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1.
Apx toxins in Pasteurellaceae species from animals   总被引：6，自引：0，他引：6  
Pasteurellaceae species particularly of porcine origin which are closely related to Actinobacillus pleuropneumoniae were analyzed for the presence of analogues to the major A. pleuropneumoniae RTX toxin genes, apxICABD, apxIICA and apxIIICABD and for their expression. Actinobacillus suis contains both apxICABD(var.suis) and apxIICA(var. suis) operons and was shown to produce ApxI and ApxII toxin. Actinobacillus rossii contained the operons apxIICA(var.rossii) and apxIIICABD(var.rossii). However, only the toxin ApxII and not ApxIII could be detected in cultures of A. rossii. The Apx toxins found in A. suis and A. rossi may play a role in virulence of these pathogens. Actinobacillus lignieresii, which was included since it is phylogenetically very closely related to A. pleuropneumoniae, was found to contain a full apxICABD(var.lign.) operon which however lacks the -35 and -10 boxes in the promoter sequences. As expected from these results, no expression of ApxI was detected in A. lignieresii grown under standard culture conditions. Actinobacillus seminis, Actinobacillus equuli, Pasteurella aerogenes, Pasteurella multocida, Haemophilus parasuis, and also Mannheimia (Pasteurella) haemolytica, which is known to secrete leukotoxin, were all shown to be devoid of any of the apx toxin genes and did not produce ApxI, ApxII or ApxIII toxin proteins. However, proteins of slightly lower molecular mass than ApxI, ApxII and ApxIII which showed limited cross-reactions with monospecific, polyclonal anti-ApxI, anti-ApxII and anti-ApxIII were detected on immunoblot analysis of A. equuli, A. seminis and P. aerogenes. The presence of Apx toxins and proteins that imunologically cross react with Apx toxins in porcine Actinobacillus species other than A. pleuropneumoniae can be expected to interfere with serodiagnosis of porcine pleuropneumonia.  相似文献   

2.
When Actinobacillus pleuropneumoniae (A. pp) is grown under iron-restricted conditions in vitro, transferrin binding proteins (Tbps) are induced. The functional transferrin receptor of A. pp is composed of two outer membrane proteins (Tbp1 and Tbp2) and shows an exquisite specificity for porcine transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron-restricted culture. It was found that Tbp2 was not expressed in iron replete medium by any serotype except serotypes 5a, 5b and 6 where a weak expression was seen. There was a weak expression of related antigens in Actinobacillus indolicus and Actinobacillus suis under iron-depleted conditions while no similar antigens were detected with the Mab in iron-starved Actinobacillus lignieresii, Actinobacillus porcinus, Actinobacillus minor, Haemophilus influenzae, and Haemophilus parasuis.Using an enzyme-linked immunosorbent assay (ELISA) based on the Mab 1.48, Tbp2 could be detected in both recombinant E. coli expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found both in the cytosol and on the outer membrane. These results indicate that the Mab 1.48-reactive epitope of Tbp2 is surface exposed when it is expressed without Tbp1 in E. coli while the inaccessibility of this epitope of Tbp2 in A. pp could be due to shading by the association between Tbp2 and Tbp1.  相似文献   

3.
The taxonomy of the members of the genus Actinobacillus associated with animals has been reviewed with focus on classification and identification including molecular based characterization, typing and identification. Out of the 22 species or species like taxa reported as Actinobacillus, 19 are associated with animals. When classified on the basis of 16S rRNA sequence based phylogenetic analysis, DNA-DNA hybridizations and phenotypic analysis, Actinobacillus sensu stricto is restricted to include A. lignieresii, A. pleuropneumoniae, A. equuli subsp. equuli, A. equuli subsp. haemolyticus (taxon 11 of Bisgaard), A. hominis, A. suis, A. ureae, A. arthritidis (taxon 9 of Bisgaard), Actinobacillus genomospecies 1 and 2 and the taxa 8 and 26 of Bisgaard. The remaining 11 species of Actinobacillus are unrelated to A. sensu stricto and should consequently be grouped with other genera or be renamed as new genera depending on new data. Identification of members of Actinobacillus at species level is possible through phenotypic characterization combined with information on host of isolation. PCR tests are available for specific detection of A. pleuropneumoniae. Only A. pleuropneumoniae is presently considered as a primary pathogen. Based on different types of RTX genes it is possible to PCR type A. pleuropneumoniae to serotype level. PCR might also be used for the specific detection of A. equuli subsp. haemolyticus. Epidemiological investigations and surveillance have so far included serotyping, multilocus enzyme electrophoresis (MLEE), ribotyping and restriction fragment length profiling.  相似文献   

4.
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   

5.
Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.  相似文献   

6.
The purpose of the study was to evaluate the clinical significance of Actinobacillus minor, Actinobacillus porcinus and Actinobacillus indolicus strains in gnotobiotic piglets. Twenty-two 6-h-old Caesarean-delivered and colostrum-deprived piglets were intranasally and orally inoculated with 2 x 10(6) colony-forming units of an A. minor (group 2; n = 9), A. indolicus (group 3; n = 5), or A. porcinus (group 4; n = 8) strain. Six other piglets were inoculated in the same way with phosphate-buffered saline solution and used as controls (group 1). All pigs were observed for clinical signs and rectal temperatures were taken until euthanasia 7 days after inoculation. At necropsy, conchae, tonsils, lungs, brains, liver, spleen and kidneys were macroscopically examined for lesions and samples were taken for bacteriology. None of the pigs developed fever. Mild ataxia was observed in one pig from group 3 for 2 days. Clinical signs were not observed in the other animals. In none of the animals were macroscopic lesions detected at necropsy. NAD-dependent Pasteurellaceae were not isolated from control animals (group 1). The A. minor, A. indolicus and A. porcinus strains were isolated from the tonsils of one, two and one pigs, respectively. Actinobacillus porcinus was isolated from the brains of the pig with central nervous symptoms and from the conchae of another pig. The inoculation strains were not demonstrated in the other samples. It was concluded that, using these inoculation routes and dose, the A. minor, A. indolicus and A. porcinus strains had low capacity to colonize the upper respiratory tract of gnotobiotic piglets and demonstrated low or no pathogenicity in such animals.  相似文献   

7.
During serological screening of a closed SPF-herd free of pleuropneumonia, more than half of the pigs were positive for complement-fixing antibodies to Haemophilus pleuropneumoniae. Actinobacillus bacteria closely related to A. suis were isolated from tonsillar tissue of 14 out of 20 slaughtered pigs submitted for pathological and bacteriological evaluation. None of the pigs had evidence of respiratory disease. Two pigs inoculated endobronchially with a selected Actinobacillus strain developed mild focal pneumonia and complement-fixing antibodies cross-reacting with H. pleuropneumoniae. Five pigs exposed and vaccinated with the Actinobacillus strain and five pigs spontaneously infected with the strain also developed complement-fixing antibodies against H. pleuropneumoniae and appeared to be less susceptible to experimental Haemophilus pleuropneumonia than pigs not exposed to the Actinobacillus infection. The agglutination test applied on serum treated with 2-mercaptoethanol detected antibodies against H. pleuropneumoniae serotype 5 but not against serotype 1 in pigs exposed to the Actinobacillus strain. Antibodies reactive with the Actinobacillus strain were also found in pigs hyperimmunized against H. pleuropneumoniae serotypes 1-5 in 2-mercaptoethanol tube agglutination test and rabbits hyperimmunized against serotypes 1,2 and 7, and strain 73567 in the immunodiffusion test. Conversely rabbits immunized against the Actinobacillus strain had antibodies against H. pleuropneumoniae serotypes 1, 3, 4, 5 and 6. It is concluded that pigs infected with Actinobacillus organisms may become false positive reactors against H. pleuropneumoniae.  相似文献   

8.
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.  相似文献   

9.
Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.  相似文献   

10.
目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。  相似文献   

11.
Thirty-seven local isolates of Actinobacillus suis-like organisms from diseased and clinically normal horses and 1 llama were compared with reference strains of A suis, A lignieresii, A equuli, A capsulatus, A hominis, A (Pasteurella) ureae, and equine A suis-like organisms (ASLO) previously described in literature. Comparison was by cultural characteristics, carbohydrate fermentation, enzyme profiles, and whole-cell protein polyacrylamide gel electrophoresis. Carbohydrate fermentation, determined by API-CH gallery, divided 36 equine ASLO isolates into 6 API-CH biotypes. The llama isolate was an additional distinct biotype. The biochemical comparisons between A suis and ASLO did not reveal remarkable and consistent differences. Enzyme analysis revealed 5 API-ZYM biotypes, one of which included the same strains as one of the API-CH biotypes and consisted in both instances of 4 esculin-negative ASLO cultures and the reference strain of A lignieresii. We conclude that the 4 strains were hemolytic variants of A lignieresii. Protein electrophoresis disclosed 15 banding patterns, 10 of which represented equine ASLO strains. The reference strains of A suis shared the pattern predominant among equine ASLO. Four of the remaining reference strains of Actinobacillus species each had a unique profile, whereas the type strain of A capsulatus and the llama isolate had similar profiles. The groupings of cultures resulting from the different testing methods had little relation to each other and to the anatomic source of the strains except the strains comprising API-CH biotype II, which originated in the equine respiratory tract, and the A lignieressi cluster.  相似文献   

12.
Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.  相似文献   

13.
Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.  相似文献   

14.
OBJECTIVE: To determine duration and rates of recovery of Actinobacillus pleuropneumoniae and Haemophilus parasuis from 4 liquid media and 2 swab specimen transport systems and compare findings with those of Escherichia coli. SAMPLE POPULATION: One strain each of A pleuropneumoniae (biovar 1, serotype 1), H parasuis (serovar 5), and E coli (serotype O149:K91:H19). PROCEDURE: Strains were incubated in brain heart infusion broth supplemented with horse serum and other nutrients or in horse serum alone, with and without nicotinamide-adenine dinucleotide in both instances, for 150 days at 4 degrees C or room temperature (21 degrees C). Similarly, strains were tested in Stuart and Amies transport systems after storage at room temperature for 8 days. RESULTS: Colony counts greater than those of the initial inoculum were observed after incubation in horse serum for A pleuropneumoniae but not for H parasuis. Overall, incubation at 4 degrees C in the 4 liquid media resulted in longer recovery duration and higher rates than at room temperature. Culture of H parasuis resulted in lower recovery rates and shorter durations of recovery than culture of A pleuropneumoniae, except for culture in horse serum. Haemophilus parasuis survived longer than A pleuropneumoniae in the transport systems, and all organisms survived longer in the Amies system. CONCLUSIONS AND CLINICAL RELEVANCE: Survival of A pleuropneumoniae and H parasuis indicated that horse serum prolongs survivability, which may result in exposure of more animals during a prolonged period. The Amies system might be a good choice for collection of clinical samples from animals, especially for recovery of H parasuis.  相似文献   

15.
Haemophilus parasuis is a colonizer of the upper respiratory tract of pigs and the etiological agent of Gl?sser's disease, which is characterized by a fibrinous polyserositis, meningitis and arthritis. Gl?sser's disease has never been reported in wild boar (Sus scrofa), although antibodies against H. parasuis have been detected. The goal of this study was to confirm the presence of this bacterium in wild boar by bacterial isolation and to compare the strains to H. parasuis from domesticated pigs. Therefore, nasal swabs from 42 hunted wild boars were processed for bacterial isolation and subsequent H. parasuis identification by specific PCR, biochemical tests and 16S rRNA gene sequencing. Two different strains of H. parasuis from two wild boars were isolated. These strains belonged to serotype 2 and were included by 16S rRNA gene sequencing and MLST analysis in a cluster with other H. parasuis strains of nasal origin from domestic pigs. During this study, Actinobacillus minor and Actinobacillus indolicus, which are NAD-dependent Pasteurellaceae closely related to H. parasuis, were also isolated. Our results indicate similarities in the respiratory microbiota of wild boars and domestic pigs, and although H. parasuis was isolated from wild boars, more studies are needed to determine if this could be a source of H. parasuis infection for domestic pigs.  相似文献   

16.
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.  相似文献   

17.
Nicotinamide adenine dinucleotide (NAD)-dependent Pasteurellaceae other than Actinobacillus pleuropneumoniae and Haemophilus parasuis are frequently isolated from the respiratory tract of pigs. The taxonomic classification and relevance for pathogenicity of these bacteria deserves further attention. In the present study, 107 of these NAD-dependent isolates from the porcine respiratory tract, primarily from lungs with pathological changes, were investigated. On the basis of phenotypic criteria, such as haemolysis, urease, catalase, and indole formation as well as other fermentative activities, 50 of the isolates were assigned to Actinobacillus minor, 36 isolates to Actinobacillus porcinus and 21 isolates to Actinobacillus indolicus. However, many isolates among the three species showed fermentative activities differing from those of the respective type strain of the species. Serotyping on the basis of heat-stable polysaccharide antigens and 16 rDNA sequencing also revealed substantial heterogeneity within each of the three species although they clustered together in three distinct groups in the phylogenetic analysis. These three groups of NAD-dependent bacteria are different from, or in a borderline position, to the existing species or genera within the family Pasteurellaceae. A considerable number of isolates of these three groups were isolated in pure cultures from pneumonic lungs. Consequently, it will be necessary to critically review the opinion, that these NAD-dependent Pasteurellaceae are only "agents colonizing the mucosa". Further, taxonomic examinations of the strains within these three groups are indispensable to testing isolates for their virulence in gnotobiotic pigs.  相似文献   

18.
Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease.  相似文献   

19.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA.  相似文献   

20.
Supernatants were obtained from 18 hr. broth cultures of Pasteurella multocida strains D82 and D62 (serotype D, toxigenic), Kobe 6 (type D, non-toxigenic), A50 and X73 (type A, non-toxigenic), Haemophilus pleuropneumoniae serotypes 1, 5 and 6, Haemophilus sp. taxon "minor group" (2 strains) and an avirulent serotype 1 strain of H. pleuropneumoniae. The supernates were filtered, pH-neutralized and tested for cytotoxicity after incubation for 18 hours in the presence of swine alveolar macrophage monolayers. Supernatants from H. pleuropneumoniae serotypes 1, 5 and 6 were cytocidal.  相似文献   

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