Effect of endobronchial challenge with Actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with a vaccine containing Apx toxins and transferrin-binding proteins

The efficacy of a subunit vaccine containing the Apx toxins of Actinobacillus pleuropneumoniae and transferrin-binding proteins was determined. Ten pigs were vaccinated twice with the vaccine. Eight control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 10(6.5) colony-forming units (CFU) of an A. pleuropneumoniae serotype 9 strain. In the vaccine group, none of the pigs died after inoculation. Only one pig of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The mean percentage of affected lung tissue was 64% in the control group and 17% in the vaccine group. Actinobacillus pleuropneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 5.0 x 10(8) CFU/g in the control group and 3.0 x 10(6) CFU/g in the vaccine group. It was concluded that the vaccine induced partial protection against severe challenge.     

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.     

Effect of oral enrofloxacin and florfenicol on pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 1

This study was carried out to compare the efficacy of two oral anti-microbials as metaphylactic medication to pigs inoculated with Actinobacillus pleuropneumoniae serotype 1. Forty-two pigs with an average weight of 22.64 kg were randomly assigned to three treatment groups: group F was given doses of 40 ppm of florfenicol, group E received 150 ppm of enrofloxacin and group C received no medication. Groups F and E received medicated feed 12 h before being inoculated and for 7 days after inoculation. All the pigs were inoculated by aerosol, with 2 x 10(7) CFU/ml of A. pleuropneumoniae serotype 1 each. The average body temperature was higher in group C than in groups E and F, between 12 and 96 h after inoculation (P < 0.05). No differences were found between groups F and E in respiration pattern, nasal secretion and general condition (P > 0.05): however, differences were found in group C for respiration pattern and general condition (P < 0.05), 12 h after inoculation. There was no mortality in groups F and E, whereas a 50% mortality was recorded in group C during the first 48 h after inoculation (P < 0.05). Necropsies and bacterial cultures were performed 12 days after inoculation. Lesions were observed in five pigs of group F (35.71%) with an average damage of 1.16%; in four pigs of group E (28.57%) with 1.24%; and in 13 animals in group C (92.85%) with 34.5% of affected lung tissue (P < 0.05). The infective agent was cultured from various organs of animals in groups F and C, but not from those in group E.     

Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity. Our search for new vaccine candidates identified antigens that are expressed during conditions that mimic infection; the outer membrane (OM) proteome of A. pleuropneumoniae serotype 5b was examined under iron restriction. Quantitative profiling by 2D-DiGE technology revealed that iron restriction induced expression of previously described transferrin binding proteins (TbpA, TbpB) plus four lipoproteins including spermidine/putrescine binding periplasmic protein 1 precursor (PotD2). Immunoproteomic analyses with antisera from na?ve animals and from infected pigs were able to differentiate antigens within the OM proteome that were specifically recognized during A. pleuropneumoniae infection. Immunoblots of iron-restricted profiles detected PotD2, heme-binding protein A (HbpA), and capsule polysaccharide export protein (CpxD) as well as surface antigens TbpA, TbpB, and OmlA. These data identify OM proteins that demonstrate immunogenicity and upregulation under conditions mimicking infection, providing emphasis on lipoproteins as an important class of antigens to exploit for vaccine development for A. pleuropneumoniae.     

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida

Objective To compare the sensitivity and specificity of six serological enzyme‐linked immunosorbent assays (ELISAs) based on serovar‐independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross‐reactivity in disease‐free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. Design Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. Procedure A 39‐kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N‐terminus (ApxIVA‐N, ApxIVA‐NP, ApxIVA‐NPS) or C‐terminus (ApxIVA‐C, ApxIVA‐CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. Results The 39‐kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross‐reactivity following P. multocida challenge. ELISAs based on ApxIVA N‐terminus antigens were significantly more sensitive than C‐terminus antigens for the detection of App‐induced disease. Although ApxIVA‐N and ApxIVA‐NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App‐induced disease (90–93%). Affinity purified ApxIVA‐NP antigen had marginally better specificity than ApxIVA‐N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross‐reactivity. In disease‐free pigs, the specificity of the ApxIVA‐NPS ELISA may be adversely affected by nasal carriage of apparently low‐virulence App strains. Conclusions ApxIVA‐N‐based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low‐virulence App. The 39‐kDa antigen is only of merit in exclusion of App disease by negative serology.     

Effect of tulathromycin on the carrier status of Actinobacillus pleuropneumoniae serotype 2 in the tonsils of pigs

The effect of a single or double dose of tulathromycin was evaluated in pigs carrying Actinobacillus pleuropneumoniae serotype 2 in their tonsils. Twenty-nine pigs from a reinfected specific pathogen-free-herd were selected from animals testing positive in an A pleuropneumoniae serotype 2-specific pcr test on tonsil scrapings and they were divided into three groups. The pigs in group 1 were treated subcutaneously with 2.5 mg/kg tulathromycin on day 0, the pigs in group 2 were treated with 2.5 mg/kg tulathromycin on days 0 and 4, and the pigs in group 3 were left untreated as controls. The pigs were tested by pcr on tonsil scrapings on days 0, 4, 11 and 33, and on day 33 all the animals were euthanased. There were no significant differences between the numbers of PCR-positive animals in the three groups on any of the sampling dates.     

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.     

Experimental infection of SPF pigs with Actinobacillus pleuropneumoniae serotype 9 alone or in association with Mycoplasma hyopneumoniae

The purpose of this study was to compare in SPF pigs, the pathogenicity of an Actinobacillus pleuropneumoniae serotype 9 strain 21 (isolated from the palatine tonsils of a healthy gilt on a French nucleus pig farm, with no clinical signs or lung lesions but a highly positive reaction to A. pleuropneumoniae serotype 9 antibodies) with a pathogenic A. pleuropneumoniae strain 4915 serotype 9 (isolated in France from an outbreak of porcine pleuropneumonia). The pathogenicity of one Mycoplasma hyopneumoniae strain alone or associated with A. pleuropneumoniae strain 21 was also compared. Eight groups of 7 pigs were infected (at 6 or 10 weeks of age) and a control group was kept non-infected. Results showed that sensitivity to A. pleuropneumoniae was related to the age of the pig (6 weeks vs 10 weeks) whatever the strain. Surviving pigs infected at 6 weeks of age developed severe clinical signs, lung lesions typical of A. pleuropneumoniae and they seroconverted. In contrast, symptoms and lung lesions were almost non-existent in pigs infected with strain 21 at 10 weeks of age, but a seroconversion was observed with very high ELISA titres. These results were in accordance with those observed in the nucleus pig farm. Infection with M. hyopneumoniae alone induced typical mycoplasmal symptoms, pneumonia and seroconversion. Symptoms and lung lesions were the most noticeable in pigs infected with M. hyopneumoniae at 6 weeks of age and with A. pleuropneumoniae 4 weeks later. Our results show that the presence of A. pleuropneumoniae serotype 9 in a pig herd may be clinically unnoticed and that M. hyopneumoniae may potentiate A. pleuropneumoniae infection.     

血清3型猪传染性胸膜肺炎放线杆菌的分离与鉴定

2005年3月,广东省惠州市某猪场发生了一起疑似猪传染性胸膜肺炎放线杆菌引起猪只死亡的病例,经细菌分离培养、生化鉴定、PCR、血清学鉴定,确诊该病原为血清3型猪传染性胸膜肺炎放线杆菌。     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.     

A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions

The study describes a polymerase chain reaction (PCR) assay for the detection of Actinobacillus pleuropneumoniae. The test is based on the amplification of the omlA gene coding for an outer membrane protein of A. pleuropneumoniae. To test the specificity of the reaction, 19 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR assay. The detection threshold of the test was 10(2) A. pleuropneumoniae CFU/assay. The test was then applied to the detection of A. pleuropneumoniae from tonsillar biopsies and tracheobronchial lavage fluids of pigs without a culture step. The detection of A. pleuropneumoniae in these samples was performed by PCR, by conventional culture and by bacteriology with immunomagnetic beads. The number of samples that were found positive by PCR was almost three times higher than the number of samples from which A. pleuropneumoniae was isolated by both bacteriological techniques. The detection of A. pleuropneumoniae in these samples allowed us to demonstrate its aerosol transmission to pigs under experimental conditions. The trial involved 18 specific pathogen free pigs. Six pigs, infected with A. pleuropneumoniae, were located in a unit A, together with four non-infected animals (contact pigs). Eight non-infected pigs (reporter pigs) were located in a unit B, adjacent to A. We detected A. pleuropneumoniae in samples from infected animals but also from 'contact' (unit A) and 'reporter' (unit B) pigs. The results of this study show that the simple preparation of the samples followed by the PCR assay may be a useful tool for epidemiological studies.     

Antibody production and tissue irritation in swine vaccinated with Actinobacillus bacterins containing various adjuvants

The effects that Actinobacillus pleuropneumoniae bacterins containing paraffin (mineral) oil, Al(OH)3, or lecithin-base oil adjuvants had on antibody production and muscle irritation were studied. Four hundred and thirty-two pigs were vaccinated twice (864 injection sites) at a 30-day interval. To assess effects of skin surface contamination on tissue reaction, 247 of the injections were made through skin soiled with manure. Serum samples for antibody determination and tissue samples were obtained at slaughter (15 to 90 days after vaccination). Bacterins containing Al(OH)3 or 5% lecithin-base oil as an adjuvant resulted in minimal tissue irritation. Bacterins containing paraffin oil or greater than 20% lecithin-base oil were more irritating to muscle, often resulting in a granuloma or an abscess at the injection site. Pigs given bacterins containing Al(OH)3 adjuvant had lower antibody titers than did pigs given bacterins containing paraffin oil or lecithin-base oil adjuvants.     

Identification of the heat-labile hemolysin of Actinobacillus pleuropneumoniae serotype 1.

The heat-labile hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was partially purified using ammonium sulfate precipitation and gel permeation chromatography. This partially purified material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nylon filters. The filters were treated with convalescent pig serum and subsequently with CM-5 culture supernatant containing active hemolysin. A 104 kd peptide was identified as the hemolysin because it bound antibodies in convalescent pig serum which cross-linked active hemolysin. The same 104 kd protein when injected into a rabbit produced neutralizing antibodies to the CM-5 hemolysin in culture supernatant.     

Early in vivo interactions of Actinobacillus pleuropneumoniae with tonsils of pigs.

Twenty gnotobiotic piglets were inoculated with 5 x 10(8) colony forming units of an Actinobacillus pleuropneumoniae biotype 1-serotype 9 strain onto their tonsils. Five other piglets (controls) were inoculated with phosphate-buffered saline solution. Pigs were euthanized at 30 min, 90 min, 180 min, 6 h, 9 h, 12 h or 24 h after inoculation. At necropsy, samples were taken from the tonsils for bacteriological, histological, immuno-histochemical and electron microscopical examination. A. pleuropneumoniae was isolated from tonsils of all the infected pigs, but not from tonsils of the control pigs. Early after inoculation bacteria were mainly associated with the stratified squamous epithelium and detached epithelial cells. Vacuolization and desquamation of the epithelium was observed and many transmigrating neutrophils were present. At later times after inoculation, bacteria were found closely associated with the crypt-walls and with detached cells present in the crypts. A strong neutrophil migration was observed mainly in the deeper parts of the crypts. It is concluded that attachment of A. pleuropneumoniae to tonsillar epithelial cells probably constitutes a first step in establishing bacteria at this body site.     

Serological characterization of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain.In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses.In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Protective efficacy of conjugate vaccines against experimental challenge with porcine Actinobacillus pleuropneumoniae.

In an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP) by neutralizing the effects of three virulence factors of A. pleuropneumoniae--the capsular polysaccharide (CP), the lipopolysaccharide (LPS), and the hemolysin protein (HP)--two subunit conjugate vaccines were prepared by covalently coupling the CP to the HP and the LPS to the HP. The CP, LPS, and HP were isolated from A. pleuropneumoniae, strain 4074, serotype 1, and the protective efficacy of the conjugate vaccines in swine experimentally infected with A. pleuropneumoniae was evaluated. Following a booster vaccination, a significant (P < 0.05) IgG antibody response to the CP, LPS, and HP was detected in the vaccinated pigs. The pigs vaccinated with the CP-HP and LPS-HP conjugates exhibited significantly less mortality (P < 0.05) and significantly greater weight gain (P < 0.001) than unvaccinated pigs. Vaccinated pigs exhibited significantly fewer and less extensive gross pulmonary lesions (P < 0.001) when compared with unvaccinated pigs. Thus, on the basis of mortality, weight gains, and pulmonary lesion formation, the two conjugate vaccines used in conjunction with one another provide noticeable protective efficacy against SPAP.     

Production and characterization of monoclonal antibodies to Actinobacillus pleuropneumoniae serotype 5b.

Monoclonal antibodies specific for capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of A. pleuropneumoniae serotype 5b were generated by hybridoma cells and selected by indirect ELISA of culture supernatants with purified and structurally defined LPS and CPS preparations and their synthetic conjugates. It was shown in this study that at least one monoclonal antibody, 3B4, presented 100% specificity and recognized all A. pleuropneumoniae serotype 5 field strains tested in a dot-ELISA assay.     

Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions: emphasis on tonsillar colonisation and passively acquired colostral antibodies

The objectives of this study were to elucidate at which age tonsillar colonisation by Actinobacillus pleuropneumoniae occurs in pigs and relate this occurrence to the presence of colostral antibodies to A. pleuropneumoniae. The infection patterns were studied in an isolated cohort of pigs, which consisted of the offspring from five sows originating from a conventional pig herd. The sows were transferred to isolated research facilities before farrowing. A. pleuropneumoniae was detected on the tonsils of all sows. After a nursing period of 3 weeks, the pigs were weaned and reared isolated from other pigs until slaughter. The pigs were examined repeatedly for the presence of A. pleuropneumoniae on the tonsils and for antibodies to A. pleuropneumoniae using bacteriological and serological techniques, respectively.A. pleuropneumoniae was detected in the tonsils of one pig as early as 11 days after birth, showing that A. pleuropneumoniae can be transmitted from sow to offspring during a 3-week nursing period. The cumulative proportion of pigs carrying A. pleuropneumoniae in their tonsils increased significantly between the age of 4-12 weeks. This age period corresponded to the age at which the proportion of pigs with detectable levels of colostral antibodies to the different serotypes of A. pleuropneumoniae was declining. Since these two events take place in the same age period, we expect a possible biological association between the level of the passive immunity and the degree of tonsillar colonisation. The median duration of tonsillar colonisation was estimated to approximately 7-8 weeks.