Genetic diversity and toxin gene distribution among serovars of Actinobacillus pleuropneumoniae from Australian pigs

Objective

To explore the diversity among isolates of the Actinobacillus pleuropneumoniae serovars most common in Australia (serovars 1, 5, 7 and 15) and to examine the Apx toxin profiles in selected representative isolates.

Design

A total of 250 isolates selected from different farms were examined for their genotypic profiles and a subset of 122 isolates for their toxin profiles.

Methods

The isolates of serovars 1, 5, 7 and 15 selected for this study came from different farms and different Australian states and were submitted for serotyping to the reference laboratory. The overall diversity of the strains was explored with the enterobacterial repetitive intergenic consensus (ERIC) PCR and the presence of the toxin genes was investigated with a toxin PCR assay.

Results

Some degree of variation was observed in the ERIC‐PCR pattern within all four serovars, ranging from 38% to 61% genetic diversity. When looking at the toxin gene profile and, therefore, the predicted ability to produce the expected toxin pattern, one isolate each of serovars 1 (n = 20) and 7 (n = 47) and 17 isolates of serovar 15 (n = 40) showed variation to the expected gene profile.

Conclusion

The variations in toxin gene patterns, as detected by PCR, found in this study could be related to significant changes in the gene sequence or total absence of the gene. Variation in toxin gene sequences has been observed in other countries. This variation in the toxin profile could also explain possible variation in pathogenicity observed in the field.     

Granulomatous lymphadenitis associated with Actinobacillus pleuropneumoniae serotype 2 in slaughter barrows

This study evaluated the occurrence of granulomatous lymphadenitis and its association with Actinobacillus spp. in 151 653 slaughtered pigs. Markedly enlarged pulmonary hilar, mediastinal, mandibular or hepatic lymph nodes were detected in 6 castrated males. The cut surfaces showed multifocal yellow-white lesions. Histologically, gram-negative bacilli were visible in the centers of the lesions with asteroid bodies, epithelioid cells, and multinucleated giant cells. Dense fibrous connective tissue surrounded these granulomatous lesions. Immunohistochemically, the organisms reacted with polyclonal antibodies against Actinobacillus pleuropneumoniae serotype 2 in all 6 barrows. The organism was isolated from the lymph nodes of all 6 animals. The results indicate that the granulomatous lymphadenitis was associated with A. pleuropneumoniae serotype 2 and the disorder had a tendency to occur in slaughter barrows.     

Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae). Serotypes 8, 3 and 6. Serological response and cross immunity in pigs

Immunity obtained by vaccination with Haemophilus pleuropneumoniae is type specific and protection will only be obtained against the serotype contained in the vaccine. Serotype 8 is closely related to serotypes 3 and 6 and the objective of the present study was therefore to examine if cross immunity between the three serotypes could be obtained at vaccination.     

Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds

Eight Actinobacillus pleuropneumoniae biotype 2 strains were isolated in pure culture from lungs of pigs originating from two Danish herds with growing and finishing pigs. The antigenic properties were studied by indirect haemagglutination (IHA) and immunodiffusion (ID) tests using soluble surface antigens and by enzyme-linked immunosorbent assays (ELISA) using capsular enriched fractions and LPS. In all tests the strains proved antigenically homogeneous and serologically distinct from the known biotype 1 and 2 serotypes. Thus, the strains represent a new serotype which is provisionally proposed as biotype 2 serotype 14.     

Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec.

A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains of A. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.     

Serological characterisation of Actinobacillus pleuropneumoniae isolated from pigs in 1993 to 1996

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.     

Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs.

Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.     

Infections with Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in fattening pigs. Influence of piglet production systems and influence on production parameters

Two hundred and sixty-four feeder pigs from an age-segregated herd (A-pigs) and 264 feeder pigs from a continuous production system (C-pigs) were transferred into identical but separate rooms in a fattening herd employing all-in all-out production. On arrival, none of the A-pigs and 39% of the C-pigs were seropositive to Mycoplasma hyopneumoniae (M. hyo). At slaughter 30% of the A-pigs and 99% of the C-pigs were seropositive to M. hyo. Pigs with acute swine enzootic pneumonia (SEP) at slaughter and pigs that seroconverted to M. hyo late in the rearing period showed a lower growth rate compared with pigs with chronic SEP or pigs that seroconverted to M. hyo early or not at all. No A-pigs and 12% of the C-pigs were seropositive to Actinobacillus pleuropneumoniae 2 (A. pp 2) at arrival to the fattening herd. At slaughter, 10% of the A-pigs and 13% of the C-pigs were seropositive to A. pp 2. On arrival, the prevalence of pigs seropositive to A. pp 3 was lower among A-pigs than C-pigs. During the fattening period the situation was reversed. The prevalence of pleuritis at slaughter was low (2.7-4.2%) and there were no associations between pleuritis at slaughter and developments of antibodies to A. pp 2 or 3. However, pigs with pleuritis developed antibodies to M. hyo to a greater extent than pigs without pleuritis. Pigs with pleuropneumonia at slaughter and pigs that seroconverted to A. pp 2 or 3 had, during certain periods of the rearing, higher growth rates compared with pigs without pleuropneumonia or pigs that did not seroconvert to A. pp 2 or 3.     

Decay of acquired colostral antibodies to Actinobacillus pleuropneumoniae in pigs

The main objective of this study was to estimate the decay of acquired colostral antibodies to Actinobacillus pleuropneumoniae serotype 2 in pigs. Data were obtained from pigs in an isolated cohort of 47 pigs born to five sows seropositive to A. pleuropneumoniae serotype 2. The pigs were examined serologically at 18 different times from birth until an age of about 22 weeks, using an A. pleuropneumoniae serotype 2-specific blocking enzyme-linked immunosorbent assay. Antibody concentration was expressed as an OD% derived from the optical density of the sample and the median from eight wells without serum on the same plate. A non-linear mixed model assuming a constant rate of decay (half-life) was specified and fitted to the serological data. To estimate the between-pig variability of different components, between-pig random effects of each component of the model were estimated. The estimated average half-life of acquired colostral antibodies was approximately 2 weeks, but there was a considerable variation between pigs (half-life ranged from 1-3 weeks). The duration until acquired colostral antibodies were no longer detectable ranged from 2 weeks to 2 months postpartum among the pigs in the study, mainly depending on the initial level of acquired colostral antibodies to A. pleuropneumoniae serotype 2.     

Efficacy of an Actinobacillus pleuropneumoniae bacterin against serotypes 1, 3, 5 and 9.

A trial was performed in a swine research facility to ascertain the protection provided by a polyvalent Actinobacillus pleuropneumoniae (APP) bacterin containing serotypes 1, 3, 5 and 9. The test animals consisted of 60, eight-week-old, piglets, which were randomly divided into four main groups. The four main groups were further divided into three sub-groups (I, II, III) of five pigs each. Subgroup I was vaccinated intramuscularly, sub-group II was vaccinated subcutaneously, and sub-group III served as the unvaccinated control group. Each main group was challenged with a single APP serotype (1, 3, 5 or 9). Criteria for evaluation of the bacterin efficacy were mortality, lung lesions, pleural adhesions, and isolation of APP from tonsil or lung. Significant effects of vaccination over nonvaccination were reduced mortality, lung lesions, pleural adhesions, and isolations of APP from tonsil and lung. There were no significant differences between the intramuscular and subcutaneous routes of vaccination. It was concluded that the four-way APP bacterin used in this study provided satisfactory protection against homologous challenge. Evidence of protection was lower mortality and lung lesions and increased daily weight gains in vaccinates as compared with controls.     

Blood lymphocyte subsets in pigs vaccinated and challenged with Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae bacterins do not induce protection in pigs while infection with low doses of the CM5 strain of A. pleuropneumoniae given by aerosol induces complete protection. To evaluate possible correlates of protection in blood lymphocyte subset phenotypes, pigs were treated with a commercial bacterin given intramuscularly, low dose (10(5)cfu/ml) aerosol infection with CM5 or control treatments of the bacterin adjuvant or phosphate buffered saline. All pigs were challenged with a high dose (10(7)cfu/ml) of A. pleuropneumoniae. Lymphocytes and sera were collected prior to and following primary and secondary immunizations and challenge, for evaluation of B- and T-cell markers and antibody to four A. pleuropneumoniae antigens. IgM(micro)+ B-cells were increased following primary exposure to antigen in the bacterin-vaccinated group only. An increase in CD4+ cells in the LD aerosol-infected group was apparent following secondary exposure to antigen. These early changes suggest little difference in lymphocyte populations between treatment groups, however, greater differences were observed following high-dose challenge; CD4+ lymphocytes were increased significantly in both bacterin and LD-challenged groups (p<0.05) while CD8+ cells decreased in the LD-group at this time period. Consequently, there were significant differences (p<0.05) in the CD4:CD8 ratio after high-dose challenge compared to earlier time points and control groups. Variation in cellular expression of SLA-DR and DQ was observed but trends correlating to treatment group were not evident. Complete protection or lack of protection associated with LD challenge or immunisation resulted in significant differences in B-cell frequencies and CD4:CD8 ratio phenotypes in pigs, but only changes in CD4:CD8 ratios appeared relevant to protection.     

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.     

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from pigs in Korea using new standardized procedures

The in vitro susceptibilities of 76 isolates of Actinobacillus pleuropneumoniae collected from pigs with pleuropneumonia were tested with 12 commonly used antimicrobial drugs by an agar dilution minimal inhibitory concentration procedure according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Field isolates had low MICs for ceftiofur, danofloxacin and penicillin. No correlation of antimicrobial resistance was related to serotype.     

Molecular serotyping and antimicrobial resistance profiles of Actinobacillus pleuropneumoniae isolated from pigs in South Korea

Background: Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP).

Objective: Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined.

Methods: Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect β-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR.

Results: Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains).

Conclusion: Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.