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1.
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.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
Investigations on different Ornithobacterium rhinotracheale "ORT" isolates   总被引：1，自引：0，他引：1  
The aim of the present investigation was to determine the antigenic relationship between different Ornithobacterium rhinotracheale (ORT) isolates and to serotype field isolates obtained from turkey and chickens. Different antigen extractions (heat-stable, proteinase K-stable [lipopolysaccharide], and sodium dodecyl sulfate [SDS] extractions) were prepared from each serotype (A, B, C, D, E, and G) as well as from 21 ORT field isolates and examined in agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) tests. The field isolates were cultured from turkey (16 isolates) and chicken (5 isolates) flocks showing respiratory manifestations. Monospecific reactions were obtained with heat-stable as well as proteinase K-stable antigens prepared from serotypes A, C, D, E, and G in AGP tests. On the other hand, with the same antigen preparations from a strain of serotype B in AGP tests, cross-reactions with antisera prepared against serotypes A and E could be detected. The cross-reactions were observed mostly between 48 and 72 hr. In applications of SDS-antigen preparations in AGP tests, cross-reactions between all serotypes except serotype C were detected between 24 and 72 hr. Testing all antigen preparation in ELISA, different cross-reactions were observed and the evaluation of the results is very difficult. Serotyping of the field isolates in AGP tests by using heat-extracted antigens showed after 24 hr that 10 out of 16 isolates from turkey belonged to serotype B, five to serotype A, and one to serotype E. Results obtained after 48-72 hr revealed cross-reactions between serotype B and E in 11 cases and between A and B in two cases. All five isolates obtained from chicken reacted after 24 hr only with serum against serotype A. After 48-72 hr, two isolates showed cross-reaction with antiserum against serotype B. Similar results were obtained with proteinase K-stable antigen.  相似文献   

10.
The aim of the study was to investigate the prevalence of Actinobacillus pleuropneumoniae infections in market weight pigs in Thailand. ELISA systems employing purified lipopolysaccharide antigens were used to detect antibodies in 549 serum samples collected from pigs of 22 herds. Relevant cut-off values were established from three herds defined seronegative. Serum antibodies were detected to all serotypes except serotype 10. Almost 60% of the samples were seropositive to at least one serotype and 45% of the pigs were seropositive to more than one serotype. Antibodies to the cross-reacting serotypes 1, 9 or 11 were found in 29% of the pigs. Other common serotypes included the cross-reacting serotypes 3, 6 or 8 (26% seropositive pigs) and serotype 5a (also 26%). Antibodies to serotypes 2, 5b and 12 were low in prevalence (<10%). Three herds were regarded to be seronegative and six to have a low pathogen load with respect to the prevalence of seropositive pigs. The remaining 13 herds had a high incidence of pigs with antibodies to A. pleuropneumoniae, dominated by serotypes 1-9-11 and 5a (n = 6), serotypes 3-6-8, and 5a (n = 4) or 1-9-11, 3-6-8, 5a and 4-7 (n = 3). A low pathogen load with respect to A. pleuropneumoniae, as well as small herd size and age-segregated rearing, tended to improve the performance of growers.  相似文献   

11.
PCR specific for Actinobacillus pleuropneumoniae serotype 3   总被引：1，自引：0，他引：1  
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.  相似文献   

12.
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.  相似文献   

13.
A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expceted hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.  相似文献   

14.
A total of 77 field isolates and 15 reference strains of the porcine respiratory pathogen Actinobacillus pleuropneumoniae were tested for their ability to form biofilms in a polystyrene microtiter plate assay. More than half of all field isolates, which included strains representing serotypes 1, 5 and 7, but only two reference strains (serotypes 5B and 11) exhibited biofilm formation. Strains that formed biofilms in microtiter plates also formed thick biofilms at the air-liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth. Our findings indicate that biofilm formation is a prevalent phenotype among A. pleuropneumoniae field isolates, and that this phenotype may have been previously overlooked because of its tendency to be lost upon subculturing in broth. Biofilm formation may have relevance to the colonization, pathogenesis and transmission of this bacterium.  相似文献   

15.
The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.  相似文献   

16.
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.  相似文献   

17.
Serological tests such as agglutination, coagglutination, precipitation and indirect haemagglutination were used to study the antigenic relationship of reference and field strains of Actinobacillus (Haemophilus) pleuropneumoniae of serotype 6 with reference strains of other serotypes. Both cell-associated particulate and cell-free soluble antigens prepared from unheated and heat-treated bacterial suspensions of reference and field strains of serotype 6 were used in the studies. Species-specific, common antigenic determinants associated mainly with heat-treated particulate antigens of serotype 6 were cross-reactive in tube agglutination tests with almost all the serotypes. The species-specific antigens were of a minor nature because the cross-reactivities were abolished in both 2-mercaptoethanol agglutination and coagglutination tests. Cell-free saline extracts of both unheated and heat-treated suspensions of serotype 6 strains possessed epitopes specific for serotypes 3, 5 and 8 in addition to their own specific determinants. The epitopes were dominant because the reactions of strains of serotype 6 with antisera against serotypes 3, 5 and 8 persisted in almost all the serological tests used. Serotype 6 strains were antigenically closer to serotype 8 than to serotypes 3 or 5. A combination of serological tests such as coagglutination followed by 2-mercaptoethanol tube agglutination and, or, immunodiffusion tests differentiated serotype 6 strains from those of other cross-reacting serotypes.  相似文献   

18.
Immunological properties of Actinobacillus pleuropneumoniae hemolysin I   总被引：8，自引：0，他引：8  
The 105 kDa hemolysin I protein from Actinobacillus pleuropneumoniae serotype I type strain 4074 (HlyI) was shown by immunoblot analysis to be the predominant immunogenic protein if convalescent field sera or sera from pigs experimentally infected with A. pleuropneumoniae serotype 1 were used. SDS gel- and immunoblot-analysis using total culture, washed cells or culture supernatant showed that HlyI is essentially secreted and is not found attached to the bacteria. Proteins in the 105 kDa range that react strongly with anti-HlyI antibody, are produced by all serotypes and are presumed to be their hemolysins. Sera from pigs experimentally infected with each of the 12 serotypes strongly reacted with HlyI. In addition, some sera from pigs that were confirmed to be negative for A. pleuropneumoniae, also reacted with HlyI as well as with related proteins from Actinobacillus rossii and Actinobacillus suis. These two species produce proteins in the 105 kDa range which cross-react strongly with HlyI. They could be the source of the immunological reactions of the A. pleuropneumoniae-negative sera with HlyI. However, no cross-reactions could be found between HlyI and the Pasteurella haemolytica leukotoxin, the Escherichia coli alpha-hemolysin or related proteins from various hemolytic E. coli strains isolated from pigs. The immunological cross-reactions of HlyI with related proteins from A. rossii, A. suis and possibly from other bacterial species may create uncertainty in interpretation if HlyI is used as the antigen in serodiagnosis of A. pleuropneumoniae.  相似文献   

19.
The antigenic differences between strains of serotype 2 of both biotypes I and II of Actinobacillus pleuropneumoniae were studied by using several serological techniques. Monoclonal antibodies (MAbs) against A. pleuropneumoniae biotype I serotype 2 were produced by fusion of spleen cells of BALb/c mice immunized with whole-cell (WC) suspension with SP2/O-Ag14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) using WC as antigen. MAbs MK-7 and MK-10 identified multiple bands of lipopolysaccharide in Western-blot. Treatment of WC with proteinase K and sodium periodate indicated that both MAb binding sites were carbohydrates in nature. In both ELISA and Western-blot, MAbs MK-7 and MK-10 recognized only biotype I serotype 2 isolates. Neither MAb MK-7 nor MK-10 reacted with reference strains of remaining serotypes of A. pleuropneumoniae and other Gram-negative bacteria tested. The results obtained with various serological tests showed that strains of serotype 2 biotype I shared antigenic determinants with strain N-282 of serotype 2 biotype II, but not with strain N-273 of serotype 1 biotype II. It is suggested that data obtained from this study may be helpful in the development of specific serotyping and serodiagnostic reagents of A. pleuropneumoniae strains.  相似文献   

20.
Variations in virulence among strains of different serotypes of Actinobacillus pleuropneumoniae were detected on intraperitoneal or intranasal inoculation in mice. In general, strains of serotypes 1, 5, 9, 10 and 11 were found to be highly virulent and those of serotypes 2, 3, 4, 6, 7, 8 and 12 to be less virulent. However, a few strains of serotype 5 caused low mortality in mice while some strains of serotype 3 and 7 were found to be highly virulent. Highly virulent strains of A. pleuropneumoniae were invasive and appeared in the blood within 3 to 6 h of intranasal inoculation. The type specific antigen as detected by the coagglutination test was distributed in lungs, liver, heart and spleen after intraperitoneal inoculation whereas it was mostly concentrated in the lungs after intranasal inoculation. Lowest concentration of boiled whole-cell suspension of A. pleuropneumoniae showing limulus amebocyte lysate activity was variable and independent of the serotype. Mortality caused by boiled whole cell suspension was also variable and serotype independent.  相似文献   

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