Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.

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.     

Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis

The aim of this study was to compare the use of indirect haemagglutination (IHA) and gel diffusion (GD) tests for serotyping Haemophilus parasuis by the Kielstein-Rapp-Gabrielson (KRG) scheme. All 15 serovar reference strains, 72 Australian field isolates, nine Chinese field isolates, and seven isolates from seven experimentally infected pigs were evaluated with both tests. With the IHA test, 14 of the 15 reference strains were correctly serotyped-with serovar 10 failing to give a titre with serovar 10 antiserum. In the GD test, 13 reference strains were correctly serotyped-with antigen from serovars 7 and 8 failing to react with any antiserum. The IHA methodology serotyped a total of 45 of 81 field isolates while the GD methodology serotyped a total of 48 isolates. For 29 isolates, the GD and IHA methods gave discordant results. It was concluded that the IHA is a good additional test for the serotyping of H. parasuis by the KRG scheme if the GD methodology fails to provide a result or shows unusual cross-reactions.     

Serological characterization of Haemophilus parasuis isolates from China

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.     

Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

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).     

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

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.     

Serological characterization of Danish Haemophilus parasuis isolates

A total of 103 Danish Haemophilus parasuis field isolates was collected from diseased pigs in connection with routine diagnostics. The isolates were serotyped using indirect haemagglutination (IHA) and for 57 of the isolates the serotyping was also performed by immunodiffusion. Serovar 5 was the most prevalent (36%), followed by serovar 4 (13%) and serovar 13 (22%), whereas 15% of the strains were nontypeable by IHA. Serovars 1, 2, 6, 7, 9, 12, 14, and 15 were only represented by a small number of isolates. Most of the Danish isolates belong to serovars, which earlier have been shown to be virulent. The strains could be divided into two groups depending on whether they were isolated from cases with systemic disease (polyserositis, arthritis or meningitis) or if they only were found in the lower respiratory tract. The most marked differences were observed for serovar 4, which had a higher prevalence in respiratory disease compared to systemic infection, and for the nontypeable isolates, which were mainly found in cases of systemic infection.     

Serological characterisation of Australian isolates of Actinobacillus pleuropneumoniae

SUMMARY: Using hyperimmune rabbit antiserums to 8 reference serovar strains, a total of 51 Australian isolates of Aclinobacillus pleuropneumoniae were serotyped by either a rapid slide agglutination test or a gel diffusion test. The results were: serovar 1 — 24 isolates; serovar 2 — 6 isolates; serovar 3 — 5 isolates; serovar 7 — 15 isolates; nontypable — 1 isolate. The rapid slide agglutination test was suitable for screening field isolates, as 73% of those tested reacted with only 1 of the 8 antiserums and were assigned to a particular serovar of the basis of this test alone.     

Isolation of Actinobacillus pleuropneumoniae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.     

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.     

Serological classification of Japanese isolates of Haemophilus paragallinarum using two serovar-specific monoclonal antibodies

A serological classification of 106 Japanese isolates of Haemophilus paragalinarum recovered from 1960 to 1984 was performed by dot-blotting and hemagglutination-inhibition (HI) tests using two serovar-specific monoclonal antibodies (MAbs), E5C12D10 and F2E6. By the dot-blotting test, 49 of the isolates were serovar A and 55 isolates were serovar C, and the two remaining isolates did not react with either MAb. These two nontypable strains had no hemagglutinating activity against chicken erythrocytes and were nonpathogenic to chickens. Although 49 serovar A isolates were serotyped by the HI test, only 23 of the 55 serovar C isolates could be serotyped. The remaining 32 isolates could not be serotyped because no or low hemagglutinating activity could be detected. Our results indicate that H. paragallinarum serovars A and C have both been present in Japan since 1960, with serovar A isolates being dominant before 1970 and serovar C isolates more prevalent than serovar A since 1970.     

An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.     

Hemagglutinin serotyping of <Emphasis Type="Italic">Avibacterium paragallinarum</Emphasis> isolates from Ecuador

Avibacterium paragallinarum is the causative agent of infectious coryza, an acute respiratory disease of chickens. In this study, a total of 28 isolates of A. paragallinarum from Ecuador were serotyped by the hemagglutinin scheme which recognizes nine serovars. Out of 28 isolates, 17 isolates belonged to serovar A-3, and five isolates to each serovars B-1 and C-1, whereas one isolate was non-typeable. This is the first report of A. paragallinarum serovar A-3 outside Brazil and serovar C-1 outside Japan.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

Detection of strains of Haemophilus pleuropneumoniae using the coagglutination test

Three tests were used for the serological identification of the strains of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) isolated from pigs and coming from 66 sites where pleuropneumonia, caused by Haemophilus, occurred in pigs. The coagglutination test was found to be the best for the identification of the causal agent; the ring precipitation test was somewhat less sensitive, and worse results were obtained when rapid slide agglutination was used. Of all the field isolates of H. pleuropneumoniae, serovar 2 occurred most frequently (56%), followed by serovar 1 (39%); one strain was identified as serovar 7. Two strains have remained unidentified. The serological identification of the strains was performed on the basis of their comparison with eight type serovars of H. pleuropneumoniae.     

Characterisation of porcine haemophili isolated from Australian pigs between 1988 and 1992

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.     

Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine

Objective To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15.
Design The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs.
Results With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15).
Conclusions Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.     

Immunogenicity and haemagglutination of recombinant Avibacterium paragallinarum HagA

Inactivated vaccines of Avibacterium paragallinarum provide protection and reduce the economic losses caused by infectious coryza. However, inactivated bacterins provide protection only against the Page serovars included in the vaccine. In this study, we investigated the immunological properties of a functional recombinant haemagglutinin protein (rHagA) derived from a Taiwan isolate strain A9 as the immunogen for vaccination. The rHagA subunit vaccine protected 71% of immunized chickens against 10(10) colony-forming units (CFU) of viable A9. Vaccinated chickens which showed no clinical signs of coryza developed haemagglutination inhibition (HI) titers of 1:10 or greater. Haemagglutination (HA) of serovars A and C was not affected by the presence of rHagA specific antiserum. The HA of rHagA could only be induced against formaldehyde-fixed chicken red blood cells (FA-RBCs). These results suggested that HagA is a moderate immunogen and might not be a major haemagglutinin in vivo. However, HagA might be involved in haemagglutination when treated serovar C aggregates fixed RBCs in vitro.     

Isolation of serovar C-3 Haemophilus paragallinarum from Zimbabwe: A further indication of the need for the production of vaccines against infectious coryza containing local isolates of H. paragallinarum

Various isolates of Haemophilus paragallinarum, collected from a severe outbreak of infectious coryza in poultry from Zimbabwe, were serotyped and were found to belong to serovar C-3. Previously, isolates were serotyped using polyclonal antiserum produced against serogroup reference strains (0083 for serogroup A, 0222 for serogroup B and Modesto, or H-18 for serogroup C) of H. paragallinarum. In this case, polyclonal antiserum produced against these reference isolates were used, as well as polyclonal antiserum that has been raised specifically against the serovar C-3 isolate 46 C-3. When using the latter serum at a 1 in 50 dilution, no cross-reaction with other members of serogroup C were found. The severity of the disease outbreak in Zimbabwe, the vaccination history of the infected flocks on the sites and the isolation of the uniquely southern African serovar C-3, further highlights the need for vaccines composed of local isolates to control infectious coryza in regions where vaccination failures occur.     

The comparative pathogenicity of four serovars of Actinobacillus (Haemophilus) pleuropneumoniae

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.