perform well when they are provided with enough data (Nei & Kumar, 2000). The division of H. parasuis isolates into two branches was strongly supported by high bootstrap values. One branch (branch 2, Fig. 2) showed an association with pathogenic isolates. This branch included all strains of UPGMA cluster F, with only one exception, and it could be indicative of a divergent lineage with increased virulence. The existence of this highly virulent cluster was already suggested, although less obviously, by our study of H. parasuis strains by partial sequencing of hsp60 (Olvera et al., 2006). On the other hand, branch 1 (Fig. 2) did not show an association with disease. The majority of strains in this group were of nasal origin, and only some of the isolates seem to be potentially virulent. Even though branch 2 appears to be very consistent, it has to be taken into account that recombination has a major impact on phylogenetic reconstructions and these results have to be interpreted carefully.
In conclusion, H. parasuis strains were classified by MLST and two clusters were statistically associated with nasal and clinical isolation, respectively. After NJ analysis, the isolates in the disease-associated cluster were found to be clearly divergent from the remaining H. parasuis isolates, forming a different lineage.
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ACKNOWLEDGEMENTS |
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Received 27 June 2006;
revised 24 August 2006;
accepted 29 August 2006.
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