Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping

During the period of 2001-2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1-12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods.     

Serotype and apx genotype profiles of Actinobacillus pleuropneumoniae field isolates in Korea.

A total of 100 field isolates of Actinobacillus pleuropneumoniae isolated from lung tissues of pigs with severe pleuropneumonia were serotyped by slide agglutination and precipitation tests. Polymerase chain reactions for apxICA, apxIICA, apxIIICA, apxIBD and apxIIIBD genes were used to determine their genotype prevalence. Serotypes 2 (56 isolates), 5 (28 isolates) and 6 (11 isolates) were the most common; only two isolates belonged to serotype 7, and three were untyped. Among the 97 isolates identified by serotype, 70 had the same apx genes as their respective serotype reference strains, but 27 did not have any of the apx genes present in the corresponding serotype reference strain. Among these 27 isolates, 10 were serotype 2, 12 were serotype 5, three were serotype 6 and two were serotype 7.     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Biochemical and serological identification of strains of Haemophilus pleuropneumoniae

Eighteen field isolates of Haemophilus pleuropneumoniae were studied biochemically and serotyped using the complement fixation test (CFT), agglutination test and the immunodiffusion test. Three biochemical tests (V-dependency, CAMP-reaction and urease activity) were found to be very useful for the biochemical characterization of the H. pleuropneumoniae. Haemolysis on blood agar plates, although present, was not sufficiently pronounced in all cases to warrant absolute dependence on this characteristic. Serological typing revealed the isolates belong to Serotypes 1 and 5. The immunodiffusion test proved to be the most serotype specific, while a marked cross-reaction was observed with the CFT.     

Serotyping of Haemophilus pleuropneumoniae by rapid slide agglutination and indirect fluorescent antibody tests in swine

One hundred and forty-one isolates of Haemophilus pleuropneumoniae from Iowa and Illinois swine were characterized morphologically and biochemically and serotyped by rapid slide agglutination (RSA) and indirect fluorescent antibody (IFA) tests. Hyperimmune antisera were produced in rabbits using inactivated whole-cell suspensions of the reference strains for H pleuropneumoniae serotypes 1 to 7 and strain 202, representing the taxon "minor group." Cross testing of the reference strains and reference antisera indicated the antisera to be essentially serotype-specific, although reactivity of some antisera with heterologous strains was observed. Cultures of the 141 isolates formed adherent or smooth colonies or mixtures of these colony forms. Adherent and smooth colony types were found in all serotypes identified. Microscopic and biochemical characteristics of all isolates were typical of those previously described for H pleuropneumoniae. The overall incidence of H pleuropneumoniae serotypes was serotype 5, 55.3%; serotype 1, 34.0%; serotype 7, 7.8%; and nontypeable, 2.8%. Comparing the 2 test procedures, 87.2% of the isolates could be typed by RSA, and 66.0% could be typed by IFA. Cross-reactions between serotype 4 antisera and serotype 5 and 7 isolates were common with the IFA test. The reactions with serotype 7, but not serotype 5, were eliminated by cross adsorption of serotype 4 antisera. There was good correlation between the 2 test procedures, but RSA was judged to be more specific and sensitive than IFA.     

PCR specific for Actinobacillus pleuropneumoniae serotype 3

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.     

PCR-based identification of serotype 2 isolates of Actinobacillus pleuropneumoniae biovars I and II

A genetic typing method utilizing PCR for the identification of Actinobacillus pleuropneumoniae serotype 2 isolates has been developed based on the in vitro amplification of a 1.4 kb DNA segment of the serotype 2 capsular polysaccharide genes cps2AB. The assay was tested with all serotype reference strains and a collection of 92 different A. pleuropneumoniae strains of all 15 serotypes of both biovars I and II, originating from 18 different countries worldwide. The cps2 based PCR identified the serotype 2 reference strain and all 12 serotype 2 collection strains contained in this set. DNA was not amplified from the remaining A. pleuropneumoniae reference and collection strains, indicating the PCR assay was highly specific. Furthermore, the PCR method detected all 31 A. pleuropneumoniae serotype 2 field isolates from diseased pigs that were identified in parallel as serotype 2 by agar gel diffusion. The serotype 2 PCR assay proved to be highly specific and reliable for the identification of serotype 2 isolates of A. pleuropneumoniae.     

Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test.

A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS-PAGE and Western blotting. A total of 205 A. pleuropneumoniae, strains including all 12 serotype reference strains and 13 strains representing 8 common bacterial species associated with swine or related to A. pleuropneumoniae, were tested by mixing 25 microL of polystyrene reagent with the same volume of a dense suspension of bacterial cells grown for 18 h. All A. pleuropneumoniae strains had been previously serotyped using standard procedures. The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found. The sensitized polystyrene particles were stable for at least 6 mo.     

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.     

Serotyping of Haemophilus pleuropneumoniae in the Netherlands: with emphasis on heterogeneity within serotype 1 and (proposed) serotype 9

Four hundred and forty-three Dutch field isolates of Haemophilus pleuropneumoniae were serotyped by rapid slide agglutination (RSA) using specific antisera against serotypes 1 to 5 and against the recently proposed types 6 to 9. The predominant serotypes were 9 (49%) and 2 (32%). Serotypes 1, 3, 5, 7 and 8 were isolated in small numbers: together they accounted for 3% of the total. Five percent of the isolates were not typable either due to autoagglutination or because they were not agglutinated by any of the available antisera. The remaining 49 strains (11%) agglutinated in more than one antiserum and could therefore not be properly classified. Forty-four of these 49 strains agglutinated in both anti type 1 and anti type 9 serum. Antigenic relationships between serotype 1, serotype 9 and isolates reacting with both antisera were studied using immunodiffusion and RSA with adsorbed sera. Serotype 9 strains appeared not to be a homogenous group. Isolates agglutinating exclusively in anti type 9 serum can be divided into two groups: one closely related and another hardly related to serotype 1. Serotype 9 reference strain 13261 belongs to the latter. Type 1 + 9 strains have antigens in common with serotypes 1 and 9, but they also have their own specific antigenic material. Such strains are proposed as a new serotype 10.     

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes

The detection of the apxlV gene in lung tissues from pigs experimentally infected with the 12 major A. pleuropneumoniae serotype (1 to 12) reference strains was studied by in situ hybridization using a non-radioactive digoxigenin-labeled DNA probe. In situ hybridization produced a distinct positive signal in all pigs inoculated with the 12 A. pleuropneumoniae serotypes. Positive hybridization typically exhibited a dark-brown to black reaction product in intracellular and extracellular locations, without background staining. A strong hybridization signal was seen in degenerated alveolar leukocytes ("oat cells") adjacent to the foci of coagulative necrosis and in the alveolar spaces. The in situ hybridization methodology developed for the detection of the apxIV gene is a valuable tool for the diagnosis of porcine pleuropneumonia caused by A. pleuropneumoniae when only formalin-fixed tissues are submitted for diagnosis.     

Differentiation of twelve Actinobacillus pleuropneumoniae serotypes by outer membrane lipoprotein gene-based restriction fragment length polymorphism

Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950-base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR-based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR-based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.     

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.     

Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.     

猪胸膜肺炎放线杆菌PCR快速分型系统的建立及应用

根据外膜蛋白基因中间部分的差异,毒素基因和荚膜基因的型特异性,建立3个多重PCR的分型体系,可将App1～12型分成11个模式,除9型和11型外完全区分。此分型体系用于3株野毒株的分型,得出的结果和血清学分型一致。对人工发病猪扁桃体、肺脏、鼻拭子各12份分型结果与已知血清型一致。将该分型系统直接用于临床病料的鉴定并分型,实验从35份临床可疑病料中检测出2份阳性,均为7型,从健康猪扁桃体350份中检测出5份阳性,一份为4型,一份为12型,一份为3型,2份为7型。     

Analysis of southern Ontario Actinobacillus (Haemophilus) pleuropneumoniae isolates by restriction endonuclease fingerprinting.

Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonuclease fingerprinting (REF) analysis using the enzymes BamHI and HindIII. Restriction fragments were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Except for serotypes 1 and 9, reference strains of A. pleuropneumoniae serotypes 1 to 10 had clearly distinguishable REF profiles. Analysis of REF profiles of southern Ontario field isolates revealed limited heterogeneity amongst isolates of serotype 1 or serotype 5. The REF profiles of the serotype 7 isolates studied showed greater variation. Heterogeneity could not be correlated with the presence of plasmids nor with antibiotic resistance. Limited heterogeneity could also be detected amongst REF profiles of A. pleuropneumoniae isolates recovered from a closed herd suggesting that there is a small amount of genetic variation within clonal populations.     

我国鸭疫里氏杆菌血清型的鉴定

１９９７年１月－１９９８年３月,从北京市２０个商品鸭场自然病死的北京白鸭和河南省与上海市部分鸭场的樱桃谷鸭分离到２７６株鸭疫里氏杆菌,采用凝集试验和琼脂扩散沉淀试验,对其进行了血清型的研究。其中７０株细菌为１型,６４株为２型,其余１４２株怀１,２,型参考菌株的抗血清发生反应。     

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.     

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.     

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.