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The Veterinary Journal 174 (2007) 522–529

TheVeterinary Journal

Review

Update on the diagnosis of Haemophilus parasuisinfection in pigs and novel genotyping methods

Alex Olvera a, Joaquim Segales b, Virginia Aragon a,*

a Centre de Recerca en Sanitat Animal (CReSA), Campus de Bellaterra, Universitat Autonoma de Barcelona, Barcelona, Spainb Departament de Sanitat i d’Anatomia Animals and Centre de Recerca en Sanitat Animal (CReSA), Facultat de Veterinaria,

Campus de Bellaterra. Universitat Autonoma de Barcelona, Barcelona, Spain

Accepted 20 October 2006

Abstract

Haemophilus parasuis causes Glasser’s disease as well as a number of other diseases in pigs. The diagnosis of H. parasuis-associateddisease is usually established by clinical signs, pathological findings and bacterial isolation but diagnosis is complicated by the existenceof non-virulent strains and the early colonisation of the upper respiratory tract of healthy piglets. Moreover, several strains can be foundon a farm and even within a single animal so it is important to determine the specific strain that is causing the clinical outbreak. Recently,genotyping methods have been developed with the goal of correlating genotype with the degree of virulence of H. parasuis strains. Theassociation between genotype and virulence in H. parasuis is challenging due to the lack of knowledge of the complete genomic sequenceand virulence factors of this bacterium.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Haemophilus parasuis; Glasser’s disease; Pigs; Genotyping; Diagnosis

1. Introduction

Pigs can be colonised by different microorganismsbefore weaning (Pijoan and Trigo, 1990), but some of those‘early coloniser agents’ are potentially pathogenic (Pijoanet al., 1997). Haemophilus parasuis, Streptococcus suis andActinobacillus suis have emerged as significant pathogensfor the pig industry, especially in high health status farms.Moreover, infections with H. parasuis and S. suis are con-sidered to be two of the most common and costly infectiousagents for pig herds; in fact, the terminology ‘suis-ide dis-

eases’ has been used to describe the serious impact of thesebacteria (MacInnes and Desrosiers, 1999). Therefore, thecorrect diagnosis of infection by these agents is essentialto establish the appropriate control measures.

Fibrinous polyserositis and arthritis caused by H. para-suis (Glasser’s disease) is usually diagnosed on the basis of

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doi:10.1016/j.tvjl.2006.10.017

* Corresponding author. Tel.: +34 93 581 4494; fax: +34 93 581 4490.E-mail address: virginia.aragon@cresa.uab.es (V. Aragon).

herd history, clinical signs, necropsy findings and bacterialisolation/detection (Oliveira and Pijoan, 2004b; Rapp-Gabrielson et al., 2006). Coupled with classical diagnosticmethods, different genotyping techniques have been devel-oped recently to characterise H. parasuis strains (de laPuente Redondo et al., 2003; Oliveira et al., 2003; delRio et al., 2006; Olvera et al., 2006). However, these recentdevelopments are still based on the ability to isolate H.parasuis, which represents a significant limitation due tothe fragility and fastidious growth requirements of thebacterium (Rapp-Gabrielson et al., 2006).

The present review describes the diagnosis of H. parasuis

infection, with special emphasis on the most advancedmethods of genotyping.

2. Clinical and pathological diagnoses

Glasser’s disease, caused by H. parasuis, is presentworldwide and was initially considered to be of sporadic

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occurrence, affecting 1–4 month-old pigs and especiallylinked to stressful conditions (Nielsen and Danielson,1975). However, adoption of new production technologies(mainly multiple site production systems based on a singleorigin of the pigs) and establishment of high health andspecific pathogen free (SPF) herds, together with theemergence of respiratory and systemic syndromes (espe-cially porcine reproductive and respiratory syndrome inthe early 1990s), have contributed to an increase in prev-alence and severity of the disease (Rapp-Gabrielson et al.,2006). It is known that the immune status of the herd is adeterminant of the pathogenic outcome of the infection(Nielsen and Danielson, 1975), but the potential heteroge-neity in virulence among H. parasuis strains (Kielstein andRapp-Gabrielson, 1992; Oliveira and Pijoan, 2004a) alsoseems to be a significant determinant for diseasedevelopment.

Different clinicopathological outcomes have beendescribed in H. parasuis infection (Hoefling, 1994): Glas-ser’s disease (fibrinous polyserositis and arthritis), septicae-mia without polyserositis, and respiratory distressassociated with catarrhal purulent bronchopneumonia.Although it has only been described once, H. parasuis

has also been linked to acute myositis of the masseter mus-cles in gilts (Hoefling, 1991) and panniculitis of the ears ingrowing-finishing pigs (Drolet et al., 2000).

Peracute disease may result in sudden death withoutgross lesions of polyserositis. Petechial haemorrhages insome tissues and septicaemia-like microscopic lesions, suchas disseminated intravascular coagulation and micro-haemorrhages, are the most striking findings (Amanoet al., 1997). When lesions of fibrinous polyserositis andpolyarthritis develop in pigs with acute presentation, clini-cal signs may include high fever (41.5 �C), severe coughing,abdominal breathing, swollen joints, and central nervoussystem (CNS) signs, such as lateral decubitus, paddlingand trembling (Nielsen and Danielson, 1975; Vahle et al.,1995; Solano et al., 1997). These signs can be seen togetheror independently. Chronically affected animals may have areduced growth rate as a result of severe fibrous polysero-sitis and arthritis. Dyspnoea and coughing not associatedwith Glasser’s disease have been described together withH. parasuis isolation from lungs with catarrhal-purulentbronchopneumonia and even fibrino-haemorrhagic pneu-monia (Little, 1970; Dungworth, 1991; Narita et al.,1994).

Clinical and pathological diagnoses must be consideredas tentative, since there are a number of diseases that maydisplay clinical signs or lesions similar to H. parasuis

infections. Therefore, a complete differential diagnosisshould include septicaemic bacterial infections caused byStreptococcus suis, Mycoplasma hyorhinis, Erysipelothrix

rhusiopathiae, Actinobacillus suis, Salmonella enterica sero-var Choleraesuis var. Kunzendorf and Escherichia coli(Rapp-Gabrielson et al., 2006). A conclusive diagnosisof H. parasuis infection requires further laboratorytesting.

3. Laboratory diagnosis

3.1. Bacterial isolation

Although Glasser found an association between fibrin-ous serositis and polyarthritis in pigs and a small Gramnegative rod as early as 1910 (Glasser, 1910), the causativeagent was isolated for the first time by Hjarre and Wramby(1943). Since then, the ‘gold standard’ for the diagnosis ofGlasser’s disease continues to be the isolation of H. para-

suis from the lesions of pigs showing the clinical signsand pathological findings of the disease.

H. parasuis is a fastidious bacterium and its isolation inpure culture from diseased animals is usually difficult, par-ticularly if complicated due to antibiotic treatments. How-ever, isolation of the microorganism is important, since thisallows further testing, such as antimicrobial sensitivity ortyping, which are useful for disease control.

Appropriate samples for H. parasuis isolation should beobtained from pigs showing clinical signs characteristic ofacute infection that have not been treated with antimicro-bials. Although pigs showing respiratory distress and swol-len joints are the best candidates for sampling, pigs withCNS signs should also be considered. Samples can be takenwith swabs and placed in transport systems with Amiesmedium (del Rio et al., 2003), refrigerated and transportedto the laboratory as fast as possible (<2 days). The diagno-sis of Glasser’s disease presents significant challenges dueto the early colonisation of healthy piglets by H. parasuis

and the existence of strains that have proven non-virulentin experimental challenges (Kielstein and Rapp-Gabriel-son, 1992).

The diagnostic interpretation of lung isolates is espe-cially complicated. H. parasuis can be involved in pneumo-nia but, equally, the presence of the bacterium in the lungcould be a consequence of postmortem invasion from theupper respiratory tract where H. parasuis is commonlyfound (Harris et al., 1969; Moller and Kilian, 1990). There-fore, the best samples for bacterial isolation are body fluidsin cases of fibrinous polyserositis, including cerebrospinalfluid when CNS signs are present (Solano et al., 1997;Vahle et al., 1995). Lung tissue may be submitted whenfibrinous pleuritis is observed, but isolation from lungparenchyma will not be conclusive that the strain isolatedwas the cause of the observed fibrinous polyserositis andarthritis.

H. parasuis is a small pleomorphic Gram negative bacte-rium in the family Pasteurellaceae, which requires V factor(nicotinamide adenine dinucleotide, NAD) but not X fac-tor (haemin) for growth. In the laboratory, H. parasuis

grows on enriched chocolate agar but not on blood agar.However, it can also be cultured on blood agar with aStaphylococcus aureus nurse or feeder streak as a sourceof V factor. H. parasuis requires 1–3 days to produce smallbrown to grey colonies in chocolate agar plates. Somestrains produce colonies of different sizes, but the signifi-cance of this phenomenon is not known. When a liquid cul-

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ture is needed (e.g., for biochemical tests), H. parasuis canbe cultured in liquid PPLO (Mycoplasma) broth supple-mented with NAD.

Closely related species, such as Actinobacillus minor,Actinobacillus indolicus and Actinobacillus porcinus, whichcan be also isolated from the respiratory tract of pigs,can be differentiated by simple biochemical tests, specifi-cally indole production and urease and catalase activities(Kielstein et al., 2001). The discrimination of H. parasuis

from these species is important from a diagnostic pointof view, since, even though A. minor, A. indolicus andA. porcinus are considered weakly pathogenic or non-path-ogenic for pigs (Chiers et al., 2001), they can be isolated inpure culture from pneumonic lungs (Kielstein et al., 2001).Moreover, in our experience, A. porcinus can also be iso-lated from systemic sites (Mateu et al., 2005; Olveraet al., 2006).

3.2. Detection of H. parasuis by immunohistochemistry

H. parasuis antigen has been detected in formalin-fixed,paraffin-embedded tissues by immunohistochemistry (IHC)(Amano et al., 1994; Segales et al., 1997; Vahle et al., 1997).This method has been shown to be at least as sensitive asbacteriological cultures and is especially useful in experi-mental pathogenesis studies (Segales et al., 1997; Vahleet al., 1997). The major advantage of IHC is the directvisual demonstration of bacterial antigens in affected tis-sues, which provides an association between the presenceof antigens and histopathological changes.

IHC has not however been used extensively fordiagnostic purposes, since the availability of monoclonalantibodies to H. parasuis that work on formalin-fixed,paraffin-embedded tissues is very limited and polyclonalantibodies have shown cross-reactivity with other bacteria,such as Actinobacillus pleuropneumoniae (Segales et al.,1997). Therefore, the potential of IHC to detect H. parasuis

for routine diagnostic use is limited.

3.3. Detection of H. parasuis by molecular methods

The introduction of molecular methods, mostly thepolymerase chain reaction (PCR), was a major advancefor the diagnosis of infectious diseases, in particular whendealing with slow or poorly growing microbes.

3.3.1. PCR

Due to the fastidious growth of H. parasuis, the develop-ment of a specific PCR led to an improvement in the detec-tion of this bacterium (Oliveira et al., 2001). The primerswere designed to amplify a fragment of 821 base pairs(bp) from the 16S rRNA gene. The sensitivity of thisPCR was 102 CFU/mL and proved useful for detectionof H. parasuis in clinical samples. PCR can be performedwith purified DNA from a pure broth culture of the organ-ism or directly on a single colony on agar (colony-PCR). Itis not of diagnostic value in nasal or tonsillar swabs, due to

the presence of H. parasuis in the upper respiratory tractof healthy animals and the positive non-specific reactionof A. indolicus, which is also a coloniser of the upper respi-ratory tract of pigs. Thus, the value of this PCR in livinganimals is limited.

In a recent report, Jung et al. (2004) proposed a nestedPCR to increase the sensitivity of the technique and, asan additional advantage, they established conditions forthe application of this PCR to formalin-fixed, paraffin-embedded tissues. The specific conventional PCR describedabove was followed by amplification of an internal 313 bpfragment, increasing the sensitivity of the assay to 3 CFU/mL. However, this second (nested) amplification would notbe expected to improve the specificity of the PCR, since thesequences of the nested primers have an exact match in the16S rRNA gene of A. indolicus. Unfortunately, the authorsdid not include any strain of A. indolicus to test the speci-ficity of this nested PCR.

3.3.2. In situ hybridisation

In situ hybridisation (ISH) to detect genomic sequencesof H. parasuis was developed by Jung and Chae (2004). The821 bp fragment amplified using the PCR of Oliveira et al.(2001) was labelled and used as a probe. ISH allows thedirect association of H. parasuis with tissue lesions andhas the advantage over IHC of avoiding problems of avail-ability of specific antibodies.

The agreement between ISH and nested PCR for thedetection of H. parasuis in formalin-fixed, paraffin-embed-ded tissues was 100% (Jung et al., 2004). There was noevidence of cross-reactivity of the probe with A. pleuro-

pneumoniae and Pasteurella multocida. However, the highsimilarity of the H. parasuis probe to the correspondingfragments in the 16S rRNA gene of A. indolicus (GenBanknumber AF268962, 98% identity), A. porcinus (AF268955,98% identity; AF268952, 97% identity) and A. minor

(AF268941, AF268942 and AF268946, 97% identity) doesnot definitively exclude potential cross-reactivity of thedescribed ISH technique with these bacteria. Therefore,the specificity of ISH in detecting H. parasuis for routinediagnostic use has not been fully clarified.

4. Epidemiology and typing

There is a general need for typing techniques in microbi-ology in order to characterise unambiguously the strains oforganisms. The identification of bacterial strains has differ-ent applications, such as the determination of how manystrains are implicated in a single outbreak (local epidemiol-ogy) or the relationship of particular strains with those iso-lated in different geographical areas or at distant timepoints (global epidemiology). Thus, local epidemiologycan determine if one or more strains are causing an out-break, or, in the case of persistent infection, if the treat-ment failed or a new virulent strain had been introduced.Global epidemiology studies the relationships betweendifferent clonal lines, their global distribution and the

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determinants that cause those distributions. The strengthsand weakness of a typing technique depend on its relativediscriminatory power, reproducibility, timing and cost(Foxman et al., 2005).

Differentiation of strains is of special importance inH. parasuis diagnosis and control, since it is essential to dif-ferentiate between ‘colonising’ strains and ‘disease-causing’strains. The characterisation of H. parasuis isolates is use-ful for determining the number and distribution of strainsin epidemiological studies, the differentiation of virulentand non-virulent clones and the assessment of cross-immu-nity between strains and bacterins.

Isolates of H. parasuis show high heterogeneity in viru-lence and other phenotypic and genotypic traits (Moroz-umi and Nicolet, 1986a,b; Miniats et al., 1991; Amanoet al., 1994, 1996; Blackall et al., 1997; Rubies et al.,1999; Rafiee and Blackall, 2000; Rafiee et al., 2000; Ruizet al., 2001; Oliveira et al., 2003; Angen et al., 2004;Blanco et al., 2004; Oliveira and Pijoan, 2004a; Caiet al., 2005; Olvera et al., 2006). Thus, one of the goalsof current research is to discriminate between virulentand non-virulent strains. With this goal in mind, severalstudies have been performed in an effort to associate typ-ing (classical serotyping and, more recently, genotyping)with virulence.

4.1. Serotyping

Traditionally, the classification of H. parasuis strainswas achieved by serotyping. Kielstein and Rapp-Gabriel-son (1992) defined 15 serovars of H. parasuis and demon-strated wide differences in their virulence. Oliveira et al.(2003) reported the association of serotypes 1, 2, 4, 5, 12,13 and 14 (and non-typeable isolates) with their isolationfrom systemic sites. Serotype 3 and non-typeable isolateswere isolated from the upper respiratory tract (Oliveiraet al., 2003). Unfortunately, the correlation between sero-type and virulence is not clear, with strains that belong tothe same serotype and exhibit different degrees of virulence.In addition, cross-protection between different serotypesand even within the same serotype is variable and difficultto predict (Rapp-Gabrielson et al., 1997). Moreover, sero-typing does not provide enough discrimination of isolatesfor epidemiological studies and between 15-41% of isolatesare non-typeable (Oliveira et al., 2003).

4.2. Genotyping

Molecular techniques represent a major advance for epi-demiological studies since they allow the unambiguousidentification of every isolate in a timely manner. However,they do not offer any direct functional information, andgenotypes still have to be correlated with immunologicalor virulence features using complementary data.

Genotyping is carried out by fingerprinting or sequenc-ing methods. Fingerprints, which are electrophoretic bandpatterns, can be obtained from whole bacterial genomes or

from a single gene. In whole genome techniques, band pat-terns are produced by digestion of genomic DNA withrestriction endonucleases or by PCR amplification withprimers for random targets in the genome. Single genepatterns usually employ an initial gene-specific PCR, fol-lowed by digestion of the amplicon with restrictionendonucleases.

While whole genome patterns evolve mainly by genomerearrangements, single gene patterns evolve by single pointmutations. All typing techniques have to be validated withenough number of strains to demonstrate their level of res-olution. Although information on the genomic sequence ofH. parasuis is limited, several research groups haveattempted to improve the differentiation of field strainsby genotyping techniques.

4.2.1. Restriction endonuclease pattern

The first DNA-based typing technique for H. parasuis,restriction endonuclease pattern (REP) typing, was devel-oped by Smart et al. (1988). This technique consisted ofthe digestion of highly pure genomic DNA with restrictionendonucleases and the subsequent analysis of the frag-ments by polyacrylamide gel electrophoresis. Smart et al.(1988) used REP typing to identify different strains isolatedfrom the same herd (2–4 strains per farm) and even differ-ent strains from an individual animal. Conventional farmshad a more heterogeneous population of H. parasuis

strains when compared to SPF herds. Interestingly, strainsisolated from systemic sites of diseased animals were differ-ent from those found in the upper respiratory tract ofhealthy animals.

REP typing has also been used to assess vaccination fail-ure in a Glasser’s outbreak (Smart et al., 1993). Isolatesobtained from nasal swabs and isolates from diseased ani-mals were different to those in the commercial bacterinused in the herd. The lack of cross-protection betweenH. parasuis strains is well known and the outbreak wassubsequently controlled using an autogenous bacterin(Smart et al., 1993).

4.2.2. Enterobacterial repetitive intergenic consensus PCR

Rafiee et al. (2000) applied a repetitive element polymor-phism (enterobacterial repetitive intergenic consensus,ERIC) PCR technique to randomly amplify the genomeof different H. parasuis strains. This technique uses primerswhose targets are repetitive sequences in non-codingregions (Versalovic et al., 1991). The patterns evolvemainly by deletion and insertion of different mobile ele-ments in the genome.

ERIC-PCR is especially suitable for outbreak studies,since it is fast, inexpensive and allows confirmation of thesource of infection and the number of strains involved. Ithas been used in several local epidemiological studies,which confirmed previous results by REP (Ruiz et al.,2001; Oliveira et al., 2003). A common origin for systemicisolates was identified in an outbreak affecting severalfarms (Oliveira et al., 2003).

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Interestingly, profiles from systemic isolates have indi-cated a clonal origin and have been shown to be differentfrom other non-systemic isolates or reference strains (Ruizet al., 2001). They also showed that few strains are involvedin clinical outbreaks and their fingerprints were rarely foundin isolates from the upper respiratory tract. Controversially,it was also established that the ERIC patterns of isolatesfrom systemic and pneumonic sites were related (Ruizet al., 2001). This study also confirmed the high heterogene-ity of H. parasuis already reported by REP, whole cell andouter-membrane protein profiles, multilocus enzyme elec-trophoresis and serotyping. A high genetic diversity was alsodescribed within serovar groups and non-typable strains.Genotyping was found to be a reasonable predictor of sero-type, although there is no complete agreement.

ERIC-PCR patterns have a high degree of variation,making it difficult to share typing information between lab-oratories (Foxman et al., 2005; Olvera et al., 2006). Despitethis, ERIC-PCR has been found to be more convenientthan REP typing, since REP patterns are considerablycomplex (up to 100 bands) and the implementation ofREP techniques is highly demanding.

4.2.3. Restriction length polymorphism

Recently, different restriction fragment length polymor-phism (RFLP)-PCR protocols have been developed. Thosetechniques involve the amplification of one gene by PCRand the digestion of PCR products with restriction endonu-cleases to obtain a band pattern. The main advantage isthat, if the PCR is specific, the need for bacterial isolationis eliminated and the technique can be performed directlyon clinical samples. Furthermore, RFLP-PCR is morereproducible than ERIC-PCR due to the use of higherstringency conditions. Conversely, the use of a single genemakes RFLP-PCR techniques susceptible to being affectedby lateral gene transfer.

Three RFLP-PCR schemes for H. parasuis have beendeveloped using the few currently known gene sequences.The selected genes were the transferrin binding protein A(tbpA) (de la Puente Redondo et al., 2003), the fragmentof the 16s rRNA gene amplified by the diagnostic PCRdescribed above (Lin, 2003) and the 5-enolpyruvylshiki-mate-3-phosphate synthase (aroA) (del Rio et al., 2006).The RFLP-PCR schemes for the tbpA and the 16s rRNAgene confirmed the high heterogeneity of H. parasuis andthe lack of a clear correlation between genotype and sero-type. On the other hand, some serovars are indistinguish-able by RFLP-PCR.

The aroA RFLP-PCR uses a non-species specific PCRthat amplifies this gene from H. parasuis and several mem-bers of the genus Actinobacillus so isolation and identifica-tion is strictly necessary in using this genotyping technique(del Rio et al., 2006). Curiously, some of the referencestrains of H. parasuis share RFLP patterns with those ofA. pleuropneumoniae (del Rio et al., 2006). Although thecauses for this finding are unknown, a lateral gene transferevent can not be discarded.

4.2.4. Single locus sequence typing

The use of fingerprinting methods in global epidemio-logical studies is limited as less information on the relation-ship between clusters is obtained and the results aredifficult to compare among laboratories. Recently, Olveraet al. (2006) reported the application of a partial sequence(596 bp) of the hsp60 gene for H. parasuis epidemiology. Inthe hsp60 gene sequence, 36 alleles were identified in the 91strains tested and H. parasuis strains were classified in twomain clusters. Interestingly, one of the clusters showed ahigh frequency of clinical isolates and virulent referencestrains, but no strong relationship between virulence andgenotype could be detected.

This work also included an extensive sequencing analy-sis of the 16S rRNA gene (1391–1394 bp), which registereda high level of sequence diversity at the subspecies level(Olvera et al., 2006). Therefore, 16S rRNA gene sequencescould also be used for the genotyping of H. parasuis

strains, as has been demonstrated for H. influenzae (Sacchiet al., 2005). However, hsp60 fragment sequencing is easierto perform than 16S rRNA gene sequencing and, forH. parasuis, hsp60 showed a better discriminatory power.

5. Discussion

The lack of information on the genome sequence ofH. parasuis complicates the development of improved diag-nostic and control tools. More importantly, a better under-standing of the pathogenesis and the identification of thevirulence factors of H. parasuis would provide the neces-sary knowledge for the rational design of new and effectivevaccines.

The future development of more specific diagnostic tools(i.e., amplification of H. parasuis virulence-specific genes)will allow the precise identification of virulent strains. ADNA fragment specific for H. parasuis serovar 2 has beenidentified recently, but the value of this fragment in typingfield strains has not yet been demonstrated (Del Rio et al.,2005). The method used for identification of this fragment,representational difference analysis, could be useful in theidentification of genes expressed only in virulent strains(Del Rio et al., 2005). A virulence-specific PCR will be use-ful for the analysis of nasal swabs from live animals.

Recently, several groups have performed studiesintended to determine virulence factors and mechanismsin H. parasuis (Hill et al., 2003; Lichtensteiger and Vimr,2003; Tadjine et al., 2004; Melnikow et al., 2005; Vanieret al., 2006). The production of species-specific monoclonalantibodies (which could be useful as diagnostic tools) andtheir protective role in a mouse infection model has impli-cated lipopolysaccharide in the pathogenesis of H. parasuis

(Tadjine et al., 2004). Vanier et al. (2006) demonstrated thecapacity of virulent strains of H. parasuis to invade endo-thelial cells, but the factors involved in this function needto be determined. Other studies have concentrated on geneexpression under conditions mimicking the in vivo environ-ment (Hill et al., 2003; Melnikow et al., 2005). Although

Table 1Comparison of different typing methods for Haemophilus parasuis

Typing techniques Relativediscriminatorypower

Relativereproducibility

Time required Interpretation Cost Value asepidemiologicalmarker

Serotyping Low Moderate Hours-1 day Easy Low LowREP High Moderate 1–2 days Difficult Moderate LowERIC-PCR High Low 2 days Intermediate Moderate ModerateRFLP-PCR Moderate Moderate 2 days Easy High Moderatehsp60 SLST Moderate High 2–3 days Easy High High16S rRNA gene Moderate High 2–3 days Easy High High

A. Olvera et al. / The Veterinary Journal 174 (2007) 522–529 527

some putative virulence genes have been identified, moreextensive studies are needed to determine the real role ofthese genes in the pathogenesis of H. parasuis.

The development of an efficient genetic manipulationsystem will be essential to demonstrate the role of differentgenes and factors in H. parasuis virulence. Two differentsystems have been reported for the transformation ofH. parasuis: electroporation with a native plasmid (Lanca-shire et al., 2005) and natural transformation (Bigas et al.,2005). The recombinant expression of specific genes, as wellas the production of defined mutants, will establish theroles of these genes. Thus, the identification of the essentialfactors in the pathogenicity of H. parasuis will allow thedevelopment of virulent strain-specific diagnostic methods(e.g., PCR for virulence genes) and the design of universalvaccines (e.g., subcellular vaccines).

Knowledge of the epidemiology of H. parasuis infectionsis limited by current genotyping methods (Table 1). Unfor-tunately, none of the molecular typing techniques devel-oped so far can predict the virulence or cross-immunity ofH. parasuis isolates. Multilocus sequence typing (MLST)is an attractive method because it is reproducible, easy toshare and has a high degree discrimination, but the highcost of sequencing represents a limitation for routine appli-cation. Sequence based typing will probably be the tech-nique of choice for future global epidemiological studies.Sequence typing provides high level of discrimination whileallowing the identification of relationships between differentstrains. This technique will certainly help in finding a corre-lation between virulence, cross-immunity and genotype.

6. Conclusion

The best diagnosis of H. parasuis infection is currentlyachieved when several methods are used; clinical symp-toms, pathological findings, bacterial culture and molecu-lar tests must be in agreement. It is especially importantto test the proper samples to determine the strain that iscausing the clinical problem. Nasal samples and, if possi-ble, lung samples, should not be used, and systemic samplesare most appropriate. Genomic and functional studies willhelp to elucidate cross immunity between strains and iden-tify virulent clone lines and virulent genes in order, eventu-ally, to develop more reliable disease control methods.

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