Actinobacillus Pleuropneumoniae Serotype Research Articles

No overall relationship between average daily weight gain and the serological response to Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in eight chronically infected Danish swine herds

The association between the average daily weight gain (from approximately 4 to 20 weeks of age) and the serological responses to respiratory infections was examined in a longitudinal study including 825 pigs from eight chronically infected herds. Pigs were bled every 4th week (starting from approximately 4 weeks of age), and sera were analyzed for antibodies to Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae serotypes 2, 5–7 and 12. Mixed analysis of covariance analyzed the relationship between the average daily weight gain and a categorical variable defining seroconversion as none, early or late as compared to the median time (estimated across herds) of seroconversion for the particular pathogen. The variables “gender”, “weight at an approximate age of 4 weeks” and “time” (defining the exact length of the follow-up period), were included as explanatory variables, and “litter” and “herd” were included as explanatory random variables. The individual pig was the unit of concern. The variable defining time at seroconversion was not significantly associated with the average daily weight gain, when evaluating models across all eight herds. The apparent lack of effect could be because most pigs included in the study were subclinically infected, or because a temporary negative influence of the infections is hidden due to an increased growth in the period following infection. In conclusion, at least in these eight herds, seroresponses to M. hyopneumoniae and A. pleuropneumoniae could not be used to predict the effect of the pathogens on the daily weight gain.

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79–1.00)) and specificity (0.97 (0.93–0.99)).

豚胸膜肺炎野外例における免疫組織化学染色法を用いた<I>Actinobacillus pleuropneumoniae</I>血清型1の抗体検出

豚胸膜肺炎野外例における免疫組織化学染色法を用いた<I>Actinobacillus pleuropneumoniae</I>血清型1の抗体検出

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains.

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.

Membrane vesicles released by Actinobacillus pleuropneumoniae contain proteases and Apx toxins.

Actinobacillus pleuropneumoniae serotype 1 releases vesicles containing proteases and Apx toxins into the culture medium. Vesicles were concentrated by ultracentrifugation and analyzed by electron microscopy and electrophoresis; their size ranged from 20 to 200 nm. A polyclonal antiserum raised against a purified high molecular mass secreted protease of serotype 1 recognized this protease on the surface of the vesicles by immunogold electron microscopy. Higher molecular mass polypeptides from vesicle extracts were recognized by the antiserum by Western immunoblot, indicating that the protease could form oligomers. However, these oligomers were not active against gelatin until secreted. Additionally, Apx toxins were also present in vesicles, and were recognized by Western immunoblot by an anti-serotype 1 toxins polyclonal serum. A. pleuropneumoniae antigens in vesicles were recognized by convalescent-phase pig sera from animals infected with serotype 1 or 5. The release of vesicles containing virulence factors could be a tissue damage mechanism in swine pleuropneumonia.

Membrane vesicles released by Actinobacillus pleuropneumoniae contain proteases and Apx toxins

Actinobacillus pleuropneumoniae serotype 1 releases vesicles containing proteases and Apx toxins into the culture medium. Vesicles were concentrated by ultracentrifugation and analyzed by electron microscopy and electrophoresis; their size ranged from 20 to 200 nm. A polyclonal antiserum raised against a purified high molecular mass secreted protease of serotype 1 recognized this protease on the surface of the vesicles by immunogold electron microscopy. Higher molecular mass polypeptides from vesicle extracts were recognized by the antiserum by Western immunoblot, indicating that the protease could form oligomers. However, these oligomers were not active against gelatin until secreted. Additionally, Apx toxins were also present in vesicles, and were recognized by Western immunoblot by an anti-serotype 1 toxins polyclonal serum. A. pleuropneumoniae antigens in vesicles were recognized by convalescent-phase pig sera from animals infected with serotype 1 or 5. The release of vesicles containing virulence factors could be a tissue damage mechanism in swine pleuropneumonia.

A longitudinal study of serological patterns of respiratory infections in nine infected Danish swine herds

Sixteen litters of seven pigs from each of nine Danish farrow-to-finish herds were followed to investigate the serological patterns caused by natural infection with Mycoplasma hyopneumoniae, Pasteurella multocida toxin and Actinobacillus pleuropneumoniae serotypes 2, 5–7, 12. In seven of the herds, pigs were followed as two separate cohorts started 4 weeks apart, and in two herds only one cohort was followed. A total of 999 pigs were included in the study. The pigs were blood sampled at weaning and subsequently every fourth week until slaughter. All pigs were examined for antibodies against M. hyopneumoniae (enzyme-linked immunosorbent assay), P. multocida toxin (enzyme-linked immunosorbent assay) and A. pleuropneumoniae serotypes 2, 5–7, 12 (complement-fixation tests). The most-common pattern (28%) of seroconversion was that of pigs first seroconverting to A. pleuropneumoniae serotype 2, followed by seroconversion to M. hyopneumoniae. Each herd had a dominant serotype of A. pleuropneumoniae to which most pigs seroconverted. Seroconversion to the respiratory pathogens occurred mainly in the growing-to-finishing units (8–24 weeks). The risk of seroconversion to the P. multocida toxin was very low (<20%) and occurred late. None, four and seven herds tested seropositive to PRRS and to swine influenza virus subtypes H3N2 and H1N1, respectively, when testing 10 pigs per herd (selected randomly among the study pigs) at the age of 20 weeks.

Identification of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii

Monoclonal antibodies (Mabs) against Actinobacillus pleuropneumoniae serotype 7 were produced and characterized. Three Mabs directed against surface polysaccharides were selected. One of the Mabs was directed against a capsular polysaccharide epitope (CPS) of A. pleuropneumoniae serotype 7 whereas two other Mabs reacted with different epitopes of the LPS O-chain. One of the latter reacted with the reference strain of serotype 7 and the other one with serotypes 7 and 4. These three Mabs were used to test, by Dot-ELISA, 508 field strains of A. pleuropneumoniae. None of the strains belonging to other serotypes different from serotypes 4 and 7 were positive with the Mabs. Used in combination, the CPS and one of the LPS O-chain directed Mabs were shown to be suitable for serotyping since they detected 100% of serotype 7 strains. In this study, we confirm for the first time that A. pleuropneumoniae serotype 4 is present in North America. Finally, both O-chain specific Mabs also reacted with the O-chain of Actinobacillus lignieresii. The cross-reactivity between the two species was confirmed using sera from pigs experimentally infected with A. pleuropneumoniae serotype 7 and A. lignieresii, using immunoblotting and ELISA. This is the first report of a specific cross-reactivity between the LPS of these bacterial species.

Detection of antibodies against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7 using the immunohistochemical staining.

Whole cells of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) serotype 1, 2, 5 or 7 attached to fibrins were fixed in 10% neutral buffered formalin and embedded in paraffin. The sections on a slide glass were stained by the avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The serum antibodies against A.pleuropneumoniae (serotypes 1, 2, 5 and 7) were measured by the ABC method and complement fixation (CF) test. There was good correlation between the ABC and CF tests. The present results indicate that the immunohistochemical staining is as useful as the CF test for the detection and quantification of antibody in swine sera.

Genomic relationships of Actinobacillus pleuropneumoniae serotype 2 strains evaluated by ribotyping, sequence analysis of ribosomal intergenic regions, and pulsed-field gel electrophoresis.

The aim of the present study was to examine the genomic relationship among 112 Actinobacillus pleuropneumoniae serotype 2 strains obtained throughout Europe and North America. HindIII ribotyping of the strains resulted in five ribotypes of high similarity (87-98%). Sequence analysis of the ribosomal intergenic region of strains representing each ribotype and each country showed no differences. A common ribotype was further characterized by PFGE of 12 strains representing all countries. The resultant five PFGE patterns of European strains showed a similarity of more than 91%, to which the two PFGE patterns of the Canadian strains showed a similarity of 84-86%. In conclusion, strains of A. pleuropneumoniae serotype 2 seem to be clonally related.

Comparison of ELISA and 2-META assays used in serological diagnosis of infection with Actinobacillus pleuropneumoniae serotypes 2 and 4–7 in breeding pigs in Croatia

The aim of the study was to compare the results of ELISA and 2-META assays in serological diagnosis of swine pleuropneumonia in breeding pigs of different ages. A total of 874 sera were examined for the presence of antibodies against Actinobacillus (A.) pleuropneumoniae serotype 2. The sera had been taken from 437 gilts (aged ≈6 months), 334 sows (>2 years of age) and 104 boars (1–2 years). Sixty-nine sera of the gilts and sows, and 46 sera of the boars were also examined for the presence of antibodies against A. pleuropneumoniae serotype 4–7. Sera from the youngest age group (gilts) showed the highest agreement between the results obtained by the two assays, with a kappa value of 0.633 and 0.437 for detection of antibodies against A. pleuropneumoniae serotype 2 and 4–7, respectively. The lowest agreement of the two assays was recorded in the oldest age group (sows), with a kappa value of 0.128 and 0.199 for detection of antibodies against A. pleuropneumoniae serotype 2 and 4–7, respectively. Boars were older than gilts and younger than sows. In this group of animals, agreement between the results of the two assays was inferior to that found in gilts and superior to that recorded in sows.

Effects of endobronchial challenge with actinobacillus pleuropneumoniae serotype 9 of pigs vaccinated with inactivated vaccines containing the APX toxins

The efficacy of two inactivated vaccines containing the Apx toxins of Actinobacillus pleuropneunwniae (Hemopig™, Biokema, Lausanne, Switzerland and Porcilis™ App, Intervet, Boxmeer, the Netherlands) was determined. Ten pigs were vaccinated twice with Hemopig™ and eight pigs with Porcilis™ App. Ten control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 10’ colony‐forming units (CFU) of an A. pleuropneumoniae serotype 9 strain. Increased respiratory rate and/or fever were observed in all vaccinated and control pigs after challenge. One pig of the Hemopig™ group and of the Porcilis™ App group died, whereas all pigs of the control group survived the challenge. Surviving pigs were killed at 7 days after challenge. The mean percentage of affected lung tissue was 34% in the control group, 16% in the Hemopig™ group, and 17% in the Porcilis™ App group. A. pleuropneumoniae was isolated from the lungs of all 10 control animals, from 7 of the 10 animals vaccinated with Hemopig™ and from 5 of the 8 animals vaccinated with Porcilis™ App. The mean bacterial titres of the caudal lung lobes were 1.4×106 CFU/g in the control group, 1.7×103 CFU/g in the Hemopig™ group, and 4.8×103 CFU/g in the Porcilis™ App group. In both vaccinated groups the mean number of days with dyspnoea, the mean number of days with fever, the mean percentage of affected lung tissue, and the mean bacterial titre in the caudal lung lobes were significantly lower than in the control group. Significant differences between the two vaccinated groups were not observed. It was concluded that both vaccines induced partial protection.

Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae

The genome size of Actinobacillus pleuropneumoniae was determined by pulsed field gel electrophoresis of AscI and ApaI digested chromosomal DNA. The genome size of the type strain 4074 T (serotype 1) was determined to be 2404±40 kb. The chromosome sizes for the reference strains of the other serotypes range between 2.3 and 2.4 Mb. The restriction pattern profiles of AscI, ApaI and NheI digested chromosomes showed a high degree of polymorphism among the different serotype reference strains and allowed their discrimination. The analysis of the macrorestriction pattern polymorphism revealed phylogenetic relationships between the different serotype reference strains which reflect to some extent groups of serotypes known to cross-react serologically. In addition, different pulsed fields gel electrophoresis patterns also revealed heterogeneity in the chromosomal structure among different field strains of serotypes 1, 5a, and 5b, while strains of serotype 9 originating from most distant geographical places showed homogeneous ApaI patterns in pulsed field gel electrophoresis.

Relationships Between Selected Climatic Factors in Fattening Units and their Influence on the Development of Respiratory Diseases in Swine

The correlation between climatic parameters with one another in fattening units and the influence of environmental factors on lung lesions registered at slaughter were studied in 6 integrated herds with continuous production systems. In addition, the influence of environmental parameters on the spread of Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae (serotypes 2 and 3) and on the productivity was monitored in 2 specialized fattening herds with strict batch production. The outdoor temperature was positively correlated to the indoor temperature, but negatively correlated to the relative humidity and the concentration of carbon dioxide in the stables. These indoor parameters were also correlated to each other. The concentration of ammonia was not correlated to any other climatic parameter. No correlation between the climatic parameters measured and the prevalences of pneumonia and pleuritis registered at slaughter was shown. The rapidness in spread of mycoplasmosis seemed to be more dependent of the antibody status of the pigs on arrival than on the climate of the units. In contrast, a correlation between the climatic parameters and the spread of the less contagious infection (Actinobacillus) was indicated. The influence of the climatic parameters on the daily weight gain was not ensured.

Associations between behavioural indicators of `stress' in weaner pigs and respiratory lesions at slaughter

The aim of the study was to investigate associations on the individual-pig level between eight behavioural indicators of `stress' (defined in a very general sense), in seven- to eight-week-old piglets four weeks after weaning and the occurrence of atrophic rhinitis (AR) and chronic pleuritis (CP) at slaughter. The study was designed as a cohort study, including 512 pigs from two commercial farms. With one exception Actinobacillus pleuropneumoniae serotype 2 (App2) infection was not associated with any of the behaviour patterns observed. Using random-effects logistic regression, weak statistical associations, positive as well as negative, were found between the performance of several behaviour patterns and the two pathological lesions. This suggests the existence of biological associations between oral behaviour patterns in young pigs and the health status later in life. However, the results were not consistent between specific oral behaviour patterns or between the two herds indicating that the associations found are either due to chance or reflect the existence of herd-specific confounders. Therefore, results based on studies within only one herd should not be generalized to other herds without care. Behavioural observations on seven- to eight-week-old piglets on the basis of the present investigation cannot be recommended for use as early predictors of AR or CP at slaughter. Further studies are needed in order to elucidate causal relations between behaviour and health.

Interference of peptides and specific antibodies with the function of the Actinobacillus pleuropneumoniae transferrin-binding protein.

Multiple-antigenic peptides (MAPs) containing transferrin-binding domains of the Actinobacillus pleuropneumoniae serotype 7-derived transferrin-binding protein (TfbA) (K. Strutzberg, L. von Olleschik, B. Franz, C. Pyne, M. A. Schmidt, and G.-F. Gerlach, Infect. Immun. 63:3846-3850, 1995) were constructed. It was found that the MAPs inhibited transferrin binding of the recombinant TfbA protein, whereas antibodies directed against transferrin-binding domains failed to do so.

Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease.

The urease gene cluster from the virulent Actinobacillus pleuropneumoniae serotype 1 strain CM5 was cloned and sequenced. The urease activity was associated with a 6.3-kbp region which contains eight long open reading frames (ORFs). The structural genes, ureABC, are separated from the accessory genes, ureEFGD, by a 615-bp ORF of unknown function, ureX. Homologies were found with the structural and accessory urease gene products of Haemophilus influenzae and, to a lesser extent, with those of other organisms. The urease enzyme subunits had predicted molecular masses of 61.0, 11.3, and 11.0 kDa, and the size of the holoenzyme was estimated to be 337 +/- 13 kDa by gel filtration chromatography. Urease activity was maximal but unstable at 65 degrees C. In cell lysates, the A. pleuropneumoniae urease was stable over a broad pH range (5.0 to 10.6) and the optimal pH for activity was 7.7. The Km was 1.5 +/- 0.1 mM urea when it was assayed at pH 7.7. The low Km suggests that this enzyme would be active in the respiratory tract environment, where urea levels should be similar to those normally found in pig serum (2 to 7 mM).

Experimental Vaccination of Pigs with an Actinobacillus pleuropneumoniae Serotype 5b Capsular Polysaccharide-Tetanus Toxoid Conjugate

The protective efficacy of an Actinobacillus pleuropneumoniae serotype 5b capsular polysaccharide-tetanus toxoid conjugate (Ap5bCP-TT) against homologous challenge of pigs was investigated. Four pigs were non-vaccinated controls (group A), 4 pigs were injected with adjuvant without antigen (group B) and 8 pigs were vaccinated with Ap5bCP-TT and adjuvant (group C). Pigs vaccinated with Ap5bCP-TT developed antibody responses to the capsular polysaccharide from A. pleuropneumoniae serotype 5b (Ap5bCP). After challenge, all pigs in groups A and B had severe clinical signs of disease and were euthanized. In group C, 3 out of 8 pigs showed severe symptoms and were euthanized. Five pigs in group C survived throughout the study. The post challenge observation period was 72 h. All pigs were subject to necropsy and results from gross pathological findings and microbiological examination are described. Pigs vaccinated with Ap5bCP-TT had statistically significant reduced values of the mass ratio of affected to unaffected lung tissue compared to pigs in groups A and B (p = 0.01 and p = 0.007, respectively). The results showed that Ap5bCP-TT-vaccination had considerable protective efficacy against lethality and pulmonary lesions caused by experimental infection with A. pleuropneumoniae serotype 5b.

Detection of an Actinobacillus pleuropneumoniae serotype 2 lipopolysaccharide (LPS) variant

Until now 12 serotypes of Actinobacillus pleuropneumoniae have been recognized. The specificity of the serotypes reside in the carbohydrate composition of the capsular polysaccharides and lipopolysaccharides (LPS). The LPS of A. pleuropneumoniae serotype 2 is a smooth type LPS with O-chains of linear repeating pentasaccharide units with an O-acetyl group linked to a glucose unit. A monoclonal antibody (MAb 102-G02) directed against A. pleuropneumoniae serotype 2 was characterized in enzyme linked immunosorbent assay (ELISA) and in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The MAb 102-G02 was directed against an epitope on the O-chain of the LPS and was used to defined a new LPS variant of A. pleuropneumoniae serotype 2 (referred to as A. pleuropneumoniae serotype 2X). Investigation of the reactivity of the MAb 102-G02 against an A. pleuropneumoniae serotype 2X field isolate (9008) and the Danish App-2 strain 4226 in electron microscopy, confirmed the different binding patterns.

Comparison of virulence of different Actinobacillus pleuropneumoniae serotypes and biotypes using an aerosol infection model

An aerosol infection model for inoculation of pigs with Actinobacillus pleuropneumoniae is described. With this model the virulence of three A. pleuropneumoniae biotype 1 strains representing serotypes 2, 5b and 6, and one Danish biotype 2 were compared using 13-week-old pigs for inoculation. The pigs were sacrificed 24 h after aerosol exposure and lung lesions were evaluated. In pigs exposed to aerosols of suspensions containing 10 4 CFU/ml of serotypes 2, 5b and 6, a number of 5–10 lesions of haemorrhagic necrotizing pneumonia were induced. For the biotype 2 strain the dose creating similar lesions was 10 9 CFU/ml. Repeated experiments confirmed these results showing similar virulence of serotypes 2, 5b and 6 whereas the biotype 2 strain proved less virulent. The aerosol infection model allowed a comparison of the number of A. pleuropneumoniae CFU/liter air which were necessary to induce lung lesions in susceptible pigs. This indicates that the model will be well suited for virulence studies of A. pleuropneumoniae serotypes in pigs.