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1.
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   

2.
Objective To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15.
Design The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs.
Results With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15).
Conclusions Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.  相似文献   

3.

Background

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease that is caused by Actinobacillus pleuropneumoniae (APP) and characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia, which is a severe threat to the swine industry. In addition to APP RTX-toxins I (ApxI), APP RTX-toxin II (ApxII), APP RTX-toxin III (ApxIII) and Outer membrane protein (OMP), there may be other useful antigens that can contribute to protection. In the development of an efficacious vaccine against APP, the immunogenicities of multicomponent recombinant subunit vaccines were evaluated.

Methods

Six major virulent factor genes of APP, i.e., apxI, apxII, apxIII, APP RTX-toxins IV (apxIV), omp and type 4 fimbrial structural (apfa) were expressed. BALB/c mice were immunized with recombinant ApxI ( rApxI), recombinant ApxII (rApxII), recombinant ApxIII (rApxIII) and recombinant OMP (rOMP) (Group I); rApxI, rApxII, rApxIII, recombinant ApxIV (rApxIV), recombinant Apfa (rApfa) and rOMP (Group II); APP serotype 1 (APP1) inactivated vaccine (Group III); or phosphate-buffered saline (PBS) (Control group), respectively. After the first immunization, mice were subjected to two booster immunizations at 2-week intervals, followed by challenge with APP1 Shope 4074 and APP2 S1536.

Results

The efficacy of the multicomponent recombinant subunit vaccines was evaluated on the basis of antibody titers, survival rates, lung lesions and indirect immunofluorescence (IIF) detection of APP. The antibody level of Group I was significantly higher than those of the other three groups (P < 0.05). The survival rate of Group I was higher than that of Groups II and III (P < 0.05) and the control (P < 0.01). Compared with the other three groups, the lungs of Group I did not exhibit obvious hemorrhage or necrosis, and only showed weak and scattered fluorescent dots by IIF detection.

Conclusion

The result indicates that the multicomponent recombinant subunit vaccine composed of rApxI, rApxII, rApxIII and rOMP can provide effective cross-protection against homologous and heterologous APP challenge.  相似文献   

4.
New serological tests have recently been introduced for Actinobacillus pleuropneumoniae diagnosis. No information is currently available on how these tests compare regarding the detection of antibodies from subclinically infected pigs. To answer this question, 80 pigs were randomly assigned to experimental groups infected with A. pleuropneumoniae serovars 1, 3, 5, 7, 10, 12, 15 and a non-inoculated control group. Blood samples and oropharyngeal swabs were collected prior to infection and for 7 consecutive weeks thereafter. Serum samples were tested using the Swinecheck(?) APP ELISA and the Multi-APP ELISA (University of Montreal). All pigs were euthanized at 49 days post-inoculation. Tonsil and lung samples were cultured for isolation and tested by PCR. The Multi-APP ELISA detected seroconversion 1 week earlier than the Swinecheck(?) APP ELISA with the earliest seroconversion detected at 1 week post-infection (serovar 10) and the latest at 3 weeks post-infection (serovar 1). Seroconversion at day 49 was serovar-dependent and varied from 4 (44%) positives detected in the serovar 10 group to 9 positives (100%) detected in the serovar 15 group. Thirty-one pigs were serologically positive for A. pleuropneumoniae at 49 days post-infection and only 15 still carried A. pleuropneumoniae on their tonsils based on PCR results. No cross-reactions were observed when serum samples were cross-tested using the Swinecheck(?) APP ELISA. A. pleuropneumoniae was successfully isolated from the lung of 2 pigs that developed pleuropneumonia, but was not isolated from tonsils due to heavy contamination by the resident flora. This study offers a comprehensive evaluation of the diagnostic tools currently available for detection of A. pleuropneumoniae subclinical infection.  相似文献   

5.
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   

6.
Most serotypes of A. pleuropneumoniae produce more than one toxin in vivo. To determine the value of the production of more than one toxin in the development of disease, we tested the pathogenicity of isogenic strains of A. pleuropneumoniae serotype 1 that are mutated in the toxin genes apxIA and/or apxIIA or in the transport genes apxIBD. Bacteria mutated in both apxIA and apxIIA, or in apxIBD, were unable to induce pathological lesions, thereby confirming the conclusion that ApxI and ApxII are essential for the pathogenesis of pleuropneumonia. Infection with isogenic strains lacking either ApxI or ApxII did not consistently lead to pleuropneumonia unlike the parent strain S4074. ApxII seemed at least as important as ApxI for the development of clinical and pathological symptoms. Only one of the four pigs inoculated with a mutant strain unable to produce ApxII developed mild pneumonia whereas two out of the three pigs inoculated with a mutant strain unable to produce ApxI developed more severe lesions. The results indicate that both ApxI and ApxII of A. pleuropneumoniae serotype 1 are necessary for full virulence.  相似文献   

7.
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.  相似文献   

8.
Xu F  Chen X  Shi A  Yang B  Wang J  Li Y  Guo X  Blackall PJ  Yang H 《Veterinary microbiology》2006,118(3-4):230-239
Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia, a highly contagious and often fatal disease. A candidate live vaccine strain, potentially capable of cross-serovar protection, was constructed by deleting the section of the apxIA gene coding for the C-terminal segment of ApxI toxin of the A. pleuropneumoniae serovar 10 reference strain (D13039) and inserting a chloramphenicol resistance gene cassette. The mutant strain (termed D13039A(-)Chl(r)) produced an approximately 48kDa protein corresponding to the N-terminus of the ApxI toxin, and exhibited no haemolytic activity and lower virulence in mice compared with the parental strain. The mutant was evaluated in a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant at 14 days intervals and then challenged 14 days after the last vaccination with either A. pleuropneumoniae serovar 1 (4074) or serovar 2 (S1536) or serovar 10 (D13039) reference strains. The haemolysin neutralisation titres of the pre-challenge sera were significantly higher in the vaccinated pigs than in the unvaccinated pigs. The mortalities, clinical signs and lung lesion scores in the vaccinated pigs were significantly lower than those in the unvaccinated pigs for the serovar 1 challenge. A significantly lower lung lesion score was also observed in the vaccinated pigs, compared with unvaccinated pigs, for serovar 2 challenge. Our work suggests that the mutant strain offers potential as a live attenuated pleuropneumonia vaccine that can provide cross-serovar protection.  相似文献   

9.
为鉴定胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)ApxIA毒素蛋白N端疏水区和C端Ca~(2 )结合区的免疫原性,参照apxIA基因序列(D16582)设计4条引物,用于扩增APP血清10型参考菌株(D13039)基因组DNA中长约3.2kb的apxIA基因及其N端(1.4kb)和C端(1.8kb)基因片段,经克隆测序后分别插入原核表达载体DET-32a中进行表达,表达的融合蛋白大小分别约为125Ku、65Ku和80Ku。表达产物免疫小鼠后的攻毒保护试验结果显示,ApxIA表达蛋白对APP血清10型(D13039)攻毒可提供完全保护,而对APP血清1型(4074)攻毒仅能提供部分保护,与提纯的ApxⅠ毒素蛋白免疫保护效果相当;ApxIA-N端表达蛋白免疫保护活性显著高于ApxIA-C端表达蛋白,提示ApxIA蛋白免疫活性位点多位于N端,在ApxⅠ毒素蛋白的保护性抗原活性中发挥更重要作用  相似文献   

10.
In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either ~4.0 × 10(5) colony-forming units (CFU) or ~1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.  相似文献   

11.
Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.   总被引：29，自引：0，他引：29  
We report on the re-examination of nine Australian isolates of Actinobacillus pleuropneumoniae that have been previously assigned to serovar 12. In the ring precipitation test, none of the nine isolates reacted with antisera to serovars 1-14 of A. pleuropneumoniae. Antiserum prepared against one of the Australian isolates gave no reaction with any of the 14 recognised serovar reference strains, except serovar 7. This reaction of the HS143 antiserum with serovar 7 antigen could be removed by adsorption with serovar 7 antigen. The adsorbed antiserum remained reactive with HS143 and the other eight Australian isolates. The nine Australian isolates were all shown to express ApxII and ApxIII, found in serovars 2, 4, 6 and 8, as well as the 42kDa outer membrane protein found in all serovars of A. pleuropneumoniae. The nine Australian isolates were found to possess the following toxin associated genes apxIBD, apxIICA, apxIIICA, apxIIIBD and apxIVA. The toxin gene profile of the Australian isolates is typical of A. pleuropneumoniae serovars 2, 4, 6 and 8. On the basis of the serological characterisation results and the toxin gene profiles, we propose that these isolates represent a new serovar of A. pleuropneumoniae--serovar 15--with HS143 being the reference strain for the new serovar.  相似文献   

12.
Capsular extracts of Haemophilus pleuropneumoniae, Serotype 1, were mixed with AL(OH)3 gel (3 parts extract + 1 part Al(OH)3) and used as vaccines in pigs and mice. Four preparations were tested in Experiment I: NaCl and Cetavlon (hexadecyltrimethyl ammonium bromide) extracts of both low in vitro passage (LP) and high in vitro passage (HP) culture, respectively. Four pigs vaccinated with the NaCl extract of the LP strain survived, whereas one of four from each of the remaining vaccine groups and five of six from the control group died. All vaccines induced complement-fixing antibodies. No apparent boosting of titres occurred as a result of challenge with live bacteria. Mice were vaccinated in Experiment II with NaCl and Cetavlon extracts of the LP strain. Both were protective, although the Cetavlon vaccine appeared more efficacious than the NaCl extract. The use of Al(OH)3 adjuvant improved the efficacy of the NaCl vaccine in mice. In Experiment III six gnotobiotic pigs were vaccinated with a combined NaCl and Cetavlon vaccine and seven animals were given placebo. In Experiment IV seven specific pathogen-free (SPF) pigs were given the combined vaccine and eight pigs received placebo treatment. Both of these experiments indicated that the extract vaccines did not completely protect but reduced the mortality in pigs challenged with homologous virulent H. pleuropneumoniae bacteria. The results indicate that capsular antigens of H. pleuropneumoniae have some protective immunogenic efficacy in pigs and mice.  相似文献   

13.
Serum samples were collected from 30 piglets, derived from 17 litters, whose dams had been vaccinated against leptospirosis. Microscopic agglutination test (MAT) titres against Leptospira interrogans serovar pomona varied greatly from pig to pig; there was less variation among littermates. Titres declined between 4 and 10 weeks of age, with an uncorrected half-life of 15.5 days, consistent with IgG being the main antibody class involved. Twelve pigs, 4 derived from unvaccinated sows and 8 from sows vaccinated against leptospirosis, were challenged intravenously at 8 weeks of age with leptospires of serovar pomona. Colostrum-derived antibody protected 4 out of 8 pigs, and in 1 of the remaining 4 the serological response was reduced. Three of the protected pigs showed reduced serological responses and in the fourth the response was strong, but delayed. All of the pigs derived from unvaccinated sows developed leptospiraemia and leptospiruria and showed strong serological responses. Protection by colostrum-derived antibody bore an inexact relationship to MAT titre, but a titre of 16 appeared to be sufficient for protection.  相似文献   

14.
The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.  相似文献   

15.
To differentiate pigs infected with porcine parvovirus (PPV) from those vaccinated with inactivated whole-virus vaccine, an enzyme-linked immunosorbent assay (ELISA) based on detection of a nonstructural polyprotein 1 (NS1) was developed. A threshold of 0.23 optical density units was established and the assayhad high specificity (100), sensitivity (88), accuracy (90) and positive predictive value (100) using haemagglutination inhibition as the standard method. A reproducibility test revealed that the coefficients of variation of sera within-plates and between-run were less than 10%. The assayshowed no cross-reactivitywith antibodies to porcine reproductive respiratorysyndrome virus, pseudorabies virus, foot and mouth disease virus, Actinobacillus pleuropneumoniae, Toxoplasma or Chlamydia. Sera obtained from pigs infected with PPV reacted with recombinant NS1 protein in the ELISA. Sera from pigs vaccinated with inactivated whole virus did not recognize this protein in the ELISA. In contrast, antibodies against PPV whole virus were present in both PPV-infected and vaccinated animals. Serum conversion against NS1 was first detected 10 days after infection and NS1-specific antibodies were detectable up to half a year post infection. In conclusion, the PPV-NS1 ELISA can differentiate PPV-infected versus inactivated PPV-vaccinated pigs and could be applied in disease diagnosis and surveillance.  相似文献   

16.
Pigs, asymptomatically infected with Actinobacillus pleuropneumoniae in their upper respiratory tract, can transmit the infection. Detection of such animals is indispensable to prevent the intake of the disease in a herd. This study was conducted to evaluate bacteriology, polymerase chain reaction (PCR) and serology for the detection of subclinically infected pigs. Pigs were inoculated onto the tonsils with an A. pleuropneumoniae serotype 9 strain (n=12, group 1) or phosphate buffered saline solution (PBSS) (n=5, group 2). To prevent infection of the lungs, pigs of group 1 were treated three times with sodium ceftiofur as an aerosol. A third group (n=5) was inoculated intranasally with the same strain. All animals were euthanized 30 days post-inoculation (dpi). In pigs of group 1, clinical signs were not observed. A small lung lesion was found in only one pig and A. pleuropneumoniae was isolated from this lesion. The bacterium was not isolated from the lungs of animals that did not develop lung lesions. A. pleuropneumoniae was demonstrated in tonsils of 9/12 animals using bacteriological isolation, whereas it was demonstrated in mixed bacterial cultures from tonsils of all 12 animals by PCR. In non-infected animals (group 2), clinical signs were not observed and A. pleuropneumoniae was not demonstrated in any sample. All intranasally infected animals (group 3) developed disease signs and lung lesions. High antibody titers against ApxI, ApxII and heat-stable antigens were detected in animals that developed lung lesions. Antibody titers against these antigens were low or absent in all other pigs. It was concluded that pigs carrying A. pleuropneumoniae in the upper respiratory tract generally do not show measurable antibodies in serum. Therefore, sensitive methods for the detection of the etiological agent such as PCR are required to identify carrier animals, while serological methods are not suitable.  相似文献   

17.
Actinobacillus pleuropneumoniae is a causative agent of porcine pleuropneumonia, a highly contagious endemic disease of pigs worldwide, inducing significant economic losses worldwide. Apx toxins, which are correlated with the virulence of A. pleuropneumoniae, were expressed in Saccharomyces cerevisiae and its possible use as an oral vaccine has been confirmed in our previous studies using a murine model. The present study was undertaken to test the hypothesis that oral immunization using S. cerevisiae expressing either ApxI or ApxII could protect pigs against A. pleuropneumoniae as an effective way of inducing both mucosal and systemic immune responses. The surface-displayed ApxIIA#5 expressing S. cerevisiae was selected as an oral vaccine candidate by finding on induction of higher immune responses in mice after oral vaccination. The surface-displayed ApxIIA#5 expressing S. cerevisiae and the ApxIA expressing S. cerevisiae were developed to serve as an oral vaccine in pigs. The vaccinated pigs showed higher specific IgG- and IgA-related antibody activities than the non-treated control and vector control pigs. Additionally, the induced immune responses were found to protect pigs infected with A. pleuropneumoniae according to the analysis of clinical signs and the gross and microscopic pulmonary lesions. These results suggested that the surface-displayed ApxIIA#5 and ApxIA in S. cerevisiae might be a potential oral vaccine to protect pigs against porcine pleuropneumonia. Thus the present study is expected to contribute to the development of a live oral vaccine against porcine pleuropneumonia as an alternative to current conventional vaccines.  相似文献   

18.
Actinobacillus pleuropneumoniae, a gram-negative rod of the Pasteurellaceae family, causes pleuropneumonia in pigs. Establishing A. pleuropneumoniae free herds is difficult due to the occurrence of persistently infected animals. The ApxIV toxin is expressed by A. pleuropneumoniae in vivo and an ELISA based on the toxin is used to detect infection and to differentiate between infected and vaccinated animals. In this study, we have identified a 1070bp insertion element of the IS30 family, designated ISApl1, in the A. pleuropneumoniae serotype 7 strain AP76. ISApl1 contains a 924bp ORF encoding a transposase, which is flanked by 27bp inverted repeats showing six mismatches. We investigated the occurrence of ISApl1 in other A. pleuropneumoniae strains, and its possible interference with virulence associated factors. Four insertion sites were identified in AP76: within the apxIVA toxin ORF, within a putative autotransporter adhesin ORF, upstream of a capsular polysaccharide biosynthesis gene cluster, and downstream of a beta-lactamase gene. ISApl1 is also present in some serotype 7 field isolates, but not in reference or field strains of other serotypes. In A. pleuropneumoniae AP76, the transposase gene is transcribed in vitro. The insertion in the apxIVA toxin gene remains stable after animal passage. Since this insertion should disrupt toxin expression, we tested 7 pigs infected with AP76 at day 21 post-infection. All were negative in the ApxIV ELISA but four out of seven were positive in an ApxII toxin ELISA. These results show that insertion elements can affect the detection of A. pleuropneumoniae infected animals.  相似文献   

19.
In an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP) by neutralizing the effects of three virulence factors of A. pleuropneumoniae--the capsular polysaccharide (CP), the lipopolysaccharide (LPS), and the hemolysin protein (HP)--two subunit conjugate vaccines were prepared by covalently coupling the CP to the HP and the LPS to the HP. The CP, LPS, and HP were isolated from A. pleuropneumoniae, strain 4074, serotype 1, and the protective efficacy of the conjugate vaccines in swine experimentally infected with A. pleuropneumoniae was evaluated. Following a booster vaccination, a significant (P < 0.05) IgG antibody response to the CP, LPS, and HP was detected in the vaccinated pigs. The pigs vaccinated with the CP-HP and LPS-HP conjugates exhibited significantly less mortality (P < 0.05) and significantly greater weight gain (P < 0.001) than unvaccinated pigs. Vaccinated pigs exhibited significantly fewer and less extensive gross pulmonary lesions (P < 0.001) when compared with unvaccinated pigs. Thus, on the basis of mortality, weight gains, and pulmonary lesion formation, the two conjugate vaccines used in conjunction with one another provide noticeable protective efficacy against SPAP.  相似文献   

20.
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.  相似文献   

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