Serological characterisation of Haemophilus parasuis isolates from Australian pigs

A total of 31 isolates of Haemophilus parasuis obtained from Australian pigs were serotyped by the Kielstein-Rapp-Gabrielson scheme. The isolates were assigned to serovar 1 (1 isolate), serovar 2 (1 isolate), serovar 4 (4 isolates), serovar 5 (7 isolates), serovar 9 (2 isolates), serovar 10/7 (4 isolates), serovar 12 (1 isolate) and serovar 13 (6 isolates). The remaining 5 isolates could not be assigned to a serovar. Two different serovars (5 and 13) were detected in one herd. The only 2 isolates obtained from clinically normal pigs (from the same herd) were serovar 9. The common serovars were isolated from pigs with pneumonia as well as from pigs with conditions of the Glässer's disease type. The serological heterogeneity amongst Australian isolates of H parasuis has important implications for the use of vaccines to control Glässer's disease

Establishment, validation and use of the Kielstein-Rapp-Gabrielson serotyping scheme for Haemophilus parasuis

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. Hparasuis serovars 5 and 13 remain the predominant serovars present in Australian pigs

Application of ERIC-PCR for the comparison of isolates of Haemophilus parasuis

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 Glässer's 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 Glässer's 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's 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 Glässer's disease

Comparison of sampling sites and detection methods for Haemophilus parasuis

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