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1.
Objective Investigate the diversity of serovars of Haemophilus parasuis (Hps) present in Australian pig herds. Design Nasal swabs were used to obtain multiple isolates of Hps, which were grouped first by genotyping and then by serotyping representative isolates. Procedure Swabs were taken from the nasal cavity of just-weaned healthy pigs from multiparous sows on 12 farms and from post-weaned pigs of multiparous sows on 1 farm. On 5 of the 13 farms, nasal swabs were also obtained from pigs showing clinical signs suggestive of Glässer's disease. On a further 7 farms, nasal swabs were obtained only from pigs with clinical signs suggestive of Glässer's disease. Results A total of 556 Hps isolates were genotyped, and 150 isolates were serotyped. Hps was detected on 19 of the 20 farms, including 2 farms with a long history of freedom from Glässer's disease. Isolates of Hps belonging to serovars regarded as potentially pathogenic were obtained from healthy pigs at weaning on 8 of the 10 farms with a history of Glässer's disease outbreaks. Sampling 213 sick pigs yielded 115 isolates of Hps, 99 of which belonged to serovars that were either potentially pathogenic or of unknown pathogenicity. Only 16 isolates from these 213 sick pigs were of a serovar known to be non-pathogenic. Conclusion Healthy pigs contain a range of Hps serovars, even on farms free of Glässer's disease. Nasal swabbing of both healthy and sick pigs seems a useful method of serovar profiling of farms.  相似文献   

2.
Objective To validate a polymerase chain reaction (PCR) based method, Enterobacterial Repetitive Intergenic Consensus‐PCR (ERIC‐PCR), for the fingerprinting of Haemophilus parasuis strains and to use that method to differentiate isolates from apparently related outbreaks of Glässers disease on three pig farms. Design ERIC‐PCR was evaluated by comparing 15 different strains that represented all 15 recognised serovars in the Kielstein‐Rapp‐Gabrielson (KRG) scheme for serotyping H parasuis. Next, ERIC‐PCR was used to examine 14 Australian field isolates of H parasuis; 12 collected from three farms suffering apparently related outbreaks of Glässers disease and two from two other farms with no known connection. Results The 15 serovar reference strains all gave unique, reproducible ERIC‐PCR fingerprints. The 12 isolates from the three apparently related outbreaks all gave a single fingerprint, which was distinct from any seen in the 15 serovar reference strains and the two other Australian field isolates in the studied farms. The confirmation that all 12 isolates were the same strain allowed the development of a prevention and control program that has prevented the emergence of any further outbreaks of Glässer disease on the three farms. Conclusion ERIC‐PCR is a suitable technique for the differentiation of unrelated strains of H parasuis. The finding that the 12 field isolates of H parasuis all shared the same fingerprint is strong evidence that there was a common source of infection on all three farms. This study has shown, for the first time, that ERIC‐PCR is a suitable technique for the sub‐typing of H parasuis and useful for studying the epidemiology of outbreaks of Glässers disease.  相似文献   

3.
Comparison of sampling sites and detection methods for Haemophilus parasuis   总被引：3，自引：0，他引：3  
Objective To improve the isolation rate and identification procedures for Haemophilus parasuis from pig tissues. Design Thirteen sampling sites and up to three methods were used to confirm the presence of H. parasuis in pigs after experimental challenge. Procedure Colostrum‐deprived, naturally farrowed pigs were challenged intratracheally with H parasuis serovar 12 or 4. Samples taken during necropsy were either inoculated onto culture plates, processed directly for PCR or enriched prior to being processed for PCR. The recovery of H parasuis from different sampling sites and using different sampling methods was compared for each serovar. Results H parasuis was recovered from several sample sites for all serovar 12 challenged pigs, while the trachea was the only positive site for all pigs following serovar 4 challenge. The method of solid medium culture of swabs, and confirmation of the identity of cultured bacteria by PCR, resulted in 38% and 14% more positive results on a site basis for serovars 12 and 4, retrospectively, than direct PCR on the swabs. This difference was significant in the serovar 12 challenge. Conclusion Conventional culture proved to be more effective in detecting H parasuis than direct PCR or PCR on enrichment broths. For subacute (serovar 4) infections, the most successful sites for culture or direct PCR were pleural fluid, peritoneal fibrin and fluid, lung and pericardial fluid. For acute (serovar 12) infections, the best sites were lung, heart blood, affected joints and brain. The methodologies and key sampling sites identified in this study will enable improved isolation of H parasuis and aid the diagnosis of Glässer's disease.  相似文献   

4.
An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPTM) and intratracheally (PAPTCp), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPTCp. Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPTCp yielded a serum IgA response.  相似文献   

5.
SUMMARY A total of 362 haemophili, isolated from pigs throughout Australia, were characterised by phenotypic properties. Most were identified as Actinobacillus pleuropneumoniae (296 isolates) or Haemophilus parasuis (52 isolates). The remaining isolates were identified as Haemophilus Taxon ‘minor group’ (12 isolates) and Haemophilus Taxon D (two isolates). All 296 A pleuropneumoniae isolates were serotyped by slide agglutination and/or gel diffusion, using rabbit antisera against all 12 recognised serovars. Of these, only 156 (52.7%) could be assigned to a single serovar as follows: serovar 1–85 isolates, serovar 2–4 isolates, serovar 3–2 isolates, serovar 5–10 isolates, serovar 7–51 isolates, serovar 11–2 isolates and serovar 12–2 isolates. Of the remaining 140 isolates, 91 gave cross-reactions with serovars 3 and 6, one cross-reacted with serovars 9 and 10, one cross-reacted with serovars 9 and 11 whereas 47 gave no reaction with any of the antisera.  相似文献   

6.
OBJECTIVES: To produce antisera to the 15 recognised reference strains of the Kielstein-Rapp-Gabrielson (KRG) serotyping scheme for Haemophilus parasuis, validate those sera and use them to serotype 46 Australian field isolates of H parasuis. DESIGN: Antisera were produced in rabbits and validated by cross-testing with the reference strains and re-testing 15 Australian field isolates of H parasuis that had been previously serotyped in the United States of America. The validated antisera were then used to determine the serovar of 46 Australian isolates. RESULTS: Monospecific antisera were produced for 14 of the 15 KRG serovars of H parasuis. Two Australian field isolates, confirmed previously as serovars 1 and 7, were used to produce monospecific antisera for serovars 1 and 7 respectively. The antiserum for serovar 4 gave a one-way cross reaction with the antigen of serovar 14. The typing antisera correctly typed all 15 H parasuis that had been previously typed by antisera produced overseas. The 46 field isolates were shown to belong to serovars 2 (two isolates), 4 (one isolate), 5 (18 isolates), 12 (two isolates) and 13 (four isolates). The remaining 19 isolates were non-typable. CONCLUSION: Serotyping of H parasuis isolates is now available in Australia. H parasuis serovars 5 and 13 remain the predominant serovars present in Australian pigs.  相似文献   

7.
Haemophilus parasuis causes contagious porcine Glässer's disease, which is occurring worldwide and leads to severe losses in the pig industry. To identify novel antigen candidates against this disease, 22 surface-exposed or secreted proteins were selected from the annotated H. parasuis genome by reverse vaccinology strategy. Expression of these proteins in Escherichia coli was attempted. Immunogenicity of the expressed candidates was assessed using Western blot analysis with mouse-derived antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5. Three ABC-type transporters (OppA, YfeA and PlpA) and 1 curli protein assembly (CsgG) were identified as potent immunogenic proteins. The proteins show cross-reactions when tested with sera raised against serovars 4 and 5 of H. parasuis.  相似文献   

8.
From September 2008 to December 2010, 112 Haemophilus parasuis strains were isolated from 536 pigs with clinical signs of Glässer’s disease in South China, for a frequency of 21%. The 112 strains were subjected to serovar analysis by gel diffusion (GD) and indirect hemagglutination (IHA) tests and to genotype analysis by means of pulsed-field gel electrophoresis (PFGE). With a combination of the GD and IHA results, serovars 5 and 4 were found to be the most prevalent, at 23% and 17%, respectively, followed by serovars 2 (8%), 15 (7%), 13 (6%), and 12 (5%); 20% of the strains were nontypeable. The 112 strains were genetically diverse, with 85 genotypes identified (discriminatory index 0.992). The 89 typeable isolates belonged to 15 H. parasuis serovars displaying 63 different PFGE profiles. The 23 nontypeable strains displayed 22 different PFGE profiles. These findings confirmed that 15 serovars and diverse genotypes of H. parasuis were widely distributed in southern China.  相似文献   

9.
OBJECTIVES: To clarify the serological identity of the prototype strain of a group of Actinobacillus pleuropneumoniae isolates that could not be serotyped in previous studies and to establish the serovar of 378 isolates of A pleuropneumoniae obtained from pigs in Australia over the period 1993 to 1996. DESIGN: After initial validation, QGD and IHA tests were used to characterise the prototype isolate (HS143) selected to represent the cross-reacting isolates that were found in a previous study. Next, 378 recent field isolates of A pleuropneumoniae were characterised using the existing gel diffusion serotyping technique and/or the IHA or QGD tests. RESULTS: The indirect haemagglutination test was shown to be capable of correctly recognising the reference strain for all serovars except serovar 11. While the quantitative gel diffusion test was not as effective as indirect haemagglutination, it could recognise serovar 11. When the two tests were used to examine the prototype strain (HS143) of the cross-reactive isolates, the results indicated that HS143 is serologically distinct from all 12 of the recognised serovars of A pleuropneumoniae. However, as HS143 was subsequently identified as serovar 12 by one of the leading international reference laboratories, the antiserum to isolate HS143 was used as the serovar 12 antiserum. A total of 346 of the 378 A pleuropneumoniae field isolates examined could be confidently serotyped with almost 90% of the isolates being either serovar 1 (104 isolates); serovar 7 (83 isolates) or serovar 12 (142 isolates). A range of other serovars and some cross-reactive isolates made up the remainder of the isolates. CONCLUSION: The serovar 12 antiserum produced against the international reference strain (1096) does not recognise Australian serovar 12 isolates. The antiserum raised against isolate HS143 does recognise the Australian serovar 12 isolates. The dominant serovars of A pleuropneumoniae infecting Australian pigs are (in decreasing order) serovars 12, 1 and 7.  相似文献   

10.
Haemophilus parasuis is the aetiological agent of Glässer’s disease and is also a commensal of the upper respiratory tract of pigs. Trimeric autotransporter (vtaA) genes have been identified in H. parasuis and divided into three groups on the basis of the translocator domain sequence. In this study, group 3 vtaA genes were demonstrated by PCR in all 157 H. parasuis isolates tested. Group 1 vtaA genes were associated with virulent strains; 52/54 (96%) group 1 vtaA negative field isolates were isolated from the nasal passages of healthy animals, whereas no group 1 vtaA negative field isolates were isolated from cases of Glässer’s disease. There was an association between absence of group 1 vtaA, sensitivity to phagocytosis and serum and classification of isolates into nasal cluster C by multilocus sequence typing. A multiplex PCR was developed for diagnosis of H. parasuis at the species level (group 3 vtaA positive) and to differentiate putative non-virulent strains (group 1 vtaA negative). When applied to field samples, the PCR confirmed a high prevalence of H. parasuis in conventionally farmed pigs and demonstrated that almost half of the animals carried potentially virulent strains.  相似文献   

11.
Prevalence of Haemophilus parasuis serovars among isolates from swine.   总被引：11，自引：0，他引：11  
Two hundred sixty Haemophilus spp isolates that had been obtained from the respiratory tract and other sites of swine were acquired from diagnostic laboratories, primarily in the United States and Canada. The majority of isolates (243/260) were biochemically characterized as H parasuis; however, a few isolates of taxa distinct from H parasuis (taxa "minor group," D, E, and F) were identified. Fourteen H parasuis serovars were identified, and of those previously described, the most prevalent were 5 (24.3% of isolates), 4 (16.1%), 2 (8.2%), and 7 (3.7%). Three new serovars that were also prevalent included ND4 (11.1%), ND3 (8.6%), and ND5 (6.6%). Serovars 1, 3, 6, C, D, and new serovars ND1 and ND2 were infrequently identified, and 15.2% of isolates were nontypeable. It was not uncommon to isolate multiple serovars from swine of the same herd or related herds. Distribution of serovars among isolates from the United States and Canada was generally similar; however, a higher prevalence of serovar 5 and a lower prevalence of serovars 2, ND3, and ND5 were evident in isolates from Canada. Comparison of isolates obtained from the respiratory tract of swine without polyserositis with those obtained from swine with polyserositis revealed an increased frequency of serovars 4 and 5, and a decreased frequency of serovar 2, among isolates from swine with polyserositis. However, all prevalent serovars were isolated from swine with polyserositis, and data were not indicative of an association between serovar, site of isolation, or pathogenic potential.  相似文献   

12.
OBJECTIVE: To characterize the genetic diversity of Haemophilus parasuis field isolates with regard to serovar, herd of origin, and site of isolation. SAMPLE POPULATION: Isolates of H parasuis obtained from pigs in 15 North American herds and multi-farm systems. PROCEDURE: 98 H parasuis isolates were genotyped with the enterobacterial repetitive intergeneic consensus based-polymerase chain reaction (ERIC-PCR) technique and serotyped via agar gel precipitation test. Genomic fingerprints were analyzed and dendrograms were constructed to identify strains from the same serovar group, herd of origin, or isolation site and to evaluate the genetic variability within these categories. RESULTS: Serovar 4 (39%) and nontypeable (NT) isolates (27%) were most prevalent. Thirty-four distinct strains were identified among the 98 isolates, using a 90% similarity cutoff. Strains from serovar 4 and NT isolates had high genetic diversity (12 and 18 strains, respectively). One to 3 major clusters of prevalent strains could be identified in most of the evaluated herds. Haemophilus parasuis strains isolated from the upper respiratory tract were either serovar 3 or NT isolates. Potentially virulent strains (isolated from systemic sites) were either serovars 1, 2, 4, 5, 12, 13, or 14, or NT isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Although H parasuis had high genetic diversity overall, only a few strains caused disease in these herds. The ERIC-PCR technique was more discriminative than serotyping, and a broad genetic variety was observed within particular serovar groups.  相似文献   

13.
Serological characterization of Haemophilus parasuis isolates from China   总被引：21，自引：0，他引：21  
Cai X  Chen H  Blackall PJ  Yin Z  Wang L  Liu Z  Jin M 《Veterinary microbiology》2005,111(3-4):231-236
From September 2002 to December 2004, a total of 281 strains of Haemophilus parasuis were isolated from 17 provinces of China. All these isolates were serotyped by both the gel diffusion (GD) and the indirect haemagglutination (IHA) tests. By combining the GD and IHA results, serovar 4 (24.2%) and serovar 5 (19.2%) were the most prevalent serovars, followed by serovars 13 (12.5%), 14 (7.1%) and 12 (6.8%), while 12.1% of the isolates could not be assigned to a serovar (nontypable). A comparison of the number of isolates obtained from the respiratory tract of swine without polyserositis with those obtained from swine with polyserositis revealed an increased frequency of serovar 4 and a significantly decreased frequency of serovar 13 among isolates from the respiratory tract of swine without polyserositis, whereas the frequency of isolation of serovars 5, 12, 14 and nontypable from swine with or without polyserositis were similar. Co-infection of H. parasuis and other bacterial agents was studied in 183 cases examined from June 2003 to December 2004. Streptococcus suis (30.6%; 56), Escherichia coli (21.9%; 40), Bordetella bronchiseptica (21.3%; 39) and Pasteurella multocida (14.2%; 26) were the bacterial agents frequently co-isolated with H. parasuis in China.  相似文献   

14.
Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.   总被引：29，自引：0，他引：29  
We report on the re-examination of nine Australian isolates of Actinobacillus pleuropneumoniae that have been previously assigned to serovar 12. In the ring precipitation test, none of the nine isolates reacted with antisera to serovars 1-14 of A. pleuropneumoniae. Antiserum prepared against one of the Australian isolates gave no reaction with any of the 14 recognised serovar reference strains, except serovar 7. This reaction of the HS143 antiserum with serovar 7 antigen could be removed by adsorption with serovar 7 antigen. The adsorbed antiserum remained reactive with HS143 and the other eight Australian isolates. The nine Australian isolates were all shown to express ApxII and ApxIII, found in serovars 2, 4, 6 and 8, as well as the 42kDa outer membrane protein found in all serovars of A. pleuropneumoniae. The nine Australian isolates were found to possess the following toxin associated genes apxIBD, apxIICA, apxIIICA, apxIIIBD and apxIVA. The toxin gene profile of the Australian isolates is typical of A. pleuropneumoniae serovars 2, 4, 6 and 8. On the basis of the serological characterisation results and the toxin gene profiles, we propose that these isolates represent a new serovar of A. pleuropneumoniae--serovar 15--with HS143 being the reference strain for the new serovar.  相似文献   

15.
Haemophilus parasuis is the etiological agent of Glässer’s disease characterized by fibrinous polyserositis, polyarthritis, and meningitis in young pigs. But it is difficult to develop universal serological diagnostic tools and effective vaccines against this disease because of the serovar diversity of the isolates. In this study, enterobacterial repetitive intergenic consensus-polymerase chain reaction, were performed to investigate the gene profile of 1 1 1 isolates of H. parasuis from China. And a specific common gene of H. parasuis was cloned and identified as the outer-membrane protein (OMP) P2 gene. Sequencing results of OMP P2 genes of 22 isolates showed that they had high homology and could be divided into 2 genetic types. Moreover, the OMP P2 protein was expressed in Escherichia coli expressing system. And the purified recombinant protein provided partial protection against H. parasuis infection in mice. It suggested the OMP P2 was an immunogenic protein and had great potential to serve as a vaccine and diagnostic antigen.  相似文献   

16.
Swine erysipelas vaccines are routinely used to protect pigs against peracute and acute/urticarial forms of Erysipelothrix. Between 1995 and 1998, 34 swine herds across four Australian states experienced vaccine failure. Forty-four isolates of Erysipelothrix rhusiopathiae of serovars 2, 1a, 1b and 1bx21 were recovered from 15 of these 34 vaccine breakdown herds. These isolates were characterised by restriction fragment length polymorphism (RFLP) analyses using RsaI and AluI on whole cell DNA and for the presence of plasmid DNA. Results were compared with those of 20 isolates from 16 herds unaffected by vaccine breakdown and 13 isolates representing 10 reference strains. The majority of breakdown herds possessed isolates of serovar 2 (9/15 herds), followed by serovar 1a (5 herds). No geographic predominance of a single serovar was evident. The identification of 10 RsaI profiles from whole cell DNA among the 44 isolates from 15 breakdown herds indicated that a single, new clonal lineage of E. rhusiopathiae was not responsible for vaccine failure. RsaI RFLP analyses detected a further 14 distinct profiles among 20 field strains unassociated with vaccine breakdowns, and none matched profiles of the 10 serovar reference strains for serovars 1a, 1b, 2 or 21. This technique is recommended for epidemiological studies of E. rhusiopathiae strains.  相似文献   

17.
Due to the serovar diversity in Haemophilus (H.) parasuis, it is difficult to develop a universal serological method for detection of this pathogen. Here, we report a universal plate-agglutination test for detecting H. parasuis. Diagnostic antisera were prepared by mixing antisera of serovars 4, 5, 12, 13 and 14 in the optimized ratio. The results of the plate-agglutination test showed that the diagnostic antisera could agglutinate with all 15 reference strains of H. parasuis and 74/75 clinical isolates. Further, the specificity of the method was validated with 22 bacterial strains from 12 related species.  相似文献   

18.
Haemophilus parasuis infection in pigs is characterized by fibrinous polyserositis, arthritis and meningitis. Despite the fact that traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, molecular-based methods are alternatives for species-specific tests and epidemiological studies. The aim of this study was to characterize H. parasuis field strains from different states of Brazil, employing serotyping and genotyping methods. Serotyping revealed that serovar 4 was the most prevalent (26.1%), followed by serovars 5 (17.4%), 14 (8.7%), 13 (4.4%) and 2 (4.4%), whereas 39% of the strains were considered as untypeable. AFLP with a single enzyme and PFGE were able to type all isolates tested, generating 34 and 20 different profiles, respectively, including untypeable strains. Besides the slightly higher discrimination index presented by AFLP, PFGE with Not I restriction enzyme showed a better correlation with epidemiological data, grouping strains of the same serovar, animal or farm origin. The results indicated AFLP and PFGE as valuable tools for typing H. parasuis isolates collected in Brazil.  相似文献   

19.
Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).  相似文献   

20.
The pathogenicity of 2 isolates of each of serovars 7, 3, 1 and 2 of Actinobacillus pleuropneumoniae was tested by intranasal inoculation into 60, 6-week-old large white pigs. Four dose rates varying from 0.27 to 560 x 10(6) organisms per pig with 10-fold serial dilutions were used. Surviving pigs were necropsied 7 days after inoculation. The proportion of pigs dying and developing gross lesions following infection was significantly greater for pigs given serotype 1 than for each of the other 3 serotypes, which did not differ significantly from each other. Twelve of 16 pigs given either of the 2 isolates of serovar 1 died after acute illness and 1 of 44 pigs given either of the 2 isolates each of serovars 7, 3 and 2 died. Pigs given serovar 1 showed high temperatures, severe respiratory distress, frothy haemorrhagic nasal discharge and weight loss. Lung lesions were produced in all 16 pigs given serovar 1, in 7 of 14 pigs given serovar 7, 7 of 14 pigs receiving serovar 3 and in 5 of 16 pigs given serovar 2. The lethal infections were characterised by a severe acute fibrinohaemorrhagic necrotising pleuropneumonia, whereas non-lethal cases had lung lesions ranging from necrotising purulent pleuropneumonia to abscessation. Significant differences between isolates in proportions of tissues culture positive for A. pleuropneumoniae for serovars 7 and 2, but not for serovars 3 and 1 suggested that isolates may vary in virulence within serovars, but more detailed studies are needed to clarify this point.  相似文献   

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