Bordetella bronchiseptica is a Gram-negative respiratory pathogen responsible for atrophic rhinitis and bronchopneumonia in swine. Several vaccines aimed at preventing B. bronchiseptica have been used, but a safe and efficient live vaccine for use in piglets remains elusive. In this study, we constructed an aroA-deleted B. bronchiseptica strain (QH0814) and evaluated its safety and protective efficiency in piglets. Lung lesion scores in QH0814-immunized piglets post-challenge were significantly lower than those in piglets immunized with the parent strain (P < 0.05). Immunization with QH0814 induced a vigorous immune response, especially at the mucosal surface of the respiratory tract. IgA titers in bronchoalveolar lavage fluid (BALF) and serum were significantly higher in the QH0814-immunized group compared to the inactivated-vaccine-immunized group. Piglets immunized with QH0814 were better protected than those in the inactivated-vaccine and negative control groups. The clinical symptoms, histopathological changes and immune responses elicited in the piglets were recorded. The results of this study suggest that QH0814 was able to confer complete protection against B. bronchiseptica infection and could thus be used as a candidate attenuated live vaccine against B. bronchiseptica in piglets.

The study aimed to establish a rapid and simple assay to detect Shiga-like toxin IIe(SLT-IIe).Monoclonal antibodies(McAbs) against SLT-IIe A subunit were produced by using Escherichia coli(E.coli) expressed recombinant SLT-IIe A subunit protein as antigen to immunize BALB/c mice.A competitive ELISA for detecting antibodies against SLT-IIe was developed on the basis of recombinant protein expressed by E.coli and HRP-labelled McAbs against SLT-IIe A subunit.The optimum conditions of the ELISA were developed as following: the concentration of recombinant for coating ELISA plates was 0.32 μg·mL-1;the best dilution of serum to be tested was 1∶2;the working titer for HRP-labelled McAbs was 1∶3 200;and the inhibition rate above 40% was selected as the positive judging standard.A total of 60 serum samples were detected in parallel by both competitive ELISA and vitro neutralization assay.33.8% of them were detected as positive by competitive ELISA,while 30.9% of them were positive by vitro neutralization assay.The coincidence rate of the two assays was 88.2%.The results showed that the competitive ELISA had the advantages of higher sensitivity,specificity,reproducibility,stability and easy operation.It would be very useful in diagnosis,surveillance of SLT-IIe antibodies and epidemiological survey.

According to the Bordetella bronchiseptica fimD sequence(X75811) of GenBank,we designed a pair of primer,and 1 098 bp DNA fragment of fimD was amplified from swine Bordetella bronchiseptica identificated in our laboratory.The acquired PCR products were inserted into pET-28a and expressed in BL21(DE3).Results of Sodium docecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot assay showed that the fimD gene was expressed in the form of inclusion body,and the recombinant protein was about 42 kD,and can react with the swine antisera to Bordetella bronchiseptica.The fusion protein was purified and used as coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay(ELISA).When using this ELISA to detect 668 clinical sera,30.7% positive sera were detected.Parallel examination of the same 102 serum samples was conducted by using the ELISA and micro-agglutination test(MAT),the result showed that the ELISA is more sensitivity than MAT.

Monoclonal antibodies against Pasteurella multocida toxin(PMT)were produced using Escherichia coli expressed recombinant PMT proteins as immunogens.On the basis of the recombinant protein and HRP-lablled monoclonal antibody AH12 against Pasteurella multocida toxin,a competitive ELISA was developed to detect antibodies against Pasteurella multocida toxin. Optimium conditions of the ELISA were as following:the concentration of recombinant protein for coating ELISA plates was 223 ng/mL;the best dilution of serum was 1:2;The working titer for HRP-labelled monoclonal antibody AH12 was 1:3 200;More than 50% of inhibition rate was positive judging standard.Stability test showed that both inter-assay and intra-assay coefficient variation were below 10%.Both competitive ELISA and in vitro neutralization assay were used to detect 50 known and 82 unknown sera.The results indicated that the positive coincidence and the negative coincidence of 50 known sera were 90% and 100%,respectively; 40.2% of 82 unknown sera were positive detected by competitive ELISA,while 36.6% of them were positive by in vitro neutralization assay,and the coincidence rate was 91.5%.These results showed that the competitive ELISA possessed the advantages of high sensitivity,specificity,stability and briefness.It will be practical for pig diagnosis,immunity effect evaluation and epidemiological survey.

【Objective】The epidemiological distribution,synergistic infection,antibiotic sensitivity and other biological characterization of Bordetella bronchiseptica(Bb) were determined in this paper.【Method】Up to 190 Bb strains were isolated from 2 057 lesion lungs of pigs with clinical signs of pneumonia or atrophic rhinitis while different concurrent infection bacteria species were also separated from the same samples.【Result】These microorganisms were further confirmed by corresponding specific bio-chemistry assay and polymerase chain reaction(PCR).The isolation ratios in the samples that were collected from different provinces and at the different time points(year) ranged from 7.3% to 14.1% and 7.3% to 11.8% respectively.The average isolation ratio between the years of 2003 to 2006 was 9.2%.The isolation ratios of identified co-infection bacterial species were 55.9%(57 strains),50.0%(51 strains),43.1%(44 strains),25.5%(26 strains),17.6%(18 strains)and 11.8%(12 strains) for Streptococcus,Haemophillus parasuis,E.coli,Pasterella multocida,Pseudomonas aeruginosa and Salmonella,respectively.Twenty-two Bb strains were isolated from 43 samples of lesion lungs and nasal swabs of pigs with typical clinical signs of atrophic rhinitis while 7 toxigenic Pasteurella multocida(T+Pm) strains and 6 Pseudomonas aeruginosa(Pa) strains were also separated from the same samples.Among these,7 T+Pm strains were isolated from the 7 of 43 samples,from which 7 Bb strains and 6 Pa strains were separated,simultaneously.Thirty-one Bb stains isolated in 2003 were selected from total 190 strains for determination of drug resistance,erythrocyte agglutination and induction of skin necrosis in suckling mice.The results showed that although the diverse resistance spectra to 15 antibiotics were observed,the tested strains were highly sensitive to kanamycin,gentamycin,polymyxin B,ciprofloxacin,sulperazone,agglutinated swine and sheep erythrocytes and,to different extent,produced skin necrosis in suckling mice.【Conclusion】The infection of swine caused by Bb,which was often accompanied with other co-infection bacterial species,is very prevalent in swine herds in China and Bb may play an important role in causing atrophic rhinitis of swine.

Monoclonal antibodies against FMDV vp2 protein were prepared and a competitive ELISA based on the monoclonal antibodies and vp2 protein was established. Balb/c mice were immunized with Escherichia coli expressed fusion protein. The splenocytes from immunized mice were fused with myeloma cells SP2/0. The hybridism cells were screened by indirect ELISA and limited dilution method. Two hybndoma cell Iines secreting mAbs against Asia I type foot-and-mouth disease were obtained. The titer and relative affinity of mAbs were determined by ELISA. Specificity of mAbs was analyzed by Western blotting. The ELISA titers of the ascites induced by the two hybridism cells were above 100 x 2(9).A competitive ELISA for the use of FMDV antibody detection was established using E. coli expressed fusion protein as coating antigen and HRP-labled mAb as detecting antibody. Clinical tests showed the method had 89.0 percent agreement with UBI Kit to detection of FMDV antibodies and 86.5 percent agreement with LPB- ELISA kit (Ceditest kit) for detection of antibodies against Foot-and-Mouth Disease Virus respectively.

In this study, five fragments of recombinant subunit Pasteurella multocida toxin (PMT) were constructed. Only pET28a-N1518 and pET28b-C2115 could be expressed efficiently in Escherichia coli. The molecular weight of the fusion proteins was 57 kDa and 78 kDa .Western blot confirmed that the two proteins could specifically react with antiserum against Pasteurella multocida toxin. No mice died after the intraperitoneal administration of these two proteins with the dose of 200 microg per mouse, but Vero cell was pathologically changed after administration of 896ng/mL rPMT-N. The fusion protein of rPMT-N and rPMT-C was purified, and emulsified with Freund's adjuvant in equal volumes to get subunit vaccine. Groups of Kunming mice were immunized subcutaneously twice at interval of two weeks. All mice were challenged intraperitoneally with 8.2 x 10(5) CFU HN-13 strain of T+Pm. The protection efficiency of rPMT-N, rPMT-C and crude PMT against HN-13 strain were 90%, 50% and 80%, respectively. The data revealed that the fusion protein of rPMT-N had immunogenicity and potential for developing a subunit vaccine against PAR in pigs.

OBJECTIVE:We evaluated the efficacy of the recombinant pertactin (PRN)-specific active or passive immunization against Bordetella bronchiseptica (Bb) in an aerosol challenge model established by using BALB/c mice.METHODS AND RESULTS:Mice, immunized subcutaneously with two doses of purified glutathione S-transferase (GST)-PRN protein mixed with an equal volume of Freund's adjuvant, produced robust PRN-specific IgG antibody activity. All 20 mice vaccinated with GST-PRN protein survived aerosol challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809 compared with 3 of 20 GST protein-treated controls and 4 of 20 PBS-treated controls that survived. Furthermore, we observed complete protection against intraperitoneal challenge with ten times the LD50 of virulent HH0809 strain in mice that were injected intraperitoneally with 0.5 ml rabbit anti-GST-PRN serum. No survivors were observed in mice that received either rabbit anti-GST serum or PBS alone.CONCLUSION:The recombinant PRN protein had strong immunogenicity against Bb infection.

On the basis of structural analysis of Escherichia coli Shiga-like toxin Ⅱe (SLT-Ⅱe) A subunit by TMpred software, the subunit A of SLT-Ⅱe was expressed in the soluble form which makes it easy to be purified. The strong reactivity of the recombinant SLT-Ⅱe subunit A (re-SLT-ⅡeA) was identified by Western blot. The expressed protein did not demonstrate the lethal effect to mice in mice infection experiment and cytotoxicity to Vero cells. Rabbit anti-serum against recombinant protein could neutralize, up to 90.4 % of efficacy, the cytotoxicity of natural SLT-Ⅱe to Vero cells in vitro. The mice immunized with recombinant SLT-Ⅱe A did not produce protective immunity after challenge with virulent Escherichia coli ED1 strain in mice model. The re-SLT-ⅡeA-based indirect ELISA was developed for detection of antibodies against the subunit A of SLT-Ⅱe. The ELISA could pick up the positive samples in infected pigs eight days post inoculation and 20.9% of positive samples were detected in 1 083 clinical samples while no false positive results were found in detection of nine antisera against porcine bacterial diseases.

支气管败血波氏杆菌是广泛感染多种哺乳动物,有时也感染人的一种常在病原菌.该菌属于波氏杆菌属,借其粘附素和毒素对宿主致病,引起感染动物的呼吸道疾病,并协同其他病原菌形成严重的肺部混合感染,备受人们的广泛关注.本文对该菌的形态特征、培养特性、抗原性、致病情况以及相关研究进展进行了简要概述,以提供人们对支气管败血波氏杆菌的了解.

当前在我国由仔猪下痢死亡所造成的损失十分巨大,据调查一年损失近100亿元.引起下痢的原因并不单纯,从病毒、细菌、寄生虫、中毒、营养、环境、应激等都可成为病因,而且混合感染的机会越来越多,其感染率高达45.80%.为了快速在临诊上作出初步诊断,减少损失,这里将一些常见的几种仔猪下痢病的现场诊断和防制方法简要的介绍如下,以供参考.