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Keywords:

  • dihydropteroate synthase;
  • Haemophilus parasuis;
  • representational difference analysis;
  • respiratory pathogen;
  • sulfonamide resistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA).

Methods and Results:  Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7).

Conclusion: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique.

Significance and Impact of the Study:  The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Haemophilus parasuis is a nonmotile, nicotinamide adenine dinucleotide (NAD)-dependent, small pleomorphic Gram-negative rod of the family Pasteurellaceae. This species is the causative agent of Glässer's disease, a porcine fibrinous polyserositis, polyarthritis and meningitis that has historically been considered as a sporadic, stress-associated disease of young pigs (Smart et al. 1988). However, after the establishment of specific pathogen-free herds, increased spread of the disease and increased mortality rates as a cause of Haemophilus parasuis infection have been described by Smart and Miniats (1989).

Although the pathogenicity of Glässer's disease is largely unknown, a few virulence factors have been described: lipopolysaccharide (Amano et al. 1997), capsule (Kielstein et al. 1991), outer membrane (Rosner et al. 1991; Ruiz et al. 2001) and whole-cell protein profiles (Morozumi and Nicolet 1986), fimbriae (Munch et al. 1992) and neuraminidase (sialidase) (Lichtensteiger and Vimr 1997). A total of 15 H. parasuis serovars have been identified, and an association between serovars and virulence has been reported. Therefore, serovars 1 and 5 proved to be highly virulent in pigs and guinea pigs while serovars 2–4, 6 and 7 were found to be less virulent (Rosner et al. 1991; Kielstein and Rapp-Gabrielson 1992; Rapp-Gabrielson and Gabrielson 1992). However, both virulent and avirulent strains belonging to the same serovar have been previously reported, suggesting that virulence might be associated directly with each H. parasuis strain (Rapp-Gabrielson et al. 1997).

Several molecular methods such as restriction fragment length polymorphism (RFLP) (Savelkoul et al. 1999), library screening (Baker et al. 1997) and subtractive hybridization (Straus and Ausubel 1990) have been used to identify species-specific and virulence-associated genes. Differentially display reverse transcription–polymerase chain reaction (DDRT–PCR) has also been used successfully to identify differentially expressed gene fragments associated with virulence in H. parasuis (Hill et al. 2003).

Representational difference analysis (RDA) (Lisitsyn et al. 1993), a powerful PCR-based subtractive hybridization procedure incorporating the additional feature of kinetic enrichment, has become one of the techniques of choice for the detection and cloning of genomic differences between two closely related bacterial species or isolates belonging to the same species (Tinsley and Nassif 1996). The RDA was originally applied in eucaryotic organisms to identify genetic polymorphisms in human neoplasias (Lisitsyn et al. 1993; Lisitsyn and Wigler 1995) and more recently, this method has been applied to the identification of strain-specific or species-specific sequences of a variety of Gram-positive and Gram-negative organisms other than Pasteurellaceae (Bart et al. 2000, 2001; Becker et al. 2001).

The purpose of this study was the identification of genes differentially present in H. parasuis serovar 2 (one of the most prevalent serovars in Spain (Rubies et al. 1999) compared with serovars 1, 5 (both highly virulent) and 3 (of scarce virulence).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains, plasmids, primers and growth conditions

Bacterial strains, plasmids and primers used in this study are given in Table 1. Escherichia coli TOP10 strain was grown at 37°C for 24 h on Luria–Bertani agar containing ampicillin (100 μg ml−1) and kanamycin (50 μg ml−1) when appropriate. Haemophilus parasuis and Actinobacillus pleuropneumoniae strains were grown at 37°C for 24 h on PPLO (pleuropneumonia-like organisms) agar supplemented with 0·025% NAD, 0·01%l-glutamine, 0·026%l-cysteine hydrochloride, 0·001%l-cystine dihydrochloride, 0·1% dextrose and 0·1% Tween 80.

Table 1.  Strains, plasmids and primers used in this study
Strains, plasmids and primersCharacteristics (source)
H. parasuis
No.4serovar 1 reference strain (Kielstein and Rapp-Gabrielson 1992)
SW140serovar 2 reference strain (Kielstein and Rapp-Gabrielson 1992)
SW114serovar 3 reference strain (Kielstein and Rapp-Gabrielson 1992)
Nagasakiserovar 5 reference strain (Kielstein and Rapp-Gabrielson 1992)
A. pleuropneumoniae
ATCC 27088 (Shope 4074)serotype 1 reference strain (Nielsen 1986)
ATCC 27089 (1526)serotype 2 reference strain (Nielsen 1986)
ATCC 27090 (1421)serotype 3 reference strain (Nielsen 1986)
ATCC 33378 (M-62)serotype 4 reference strain (Nielsen 1986)
ATCC 33377 (K17)serotype 5 reference strain (Nielsen 1986)
ATCC 33590 (FEMφ)serotype 6 reference strain (Nielsen 1986)
WF83serotype 7 reference strain (Nielsen 1986)
405serotype 8 reference strain (Nielsen 1986)
CVI13261serotype 9 reference strain (Nielsen 1986)
D13039serotype 10 reference strain (Nielsen 1986)
56153serotype 11 reference strain (Nielsen 1986)
8329serotype 12 reference strain (Nielsen 1986)
E. coli TOP10FmcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 rec A1 deoR araD139Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG, TOPO TA Cloning® (Invitrogen, Carlsbad, CA, USA)
pCR®II TOPOE. coli cloning vector containing and ampicillin- and kanamycin-resistance determinant (Invitrogen)
JBam125′-GATCCGTTCATG-3′
JBam245′-ACCGACGTCGACTATCCATGAACG-3′

Molecular techniques

Isolation of plasmid DNA, restriction enzyme digests and agarose gel electrophoresis were performed according to standard molecular techniques (Sambrook et al. 1989). Restriction enzyme-digested chromosomal and plasmid DNA fragments were separated on 1·5% agarose gels; fragments of interest were cut from the gel under UV illumination and further purified with a QIAquick gel extraction kit (Qiagen; Barcelona, Spain). DNA sequencing was performed at the ‘Sistemas Genómicos’ in the DNA Sequencing Core Facility using a capillary Beckman CEQ 2000 XL sequencer (Beckman Coulter; Fullerton, CA, USA) according to the manufacturer's instructions. The BLASTN computer program at the National Centre for Biotechnology Information (Altschul et al. 1990) was used for sequence analysis and alignment. The BLASTN search was used to compare nucleotide sequence of the RDA-1 fragment with the GenBank database. Multiple sequence alignments were constructed with Clustal W program (http://www.ebi.ac.uk/clustalw/).

For the extraction of bacterial genomic DNA, the strains were grown as described above and were harvested from PPLO broth. The cells were centrifuged for 10 min at 3000 g at 4°C, washed twice with TE buffer [10 mmol l−1 Tris–HCl (pH 8·0), 1 mmol l−1 EDTA], and finally lysed in buffer [0·5 mol l−1 EDTA (pH 8·0), 20% SDS, 50 mg of proteinase K per ml] for 1 h at 55°C. For RNA removal, RNase (100 μg ml−1) was added and incubated for 20 min at 37°C. Proteins were removed by adding 0·25% phenol equilibrated in TE buffer (pH 7·8). The DNA was then purified by repeated chloroform–isopropanol (24 : 1) extraction, precipitated by adding 0·1 volume of 3 mol l−1 sodium acetate (pH 5·2) and 1 volume of isopropanol. The DNA was washed twice in 70% ethanol, dried and resuspended in bidistilled water.

Representational difference analysis

The RDA technique, described initially by Lisitsyn (Lisitsyn et al. 1993) and further improved by Tinsley and Nassif (1996) was used. Haemophilus parasuis serovar 2 was used as tester and serovars 1, 3 and 5 were used as drivers. A 5 μg of each genomic DNA was cleaved with Sau3AI for 2 h at 37°C, precipitated with ethanol–sodium acetate, dried and resuspended in bidistilled water. The reaction for the preparation of the oligonucleotides adaptors consisted of 15 μl of each adapter JBam12 and JBam 24 (100 μmol l−1) (Table 1) and 7·5 μl of 10x ligation buffer. The reaction was heated for 5 min at 95°C, allowed to cool for 10 min to room temperature and was then incubated overnight at 16°C. Subsequently, DNA was ligated to oligonucleotide adapter pair by mixing 12·5 μl of tester DNA, 12·5 μl of adapters, 3 μl of 1 mmol l−1 ATP and 0·5 μl of T4 DNA ligase and incubated overnight at 16°C.

The hybridization method was accomplished in several steps to establish the optimal tester/driver ratio. Thus, control tester (tester only), control driver (driver only) and tester/driver (1 : 10 and 1 : 70) were assayed. A total of 12·5 μl of driver and 12·5 μl tester JBam ligation were combined, purified by phenol–chloroform–isoamyl alcohol and chloroform–isoamyl alcohol extraction, precipitated with ethanol–sodium acetate and redissolved in 4 μl of 1x polymerase buffer (10 mmol l−1 Tris–HCl and 50 mmol l−1 KCl, pH 8·3). The DNA was denatured at 95°C for 5 min. After the addition of 2 μl of 5 mol l−1 NaCl to the aqueous phase, the mixture was left to hybridize at 67°C for 20 h, and then was amplified by PCR. The reaction mixture for PCR consisted of 5 μl of DNA (hybrid), 2·5 μl of 10x amplification buffer, 0·75 μl of 1·5 mmol l−1 MgCl2, 0·5 μl of deoxynucleoside triphosphates (10 μmol l−1 each), and 0·1 μl of Taq DNA polymerase and was incubated at 72°C for 15 min, allowing the 3′-ends to extend. This step selects sequences found only in the tester representation, as the primer annealing site will be added at the 3′-ends of both strands of only the tester–tester duplexes, allowing exponential amplification. After incubation, 5 μl of a solution containing 5 μmol l−1 concentration of each of primers JBam12 and JBam24 ws added to the reaction mix and the PCR was run under the following conditions: one cycle for 3 min at 94°C, and 35 cycles for 1 min at 94°C, 1 min at 58°C and 3 min at 72°C, with the last cycle followed by an extension at 72°C for 10 min. The amplified DNA fragments were separated by 1% agarose gel electrophoresis.

Cloning of the PCR products

Enriched PCR products were cloned into the pCR II TOPO vector, and then transformed into E. coli TOP10 cells. Because of the concern that amplifying gel slices might amplify assorted background fragments as well as genuine different products, several colonies on each transformant plate were screened to identify a consensus insert, which should represent the desired product.

Southern blot hybridization

Briefly, to check for specificity, the PCR products from the colonies of transformants were electrophoresed through 1·5% agarose gel, DNA was then denatured in alkaline solution for 15 min at room temperature and was then transferred in alkaline transfer buffer (0·4 n NaOH with 1 mol l−1 NaCl) by capillarity to a positively charged nylon membrane. DNA was fixed in membrane with neutralization buffer [0·5 mol l−1 Tris–HCl (pH 7·2) with 1 mol l−1 NaCl] for 15 min at room temperature. Hybridization was performed using (α-32P)dCTP labelled RDA-1 fragment as probe for Southern blot analysis of Sau3AI-digested chromosomal DNA from H. parasuis serovars 1–3 and 5 following standard procedures (Sambrook et al. 1989). Probes hybridizing with genomic DNA of serovar 2 but not giving any detectable hybridization to serovars 1, 3 and 5 were considered to be specific products.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation of one DNA H. parasuis serovar 2-specific fragment using RDA

The restriction enzyme Sau3AI was used to generate DNA fragments of small size. Digested genomic DNA from H. parasuis serovar 2 was subtracted from H. parasuis serovars 1, 3 and 5. Agarose gel electrophoresis of the amplified difference products (tester/driver, 1 : 70) revealed six DNA bands ranging from 1500 to 200 bp (Fig. 1). Amplified PCR products were cloned into pCR II TOPO vector and 40 clones were obtained, but only one fragment of 369 bp in length, designated RDA-1, was shown to be specific for H. parasuis serovar 2, and no positive reaction with H. parasuis serovars 1, 3 and 5 was observed by Southern blot hybridization (Fig. 2). The others fragments turned out to be false-positives and were discarded because of the lack of relevance for not being differentially present in H. parasuis. Fragment RDA-1 was also used as probe in Southern blots with A. pleuropneumoniae serotype 1–12 reference strains. Fragment RDA-1 hybridized only to A. pleuropneumoniae serotype 7 but did not hybridize to any of the other serotypes (Fig. 2).

image

Figure 1. Agarose gel electrophoresis of RDA products between tester (Haemophilus parasuis serovar 2) and drivers (H. parasuis serovars 1, 3 and 5); lane 1, molecular size markers (Lambda DNA-HindIII digest, New England Biolabs; Barcelona, Spain); lane 2, Tester-driver (1 : 70) in agarose gel electrophoresis showing the six fragments ranging from 1500 to 200 bp

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image

Figure 2. Southern blot of RDA-1, dhps gene, with Sau3AI-digested chromosomal DNA from Haemophilus parasuis serovars 1–3 and 5 and Actinobacillus pleuropneumoniae serotypes 1–12, showing the presence of this gene solely in H. parasuis serovar 2 and in A. pleuropneumoniae serotype 7. The smear seen above the 0·8 kb band reflects partial digestion of the DNA

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Characterization of RDA-1 fragment

The RDA-1 fragment was sequenced using primers flanking the polylinker of the pCR II TOPO cloning vector. The sequence of fragment RDA-1 were subjected to a BLAST search and showed 99% similarity to sulI gene that encodes for dihydropteroate synthase (dhps) of Shigella sonnei (GenBank accession no. AF534183). This fragment also showed 99% homology to an antimicrobial resistance protein of Mannheimia haemolytica (GenBank accession no. AJ249249) and 98% homology to plasmid pTYM1 of A. pleuropneumoniae (GenBank accession no. AF303375) (Fig. 3). As expected, H. parasuis serovar 2, containing the dhps gene, was shown to be resistant to 240 μg sulfisoxazole, whereas H. parasuis serovars 1, 3 and 5 were sensitive (Swedberg et al. 1998).

image

Figure 3. Alignment of the dhps gene from Shigella sonnei (S. sonnei), Mannheimia haemolytica (M. haemol), Actinobacillus pleuropneumoniae (A. pleurop) and RDA-1 fragment from Haemophilus parasuis serovar 2. Identical base pairs are showed by an asterisk

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Differential sequences that are present in one bacteria and absent in others can be successfully isolated by RDA, even when species under investigation are highly similar in genome complexity. Differences found between two genomes can be easily converted into PCR-based markers that discriminate between taxa based on the presence or absence of amplification products.

The hypothesis that the differences in pathogenesis between members belonging to closely related species may uncover genomic differences with regard to pathogenicity (acute or chronic state) identifiable by RDA (Tinsley and Nassif 1996) has been assessed. The advantage of RDA technique to search for specific virulence related genes present in H. parasuis and absent in A. pleuropneumoniae that might account for the pathogenesis of the former organism has been used. Unlike A. pleuropneumoniae, H. parasuis does not exhibit typical virulence factors such as haemolysins, indicating that other virulence mechanisms must account for the differences in pathogenicity. A gene (dhps) encoding for dihydropteroate synthase, which is present in H. parasuis serovar 2 (associated to chronic disease) but not in serovars 1, 5 (usually associated with acute outbreaks of the disease) and 3 (scarcely virulent), has been identified (Kielstein and Rapp-Gabrielson 1992). This enzyme catalyses the condensation of 6-hydroxymethyl-7,8-dihydroterin pyrophosphate with p-aminobenzoic acid (PABA) to form an intermediate substrate that is required for the synthesis of folic acid, an essential compound in the biosynthesis of purines, pyrimidines and amino acids (Vinnicombe and Derrick 1999). Sulfonamides are structural analogues of PABA and act as antimetabolites by competing with PABA for the active site of dihydropteroate synthase enzyme (Richey and Brown 1969). Although the dhps enzymes are likely to be present in all the strains of H. parasuis and A. pleuropneumoniae, RDA technique has identified a distinct variant of sulI H. parasuis serovar 2, which confers sulfonamide resistance.

In Gram-negative bacteria, sulfonamide resistance is often associated with drug-resistant variants of the target enzyme dhps, which are plasmid-encoded (Radstrom et al. 1991). However, in Neisseria meningitidis was also reported to have a chromosomal location (Kristiansen et al. 1990). In our study, we could not isolate plasmids from H. parasuis serovar 2 that may account for the resistance to sulfonamide. This data suggest that resistance to sulfonamide may be associated to allelic variants of the chromosomal dhps gene.

Although our study did not focus its attention on field strains of H. parasuis serovar 2, preliminary data from antimicrobial susceptibility test to sulfonamide have shown that the majority of the field isolates of H. parasuis serovar 2 in Spain exhibited resistance to sulfisoxazole. This observation has obvious clinical and epidemiological consequences as H. parasuis serovar 2 is one of the most prevalent serovars in our country (Rubies et al. 1999).

The results reported here show the isolation of a H. parasuis serovar 2-specific DNA-fragment using the RDA approach. Therefore, the RDA technique has proven to be useful for the identification of genes that are differentially present in H. parasuis serovar 2.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Prof. Dr Gerald F. Gerlach for his invaluable help and advice. This work was supported by grants AGF 99-0196 and AGL 2002-04585-C02-01 GAN-ACU from the ‘Ministerio de Ciencia y Tecnología’, Spain.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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