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.     

Serotype specificity of immunological assays for the capsular polymer of Actinobacillus pleuropneumoniae serotypes 1 and 9.

The cross-reactivity of the purified polysaccharides of Actinobacillus pleuropneumoniae serotypes 1 and 9 were examined using a variety of highly sensitive assays, such as radioimmunoassay, latex agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. In addition, conventional immunodiffusion was included for comparison. Latex agglutination, utilizing affinity-purified IgG to capsule, was also used to serotype whole cells. Agglutination or precipitation tests (radioimmunoassay, latex agglutination, and immunodiffusion) indicated no cross-reactivity between the capsules of serotypes 1 and 9, and no cross-reactivity between whole cells by latex agglutination. Assays that required binding of the capsule to a solid support (ELISA and immunoblotting) did demonstrate cross-reactions between serotypes 1 and 9 capsules, although reactions with the heterologous serotype were weaker than with the homologous serotype. The cross-reactivity could not be attributed solely to nonspecific factors because similar cross-reactivity did not occur with serotype 5 or 7 capsules by any assay. Reactivity of antisera with homologous or heterologous capsule was reduced, but not completely eliminated, by adsorption with washed, live bacteria of the heterologous serotype. Thus, the assay, as well as the antigen or specificity of the antibody reagent used, may influence the results of A. pleuropneumoniae serotyping or serological tests.     

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.     

Detection of antibodies against Actinobacillus pleuropneumoniae serotype 5 using an inhibition enzyme immunoassay.

An inhibition enzyme immunoassay (EIA) for detection of antibodies against A. pleuropneumoniae serotype 5 (App-5) in pig sera, based on the inhibition of the binding of an App-5 specific monoclonal antibody was established. The monoclonal antibody (MAb 210-F11) was found to be directed against an epitope on the O-chain of App-5 LPS. In the inhibition EIA, highly purified App-5 LPS was used to coat microtitre plates. Serial dilutions of pig sera were added to the plates prior to the addition of the MAb 210-F11. The degree of binding of App-5 antibodies from pig sera was determined as the percentage inhibition of the MAb 210-F11. Pig serum from specific pathogen free (SPF) herds, from experimentally infected animals, and from acutely and chronically infected herds were tested. A serum dilution of 1/30 was found to be optimal, when using 50% inhibition as the discriminating inhibition percentage. No cross-reactivity was observed with serum from pigs infected with other App serotypes or bacteria isolated from the respiratory tract, such as A. suis and H. parasuis. The inhibition EIA will be used for surveillance of App-5 antibodies in SPF and conventional herds.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

Serodiagnosis of pleuropneumonia using enzyme-linked immunosorbent assay with capsular polysaccharide antigens of Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7.

Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7.     

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.     

猪传染性胸膜肺炎重组亚单位疫苗诱导小鼠免疫保护效果的观察

重组表达猪传染性胸膜肺炎放线杆菌6种主要毒力因子基因:apxⅠ、apxⅡ、apxⅢ、apxⅣ、apfa和omp,以重组蛋白rApxⅠ、rApxⅡ、rApxⅢ和rOMP组合免疫小鼠作为试验I组,重组蛋白rApxⅠ、rApxⅡ、rApxⅢr、OMPr、ApxⅣ和rApfa组合免疫小鼠作为试验Ⅱ组,PBS为对照组,分3次免疫小鼠,采用背部皮下多点注射,每次间隔2周,免疫剂量为0.2 mL/只,3免后1周分别以APP1型菌Shope 4074株(5×109cfu)和APP2型菌S1536株(5×1010cfu)进行攻毒试验。通过小鼠保护率与抗体效价的相关性研究、肺部病理变化及肺脏细菌的分布情况等指标进行综合评价。结果显示,试验Ⅰ组4种重组蛋白特异性抗体水平显著高于其他两组(P<0.05),对APP1型菌攻毒的保护率(9/10)明显高于试验Ⅱ组(5/10)和对照组(0/8),小鼠的免疫保护率与抗体效价之间存在显著正相关;且该组对APP2型菌攻毒的保护作用(无肺脏损伤)也明显优于其它两组(典型肺部损伤)。间接免疫荧光试验表明试验Ⅰ组对肺脏细菌的清除效果也明显优于其他两组。本试验揭示试验Ⅰ组对不同血清型APP攻击能够提供很好的交叉保护作用,从而为猪传染性胸膜肺炎新型疫苗的研制提供参考。     

猪传染性胸膜肺炎新型亚单位菌苗对小鼠的效力研究

利用大肠杆菌表达猪胸膜肺炎放线杆菌的分泌毒素ApxⅠ、ApxⅡ、ApxⅢ，提取表达产物包涵体，再加入我国流行的猪胸膜肺炎放线杆菌7型菌，和等量弗氏佐剂乳化后制成亚单位菌苗，免疫BALB／c小鼠，间隔2周加强免疫1次。每次免疫后采血检测毒素I、Ⅱ、Ⅲ的EuSA抗体和7型菌的血凝抗体，第2次免疫2周后用5LDso的猪胸膜肺炎放线杆菌1型、7型菌株进行攻毒。结果发现第2次免疫后抗体水平显著升高，用2种血清型进行攻毒后其保护力分别为83．3％和91．7％，从死亡小鼠的体内分离到了攻毒菌株，初步的动物试验表明此种新型亚单位菌苗对同型和异型的APP菌株具有较好的保护力，为进一步研制高效的亚单位菌苗打下基础。     

Characterization of an attenuated strain of Actinobacillus pleuropneumoniae, serotype 1

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 x 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.     

Studies on the interactions of two different serotypes of Actinobacillus pleuropneumoniae

In vitro and in vivo interactions of various field strains of Actinobacillus pleuropneumoniae of serotypes 1, 2, 5 and 7 were studied. There was no influence of one serotype over the other when strains belonging to two serotypes were cultivated together in vitro. In vivo interactions showed predominance of serotype 1 over other serotypes when a strain of serotype 1 was inoculated together with a strain of serotype 2 or 5 in mice. Serotype 1 strain remained predominant irrespective of whether it was inoculated before or after the inoculation of serotype 2 strain. The mortality caused by the inoculation of two strains was higher than the mortality caused by a single strain. Early mortality was observed on inoculation of strains of serotype 2, 5 or 7 along with a strain of serotype 1. Both serotypes could be detected in the blood on cultural examination of mice infected with mixed serotypes.     

The genetic organisation of the capsule biosynthesis region of Actinobacillus pleuropneumoniae serotypes 1, 6, 7, and 12

The aim of the present study was to investigate the organisation of the genes (cps) involved in biosynthesis the capsular polysaccharide (CPS) of Actinobacillus pleuropneumoniae serotypes 6, 7, and 12 and to compare these to the corresponding genes previously described in other A. pleuropneumoniae serotypes. In serotypes 6 and 7 the sequenced DNA regions comprised five and four open reading frames, respectively, designated cps6ABCDE and cps7ABCD, whereas the sequenced DNA region in serotype 12 comprised only two open reading frames designated cps12AB. At the amino acid level, CpsA, CpsB, and CpsC of A. pleuropneumoniae serotypes 2, 6, 7, and 8 contained a high degree of homology. At the amino acid level Cps6D revealed a high degree of homology to Cps8D, whereas Cps7D contained a high degree of homology to the Cps2D. The deduced gene product of the partially sequenced cps6E gene showed no homology to any deduced gene products of any cps genes of A. pleuropneumoniae investigated so far. None of the deduced gene products of the cps genes involved in encapsulation of A. pleuropneumoniae serotypes 2, 6, 7, and 8 revealed homology to the deduced gene products of the cps genes of serotypes 1, 5A, and 12. For some genes, a local homology was found to genes probably involved in teichoic acid synthesis in other bacteria. The results obtained revealed a high degree of homology among the genes involved in CPS biosynthesis for serotypes 2, 6, 7, and 8 and a different group of homologous cps genes for serotypes 1 and 12. In some serotype 7 strains, including the serotype 7 reference strain, WF83, the cps genes were not located adjacent to the genes responsible for CPS export (cpx), probably due to genetic rearrangements.     

Minimal inhibitory concentrations of antimicrobial agents against Actinobacillus pleuropneumoniae.

Forty-five isolates of Actinobacillus pleuropneumoniae were tested for susceptibility to 12 antimicrobial agents using a microdilution method for the minimal inhibitory concentration determinations. These results confirmed the high prevalence of A. pleuropneumoniae strains resistant to antibiotics as reported earlier using the disc diffusion method (Kirby-Bauer method). While 36% of the isolates were resistant to the penicillins, 47% were resistant to chloramphenicol and 68% were resistant to tetracycline. Minimal inhibitory concentrations for the resistant isolates were approximately 32 times higher than those for the susceptible isolates to the above antibacterial agents. The isolates were in general weakly susceptible or resistant to spectinomycin, lincomycin, tiamulin and spiramycin whereas most of them were susceptible to gentamicin, trimethoprim and erythromycin. The susceptibility pattern was similar throughout the 1980 to 1984 period. The 14 serotype 5 isolates were more resistant to tetracycline but less resistant to chloramphenicol and the penicillins than the 28 serotype 1 isolates.     

胸膜肺炎放线杆菌2,3,6血清型三重PCR检测方法的建立与应用

根据Gen Bank中胸膜肺炎放线杆菌(APP)荚膜多糖基因序列,设计3对引物,成功建立了检测APP 2型、3型和6型的三重PCR检测方法。该三重PCR的最低核酸检测量分别为0.25、0.5和0.25 ng/μL,对猪肺炎支原体、猪传染性萎缩性鼻炎病毒、副猪嗜血杆菌的扩增结果均为阴性。对112份自然感染病猪样品的检测结果与单一PCR检测结果完全符合。结果表明,该多重PCR方法具有很好的特异性和敏感性,可用于临床APP 2型、3型和6型的检测。     

Factors involved in immunity against Actinobacillus pleuropneumoniae in mice.

Active and passive immunization studies in mice were undertaken to examine the protective efficiency of vaccines prepared from different components of Actinobacillus pleuropneumoniae, or combinations thereof. Subcutaneous immunization using either washed formalinized whole cells, capsular polysaccharide, lipopolysaccharide or purified hemolysin I (105 kDa protein) partially protected mice against intranasal challenge with a lethal dose of homologous or heterologous A. pleuropneumoniae serotypes. However, full protection was obtained if the formalinized whole cells were supplemented with purified hemolysin. Similar protection was obtained when mice were immunized simultaneously with a sublethal dose of live cells by the intranasal route and with formalinized whole cells subcutaneously. Passive immunization using rabbit hyperimmune serum against formalinized whole cells provided almost total protection whereas hyperimmune serum against capsular polysaccharide, lipopolysaccharide or hemolysin alone provided only a partial protection. Cell mediated immunity as detected by the foot pad test may not be implicated significantly in the protein against acute A. pleuropneumoniae infection. However, humoral immune response seems to play an important role in protection. All the antigenic components examined may contribute to the protection to some extent. However, heat-labile components such as hemolysin and outer membrane proteins may play a crucial role in protection against acute challenge infection.     

Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates and their antigenic relationships with other A pleuropneumoniae serotypes

Antigenic relationship of Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates with other serotypes was studied, using tube agglutination, with and without 2-mercaptoethanol, indirect hemagglutination with and without 2-mercaptoethanol, ring precipitation, coagglutination, and immunodiffusion tests. Serotype-8 isolates possessed serotype-specific, group-specific common antigens cross-reactive with serotypes 3 and 6 and species-specific common antigens cross-reactive with other serotypes. Absorption studies were done to study the antigenic relationship of serotype 8 with serotypes 3 and 6. Rabbit antisera against whole-cell (WC) suspensions of reference strains of serotypes 3, 6, and 8 were used for absorption studies with WC and boiled WC suspensions of homologous and heterologous serotypes. Unabsorbed and absorbed sera were tested for antibodies against WC and boiled WC antigen preparations of serotype 8, using various serotests. Absorption studies revealed that serotype-8 strains possessed 2 main types of epitopes, one of which was serotype-specific and did not have cross-reactivity with other serotypes. The second type of epitopes was group specific and was cross-reactive with serotypes 3 and 6.     

Serodiagnosis of swine pleuropneumonia due to Actinobacillus pleuropneumoniae serotypes 7 and 4 using long-chain lipopolysaccharides.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 7 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the 3 antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 7 or 4 were not statistically different. The positive ELISA reaction with anti-serotype 4 sera was unexpected with the CPS, which are supposed to be serotype-specific; LPS traces present in the CPS appeared to be responsible for this reaction. In addition, sera from animals exposed to A. pleuropneumoniae serotypes 5 or 10 presented cross-reactions with the SBE and the CPS, but not with the LC-LPS. Cross-reactions were mainly due to rough LPS, as shown by immunoblotting. The LC-LPS is easily obtainable and can be used for the detection of antibodies in animals infected with A. pleuropneumoniae serotypes 7 and 4.     

The pharmacodynamic effect of amoxycillin and danofloxacin against Actinobacillus pleuropneumoniae in an in-vitro pharmacodynamic model.

The pharmacodynamic effect of amoxycillin and danofloxacin against two strains of Actinobacillus pleuropneumoniae was evaluated in an in-vitro pharmacodynamic model. For amoxycillin peak concentrations of 0.5, 1, and 4 microg ml(-1)and half-lives of 3 and 15 hours were examined. For danofloxacin peak concentrations of 0.125, 0.5, and 1. 5 microg ml(-1)and half-lives of 1.5 and 7 hours were evaluated. The initial bactericidal effect was measured as the reduction in colony count (log CFU ml(-1)) during the first three hours, and the overall pharmacodynamic effect as the area under the bacterial growth versus time curve (AUBC).The initial bactericidal effect of amoxycillin was maximal at peak concentrations of two to four times the MIC. Peak concentration and half-life only influenced the pharmacodynamic effect of amoxycillin if the antibiotic concentration fell below the MIC during the experiments, which is consistent with time >MIC as the most important parameter of pharmacodynamic effect of beta-lactam drugs.For danofloxacin maximal bactericidal effect initially was observed at peak concentrations of at least eight times the MIC. The pharmacodynamic effect was dependent on the peak concentration. The half-life only influenced the pharmacodynamic effect of danofloxacin in experiments with a peak concentration MIC ratio of less than eight. This indicated that for danofloxacin the peak concentration was the major determinant of pharmacodynamic effect.     

Serologic studies of Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype-3 and their antigenic relationships with other A pleuropneumoniae serotypes in swine

Serotype-3 strains of Actinobacillus (Haemophilus) pleuropneumoniae possess epitopes shared with almost all other Actinobacillus serotypes. Common epitopes detected in particulate antigens were heat-labile and heat-stable and were of minor nature. Additional cross-reactive epitopes were detected in soluble and particulate antigens prepared from strains of serotypes 3, 6, and 8. Cross-reactions occurring between serotype-3 antigens and those of other serotypes were of 2 types: one associated mainly with 2-mercaptoethanol (2-ME)-sensitive IgM antibodies and the other associated mainly with 2-ME-resistant IgG antibodies. The cross-reactivity between serotypes 3, 6, and 8 was associated mainly with IgG antibodies, as shown by the results of 2-ME tube agglutination, 2-ME-indirect hemagglutination, and coagglutination tests. None of the tests was entirely satisfactory for serotyping serotype-3 isolates, mainly as a result of overlapping of type-specific antigenic determinants of serotypes 3, 6, and 8 in different combinations in the same strain. A combination of tests, using particulate and soluble antigens, may be necessary for typing of serotype-3 isolates.