Serovar Reference Strains Research Articles

A new subtype of serovar 6, K6b:O3, of Actinobacillus pleuropneumoniae based on genotypic analysis

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of fifteen field isolates of Actinobacillus pleuropneumoniae, including eleven North American and four Japanese ones, reactive to antisera against serovars 3, 6, 8 and/or 15. Ten North American isolates amplified a serovar 6-indicative fragment derived from the capsular loci, whereas one North American isolate and all four Japanese isolates amplified the serovar 6-indicative fragment as well as the serovar 3-indicative fragment. The five isolates producing a 3/6 banding pattern contain a type I CPS locus, named K6b, similar to serovar 6, but with differences in the cpxABCD and cpsABC gene sequences and the length of intergenic regions (modF-cpxA, and cpsC-cpsD). The main difference found between the K6 and K6b cps genes is a loss of function of a 113 AA UDP-glycosyltransferase found in type 6b due to the amino acid substitutions in the C-terminal domain of Cps6bA. Additionally, the isolates harbor a LPS O-Ag locus highly identical to those of field and reference strains of serovars 3, 8, 15, 17 and 19 but different from that of serovar 6. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 6, that we called “K6b:O3′′, and we propose isolate EH1248 as the reference strain.

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping

ABSTRACT Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies.

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species-specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test-based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

Proposal of a subtype of serovar 4, K4b:O3, of Actinobacillus pleuropneumoniae based on serological and genotypic analysis

The aim of this study was to investigate an isolate of Actinobacillus pleuropneumoniae, named 14–760, which was serologically not classifiable among the recognised serovars of A. pleuropneumoniae. It reacted with the antisera raised against serovars 3, 6, 8, 15 and 17 in the agar gel precipitation (AGP) test, and was positive in the capsular serovar 4-specific PCR (cps4B PCR) assay. The isolate contains a type II capsule locus similar to serovar 4 but with variations in the length of four intergeneric regions (modF-cpxA, cpxD-cpsA, cpsC-a 114 bp orf, and lysA-ydeN), and three gene sequences (modF, cpsC and ydeN). The main difference found between the K4 and K4b cps genes is the additional 35 AAs found in type 4b due to a 4 bp insert in cps4bC. The LPS O-Ag locus is highly similar to that of reference strains of serovars 3, 6, 8, 15, 17 and 19. Isolate 14–760 is biovar 1 and contains solely the structural genes required for toxin ApxII production (apxIICA), and the type I secretion system (apxIBD) for the export of ApxII. Antiserum against isolate 14–760 adsorbed with antigen prepared from serovars 8, 15 or 17 reference strains remained reactive with isolate 14–760, but not with antigens prepared from serovars 1–18. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 4, that we called “K4b:O3″, and we propose isolate 14–760 as the reference strain.

Ct value-based real time PCR serotyping of Glaesserella parasuis

Glaesserella parasuis is the causative agent of Glässer's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.

Identification of serovar 1a, 1b, 2, and 5 strains of Erysipelothrix rhusiopathiae by a conventional gel-based PCR

Among the four species of the genus Erysipelothrix, Erysipelothrix rhusiopathiae is the main species that causes disease in swine and poultry and has also been isolated from human patients. Recently, E. rhusiopathiae infections in domesticated animals have increased in many countries and are also the cause of emerging wildlife disease in arctic and boreal ecosystems. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by their serovars, which are determined based on cell wall antigens. Serotyping of Erysipelothrix is important, as specific E. rhusiopathiae serovars (1a, 1b, and 2) are associated with disease in pigs, poultry, and humans. However, serotyping is laborious and time-consuming and requires a full set of serovar reference strains and strain-specific antiserum. In this study, to develop a conventional gel-based PCR assay that can detect the main disease-associated serovars of E. rhusiopathiae, the draft genome sequences of E. rhusiopathiae strains of serovars 1a, 1b, 2, and 5, the last of which is often isolated from wild animals, were analyzed. Primers were designed based on the serovar-specific sequences of the strains and tested for field strains isolated from extensive origins. Among two hundred and ninety-seven isolates of various serovar strains of E. rhusiopathiae and other Erysipelothrix species, the PCR assay identified serovar 1a, 1b, 2, and 5 strains of E. rhusiopathiae. This conventional gel-based PCR assay should be useful for serovar surveillance of E. rhusiopathiae isolates in domesticated and wild animals as well as in humans.

Antimicrobial susceptibility of Avibacterium paragallinarum isolates from outbreaks of infectious coryza in Dutch commercial poultry flocks, 2008–2017

The objective of the present study was to determine the in vitro antimicrobial susceptibility of Avibacterium paragallinarum isolates from infectious coryza outbreaks in Dutch commercial poultry, from 2008 till mid-2017. By using a broth microdilution method, minimal inhibitory concentrations (MICs) of 15 antimicrobial agents were assessed, and MIC50 and MIC90 values were determined. Additionally, isolates were subjected to different PCRs for the presence of genes that may confer antimicrobial resistance. Besides field isolates, a set of reference strains, among which the nine Kume strains and one Page serovar strain, were included in the study.For broth microdilution testing a new growth medium, recently developed for susceptibility testing of Haemophilus parasuis, was used. The medium proved to be suitable for broth microdilution susceptibility testing of NAD dependent Av. paragallinarum as well; visible growth was obtained in growth control wells and accepting a deviation of one dilution step, MIC values were reproducible. Results of 44 field isolates originating from 25 outbreaks showed relatively good susceptibility to antimicrobial agents that are recommended for the treatment of infectious coryza in the Netherlands, except for tetracycline; circa 75% of the isolates were characterized by MIC values of tetracycline of ≥16 μg/ml. In almost a quarter of these isolates with high MICs of tetracycline, tet genes were detected. For the remaining isolates with elevated MIC values, the mechanism conferring resistance remains to be studied. Of most agents, low MIC values were determined for the nine Kume and one Page serovar reference strains, as well as negative PCR results for resistance genes, being concordant with agar diffusion results reported for these strains.

Rapid detection of Haemophilus parasuis using cross-priming amplification and vertical flow visualization

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to high morbidity and mortality in naive swine populations. Accurate detection and identification of the causative agent are difficult, yet necessary, for disease control. In this study, a simple and rapid method of cross-priming amplification (CPA) with a vertical flow (VF) visualization strip was established to detect H. parasuis. The reaction can specifically identify 15 serovar reference strains and 57 clinically isolated strains of H. parasuis, with a detection limit of 14CFU. The performance of the CPA-VF assay was evaluated and compared with that of species-specific PCR by testing 62 clinical culture-positive specimens of H. parasuis. The entire process, from specimen processing to analysis of the results, can be completed in 2h without a complicated apparatus. The convenience and speed of the CPA-VF assay in this study make it a suitable choice for epidemiological investigation and point-of-care testing (POCT) for H. parasuis infection.

Characterization of Actinobacillus pleuropneumoniae field strains antigenically related to the 3-6-8-15 group from diseased pigs in Japan and Argentina

The objectives of this study were to determine the serovar of a collection of Actinobacillus pleuropneumoniae strains within the 3-6-8-15 cross-reacting group and to analyze their phenotypic and genetic properties. Based on the serological tests, forty-seven field strains of Actinobacillus pleuropneumoniae isolated from lungs with pleuropneumonia lesions in Japan and Argentina were found to be serovars belonging to the 3-6-8-15 cross-reacting group. By using a capsule loci-based PCR, twenty-nine (96.7%) and one (3.3%) from Japan were identified as serovars 15 and 8, respectively, whereas seventeen (100%) from Argentina were identified as serovar 8. The findings suggested that serovars 8 and 15 were prevalent within the 3-6-8-15 cross-reacting group, in Argentina and Japan, respectively. Phenotypic analyses revealed that the protein patterns observed on SDS–PAGE and the lipopolysaccharide antigen detected by immunoblotting of the reference and field strains of serovars 8 and 15 were similar to each other. Genetic (16S rDNA, apxIIA, apxIIIA, cps, cpx genes, apx and omlA patterns) analyses revealed that the apxIIA and apxIIIA genes of the field strains of serovars 8 and 15 were similar to those of the reference strains of serovars 3, 4, 6, 8 and 15. The results obtained in the present study may be useful for the development of more effective vaccines against disease caused by A. pleuropneumoniae by including the homologous antigens to the most prevalent serovars in specific geographical areas.

Serovar and Antimicrobial Resistance Profiles of <i>Actinobacillus pleuropneumoniae</i> Isolated in Japan from 2006 to 2011

Serovar and Antimicrobial Resistance Profiles of <i>Actinobacillus pleuropneumoniae</i> Isolated in Japan from 2006 to 2011

Development of a Multiplex Polymerase Chain Reaction Method for <i>cps</i> Typing of <i>Actinobacillus pleuropneumoniae </i>Serovars 1, 2, 5, 7, and 15

We developed a multiplex polymerase chain reaction (PCR) method using primers specific to the capsular polysaccharide biosynthesis genes (cps) to type Actinobacillus pleuropneumoniae serovars 1, 2, 5, 7, and 15. This multiplex PCR method may be useful for the typing of serovar 15, which is recently becoming more prevalent, as well as the most prevalent serovars 1, 2, 5, and 7 in Japan. Discipline: Animal health Additional key words: pig, pleuropneumonia, serotyping * Corresponding author: e-mail itohiroy@affrc.go.jp Received 7 October 2014; accepted 24 December 2014. Introduction Actinobacillus pleuropneumoniae is an etiologic agent of porcine pleuropneumonia that causes serious economic losses in the pig-rearing industry (Gottschalk 2012). Fifteen serovars of A. pleuropneumoniae have been identified to date, mainly based on the antigenic diversity of their capsular polysaccharides (Blackall et al. 2002, Perry et al. 1990). The prevalent local serovars vary among countries (Gottschalk 2012); in Japan, serovars 2 (59%), 1 (28%), 5 (8%), and 7 (3%) account for approximately 98% of the strains isolated from pigs (Asawa et al. 1995, Fukuyasu et al. 1991, 1996, Kume & Nakai 1988, Morioka et al. 2006, 2008, Suzuki et al. 1989, Yoshimura et al. 2002). However, the incidence of isolated cases of serovar 15, which is the most recently identified serovar (Blackall et al. 2002), has been recently increasing in Japan (Ito 2013, Morioka et al. 2008). Because the virulence of strains differs depending on their serovars and differences in serovars affect the effectiveness of vaccines (Gottschalk 2012), serotyping is widely performed in veterinary diagnostic laboratories. However, cross-reactions are often observed among different serovars, for example, among serovars 1, 9, and 11 (Gottschalk 2012), serovars 3, 6, 8, and 15 (Gottschalk 2012), serovars 4 and 7 (Gottschalk 2012), and serovars 7 and 15 (Blackall et al. 2002, Koyama et al. 2007), preventing the accurate and rapid typing of field strains. In the last decade, serovar-specific polymerase chain reaction (PCR) typing methods based on the sequence variation in genes involved in capsular polysaccharide biosynthesis (cps) have been developed for serovars 1-3, 5-8, 10, 12, and 15 (Angen et al. 2008, Bosse et al. 2014, Ito 2010, Jessing et al. 2003, Lo et al. 1998, Schuchert et al. 2004, Turni et al. 2014, Zhou et al. 2008). These PCR typing methods facilitate the reliable typing of A. pleuropneumoniae serovars without cross-reactions and mistyping. In the present study, we developed a multiplex PCR method for cps typing of A. pleuropneumoniae serovars 1, 2, 5, and 7, which are the most prevalent in Japan (Asawa et al. 1995, Fukuyasu et al. 1991, 1996, Kume & Nakai 1988, Morioka et al. 2006, 2008, Suzuki et al. 1989, Yoshimura et al. 2002), as well as serovar 15, isolated cases of which have been increasing (Ito 2013, Morioka et al. 2008). Materials and methods We used the A. pleuropneumoniae serovar reference strains serovar 1, 4074; serovar 2, S1536; serovar 3, S1421; serovar 4, M62; serovar 5a, K17; serovar 6, Femo, serovar 7, WF83, serovar 8, 405; serovar 9; CVJ13261; serovar 10, D13039; serovar 11; 56153; serovar 12, 8329; serovar 13, N273; serovar 14, 4906; and serovar 15, HS143, to determine suitable conditions for multiplex PCR. Seventy-

Correlation between Ureaplasma subgroup 2 and genitourinary tract disease outcomes revealed by an expanded multilocus sequence typing (eMLST) scheme.

The multilocus sequence typing (MLST) scheme of Ureaplasma based on four housekeeping genes (ftsH, rpL22, valS, and thrS) was described in our previous study; here we introduced an expanded MLST (eMLST) scheme with improved discriminatory power, which was developed by adding two putative virulence genes (ureG and mba-np1) to the original MLST scheme. To evaluate the discriminatory power of eMLST, a total of 14 reference strains of Ureaplasma serovars and 269 clinical strains (134 isolated from symptomatic patients and 135 obtained from asymptomatic persons) were investigated. Our study confirmed that all 14 serotype strains could successfully be differentiated into 14 eMLST STs (eSTs), while some of them could not even be differentiated by the MLST, and a total of 136 eSTs were identified among the clinical isolates we investigated. In addition, phylogenetic analysis indicated that two genetically significantly distant clusters (cluster I and II) were revealed and most clinical isolates were located in cluster I. These findings were in accordance with and further support for the concept of two well-known genetic lineages (Ureaplasma parvum and Ureaplasma urealyticum) in our previous study. Interestingly, although both clusters were associated with clinical manifestation, the sub-group 2 of cluster II had pronounced and adverse effect on patients and might be a potential risk factor for clinical outcomes. In conclusion, the eMLST scheme offers investigators a highly discriminative typing tool that is capable for precise epidemiological investigations and clinical relevance of Ureaplasma.

Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15.

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and impact of the study: A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Comparison of ompP5 sequence-based typing and pulsed-filed gel electrophoresis for genotyping of Haemophilus parasuis

In this study, comparison of the outer membrane protein P5 gene (ompP5) sequence-based typing with pulsed-field gel electrophoresis (PFGE) for the genotyping of Haemophilus parasuis, the 15 serovar reference strains and 43 isolates were investigated. When comparing the two methods, 31 ompP5 sequence types (STs) and 43 PFGE types were identified withompP5 STs A16 and A28 being the dominant ompP5 ST types, while no predominant PFGE type was found. The discriminatory indices were 0.95 for ompP5 sequence-based method and 0.98 for PFGE, and the two techniques were proved to be 70.7% congruent. Therefore,ompP5 sequence-based typing was a simple and inexpensive method, enabling it as a preliminary technique to research the molecular differentiation of H. parasuis. Key words: Haemophilus parasuis, ompP5-based typing method, PFGE, molecular typing.

Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector

The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glässer's disease in pigs, using a novel H. parasuis–Escherichia coli shuttle vector. A 4.2kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5×102CFU/μg of DNA. Transformation efficiency was notably increased (1.3×105CFU/μg of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen.

Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.

Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki ( H. parasuis serovar 5 reference strain, highly virulent) and SW114 ( H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.

Comparison of full gyrA, gyrB, parC and parE gene sequences between all Ureaplasma parvum and Ureaplasma urealyticum serovars to separate true fluoroquinolone antibiotic resistance mutations from non-resistance polymorphism

To determine the role of amino acid substitutions in Ureaplasma GyrA, GyrB, ParC and ParE proteins in mediating fluoroquinolone resistance. Nucleic acid sequences from gyrA, gyrB, parC and parE genes from all 14 Ureaplasma serovars were aligned. Full genome sequences for serovars 1, 3-7, 9 and 11-14 were available from the National Center for Biotechnology Information database and we sequenced the full topoisomerase genes from ciprofloxacin-susceptible reference strains of serovars 2, 8 and 10. Phylogenetic trees were constructed to analyse nucleotide sequence similarity. Deduced amino acid sequences were compared with all 33 previously reported fluoroquinolone-resistant strains to clarify true fluoroquinolone-resistance-associated substitutions. Non-resistance-associated polymorphisms were identified in GyrA (39), GyrB (26), ParC (107) and ParE (34) proteins. Phylogenetic analysis demonstrated species clustering for all genes, except parE in which serovars 4, 12, 10 and 13 formed a separate cluster more similar to Ureaplasma parvum than the remaining Ureaplasma urealyticum serovars. Examination of all previously reported fluoroquinolone-resistant strains found that one-third of identified residue substitutions could be attributed to normal species polymorphism; therefore, the mechanism of resistance for these strains is still undetermined. In particular, Glu or Asp at position 112 in GyrA and Ala or Thr at 125/136 in ParC were substitutions identified when U. urealyticum strain sequences were previously aligned with the published serovar 3 genome sequence. Combining analysis of the recently available Ureaplasma genomes with sequences from the additional serovars has enabled us to clarify which substitutions found by previous investigators could potentially be responsible for fluoroquinolone resistance.

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides ( cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.