Recent studies showed that attenuated Salmonella-derived vaccines induce both humoral and cell-mediated immunity responses [16][17][18].Particularly, S. enterica serovar Choleraesuis strain C500 is highly immunogenic and safe, and has been widely used in China for over 40 years to prevent piglet paratyphoid [19,20].In the present work, we combined two M. hyopneumoniae antigens, P97R1 and NrdF (P97R1N), into S. enterica serovar Choleraesuis C501 [21].Therefore, a recombinant attenuated S. enterica vaccine was evaluated by oral and intramuscular (IM) routes in a mouse model.The immunogenicity of S. enterica strain C501 expressing P97R1N protein was also investigated. Materials and Methods Bacterial strainsM. hyopneumoniae HNYY wild type strain isolated from tissues on primary porcine lung (Laboratory stock).Attenuated S. enterica serovar Choleraesuis strain C501 was constructed with the help of our colleagues [21].Escherichia coli

To construct a multi-subunit vaccine that could provide favorable immunologic efficacy,which provides basic data for the research of developing novel vaccines of Mycoplasmal Pneumonia of swine.The main immune original proteins of Mycoplasma hyopneumoniae p36,p46,p65 and p97R1-Nrdf were recombinantly expressed by using molecular cloning,directed mutations and restructuring expression techniques.Using the protein of p36,p46 and p65 as group Ⅰ,p36,p46,p65 and p97R1-Nrdf as group Ⅱ,using the Mycoplasma hyopneumoniae bacterin（M＋PAC） as group Ⅲ,PBS added with adjuvants as the control group to immunize BALB/c mice.Detect serum,lungs and the splenic lymphocytes which is stimulated by protein with Mycoplasma pneumoniae antibody,IFN-gamma and IL-4.The results suggested that group Ⅱ induced significantly higher antibody than group Ⅰ and group Ⅲ（P〈0.01） and also induced significantly higher IFN-γ production than group Ⅲ（P〈0.05）,however,there were no significant differences between group Ⅱ and group Ⅰ or group Ⅰ and group Ⅲ（P〈0.05）.IL-4 production was similar among the three group（P〈0.05）,but was obviously higher than the control group（P〈0.01）.The antibody level of MP antibody,IFN-γ and IL-4,in lung was group Ⅱ,group Ⅰ,group Ⅲ and the control group in proper order.The splenic lymphocytes results showed that the highest1 MP antibody and IFN-γ level were induced by group Ⅱ,while the highest IL-4 production was induced by group Ⅲ.It shows that the recombinant subunit vaccine containing p36,p46,p65 and p97R1-Nrdf provided the highest antibody level by simultaneously stimulating humoral immunity and cell immunity.Group Ⅰ provided almost the same significantly immunologic efficacy as group Ⅲ in the same way.

Background Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae are causative agents of porcine pneumonia. Over the last few years, attenuated A. pleuropneumoniae live vaccines have been shown to provide protection against A. pleuropneumoniae infection. We postulated that attenuated A. pleuropneumoniae could additionally be used as a vaccine vector for protection against M. hyopneumoniae.Methods A mutant strain of A. pleuropneumoniae, SLW36, was constructed by replacing the urease structural gene of mutant strain SLW03 of A. pleuropneumoniae with the L-lactate dehydrogenase gene (p36) of M. hyopneumoniae by transconjugation and counter selection. The urease function and the growth kinetics of SLW36 were measured. Protein expression of P36 was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and western blotting. The attenuated virulence and immunity of SLW36 were analyzed in a mouse model.Results The mutant strain SLW36 was urease negative and four-fold less virulent than the parental strain SLW03. There were no differences in expression levels of p36 at different culture time-points and the foreign gene was stable after in vitro passage. Immunoglobulin G responses against p36 antigen and M. hyopneumoniae whole-cell antigen were detected.Conclusions The mutant strain SLW36 can induce antibody against p36 and M. hyopneumoniae. The mutant strain SLW36 has the potential to be used as a live vaccine for protection against A. pleuropneumoniae and M. hyopneumoniae. Studies in pigs are needed to confirm protective levels of antibodies and to check for rare side-effects of the vaccine. Copyright (C) 2011 John Wiley & Sons, Ltd.

OBJECTIVE:The study was carried out to construct and characterize Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae and to test its immunogenicity in mice. METHOD:We made p36, p46, p65 and p97R1-Nrdf, the main immunogenic genes of Mycoplasma hyopneumoniae, to insert into the prokaryotic expression plasmid pYA3493. Then these recombinant plasmids and pYA3493 were electroporated into C500 asd-mutant, resulting in the recombinant Salmonella choleraesuis vaccine strains C36 (pYA-36), C46 (pYA-46), C65 (pYA-65), C97R1-Nrdf(pYA-97R1-Nrdf) and CpYA(pYA3493). We characterized these recombinant Salmonella choleraesuis vaccine strains and tested the immunogenicity in mice by intramuscular injection or orally immunized. RESULT:The results of the immunogenicity in mice indicated that the group orally immunized with C36, C46, C65, C97R1-Nrdf showed significantly higher Mycoplasma pneumoniae antibody than both the group orally immunized with C36, C46, C65 and the group intramuscular injected with the Mycoplasma hyopneumoniae bacterin (M + PAC) (P < 0.01). The group intramuscular injected with C36, C46, C65 showed higher IFN-gamma production than the group injected with the Mycoplasma hyopneumoniae bacterin (M + PAC) (P < 0.05), but there was no significant difference between the group orally immunized with C36, C46, C65 and the group orally immunized with C36, C46, C65, C97R1-Nrdf (P > 0.05). The highest level of IL-4 was found in the group orally immunized with C36, C46, C65; higher levels of IL-4 was observed in the group orally immunized with C36, C46, C65, C97R1-Nrdf than the group injected with the Mycoplasma hyopneumoniae bacterin (M + PAC); and the lowest IL-4 level was found in the group injected with C36, C46, C65. There were no significant differences among them (P > 0.05). The Mycoplasma pneumoniae antibody, IFN-gamma or IL-4 production of the each group was obviously higher than the control group (P < 0.01). CONCLUSION:The attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae which has immunogenicity in mice especially by intramuscular injection could probably serve as a vaccine against mycoplasmal pneumonia of swine.

The detection of pathogens with decreased susceptibility to antibacterial and elucidating the molecular mechanism of resistance are of epidemiological and clinical interest in animal husbandry. The 21 Haemophilus parasuis clinical isolates and one American Type Culture Collection reference strains of Actinobacillus pleuropneumoniae (ATCC 27090) were screened for susceptibility to nalidixic acid and enrofloxacin by the microdilution methods. The nalidixic acid MICs for all of the isolates were >= 2 mu g mL(-1) and the enrofloxacin MICs for isolates were <= 8 mu g mL(-1). In addition, the Quinolone Resistance-Determining. Regions (QRDRs) of gyrA and parC of all strains were sequenced. Strains for which nalidixic acid MICs were <= 4.0 mu g mL(-1) lacked modifications in the QRDRs of GyrA and ParC. In contrast, all strains for which nalidixic acid MICs were >= 8 mu g mL(-1) exhibited one or more amino acid changes in GyrA and ParC. Mutations in GyrA is the major resistance mechanism of Haemophilus parasuis to nalidixic acid and enrofloxacin.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.

利用正向间接血凝试验,检测了广西来宾市的5个规模化猪场的864头经产母猪的猪瘟免疫抗体水平.结果表明:在规模化猪场的864份血清中,免疫合格有797份.免疫合格率达92%;其中A场的260份血清中.有248份免疫合格.合格率为95.3%;B场的180份血清中,有162份免疫合格,合格率为90%;C场174份血清中.有157份免疫合格,合格率为90.2%;D场155份血清中,有148份免疫合格,合格率为95.5%:E场95份血清中,有82份免疫合格,合格率为86.3%.80%的猪场都达到90%的免疫合格.

【目的】构建血清10型胸膜肺炎放线杆菌弱毒菌株,为胸膜肺炎放线杆菌减毒活疫苗研究奠定基础。【方法】通过细菌接合转移和SacB负向筛选标记完成突变株的构建与筛选,用PCR、Western blot、重组位点序列对突变株进行鉴定分析。首先构建含肺炎支原体P36基因的pEICALDH重组转移质粒,并转化供体大肠杆菌（E.coliX7213）,将转化的阳性克隆子与野生型APP血清10型亲本菌混合培养6h;然后涂至含氯霉素抗性和烟酰胺腺嘌呤二核苷酸（NAD）的TSA培养基培养,挑取阳性克隆,接种至无抗性的含NAD的TSB液体培养基,培养6～8h后涂至含10%的蔗糖及NAD的TSA培养基,培养24h后挑取蔗糖抗性的克隆,即得到目的突变株。【结果】小鼠毒力试验结果表明突变株比亲本株的毒力显著降低;生长特性分析结果显示突变株与亲本株的增殖能力无显著差异;同时免疫试验结果表明突变株与安全剂量的亲本株均可诱导小鼠产生较好的免疫反应,证明apxIC基因缺失并不影响APP的免疫活性。【结论】成功构建了含猪肺炎支原体P36基因的胸膜肺炎放线杆菌血清10型突变株,所获得的突变株有望成为猪传染性胸膜肺炎弱毒疫苗株。

OBJECTIVE:To construct an attenuate Actinobacillus pleuropneumoniae serovar 10 strain apxIC-/P36+ for new vaccine development.METHODS:The mutant was constructed by transconjugation and counter-selection and then verified by PCR, western blot and sequence analysis. A transconjugation plasmid pEICALDH was constructed and transformed into donor strain Escherichia coli X7213. After mixing the donor cells with A. pleuropneumoniae acceptor cells, we cultivated the mixture for 6 hours and plated on solid medium containing chloromycetin. Then the Cm(R) positive clones were picked and inoculated into liquid medium without any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies (SucB(R)) were selected and considered as mutant.RESULTS:Compared with parental strain, the mutant have the same growth rate in vitro and reduced virulence in mice; additionally, the animal experiment indicated that the mutant strain can successfully induce as good immune response as the parental strain, despite of deletion of apxIC gene.CONCLUSION:In conclusion, we successfully constructed the attenuate strain apxIC-/P36+ of Actinobacillus pleuropneumoniae serovar 10, and this mutation system will facilitate development of live attenuated vaccines.

<正>猪支原体肺炎又称猪喘气病、猪地方流行性肺炎,是一种常被低估的、顽固性、慢性传染病,被认为是最常发生、最广流行、最难净化的原发性疾病。我国该病的发病率在50%左右,而规模化猪场约有90%感染猪肺炎支

Actinobacillus pleuropneumoniae(App),Haemophilus parasuis(Hps),Bordetella bronchiseptica(Bb),Toxigenic Pasteurella multocida(T+Pm) and Mycoplasma hyopneumoniae(Mhp) are five kinds of the most common pathogenic bacteria which can cause a series of respiratory tract diseases in swine.Five pairs of specific oligonucleotide primers were designed according to the DNA sequences of App apxIV,Hps 16S rRNA,Bb fla,toxigenic T+Pm toxA and Mhp ldh reported in NCBI website.A multiple PCR detection method was established according to improved systems and programmes on the basis of five single PCR methods.No cross reaction was detected by the multiplex PCR method between these five bacteria templates and other common bacteria templates.The sensitivity of multiple PCR was lower than 160 pg genome template.Using this multiple PCR method,bacteria could be detected from lung tissues of challenged mice with App,Hps,Bb and T+Pm respectively after being cultured for 8 h in Tryptic Soy Broth(TSB) supplemented with 5% calf serum and NAD+ factor.Simultaneous detection of pathogens in 269 clinical samples from the central area of China revealed the universal existence of App,Hps,Bb,T+Pm and Mhp.In conclusion,the multiple PCR assay showed excellent specificity,sensitivity and reduplication and could detect five pathogenic bacteria of respiratory tract in swine.We expected that this multiple PCR assay could be useful extensively for epidemiological survey and clinical differential diagnosis of five common respiratory diseases in swine.

为了测定伪狂犬病毒中和抗体与gB抗体之间的相关性,本试验用216份伪狂犬病毒中和抗体阳性血清,对每一份样本作4种不同的稀释(1∶1、1∶20、1∶40、1∶80),用Idexx公司的gB-ELISA试剂盒检测。试验结果表明:216份中和抗体阳性血清样品中,有7份gB抗体阴性,阳性检出率为97.8%(209/216);随着中和抗体效价的增高gB-ELISA OD650值降低;gB抗体阻断率随中和抗体效价的增高而增加;对同一份血清样本作1∶1、1∶20、1∶40、1∶80稀释,测定结果发现,随着稀释度的增加,gB抗体阻断率逐渐降低,而gB-ELISAOD650值逐渐升高。

This paper described a multiplex RT-PCR to rapidly diagnose the co-infection in samples from pig with reproductive failure symptoms.Three sets of primers were designed,respectively,targeting to the conserved sequences in E2 gene of classical swine fever virus(CSFV),a domain encompassing partial ORF6,ORF7 and 3-terminus UTR of porcine reproductive and respiratory syndrome virus(PRRSV) and E gene of Japanese encephalitis virus(JEV).The expected sizes of RT-PCR amplicons were 288 bp for CSFV,430 bp for PRRSV and 1 015 bp for JEV,respectively.RNA was extracted from pure culture of the viruses,followed by the synthesis of cDNA that was used as templates for method development.Through optimization by using three sets primers simultaneously,the detection limit of RNA template in the RT-PCR was 0.27 ng for CSFV,0.049 ng for PRRSV and 0.067 for JEV.No false positive results were produced in the case of transmissible gastroenteritis virus(TGEV),porcine parvovirus(PPV),pseudorabies virus(PrV) and porcine circovirus type 2(PCV-2).There were 174 samples,including sera,fetuses and lung tissues which were collected from the pigs that mainly presented high fever,were subjected to our in-house RT-PCR.PRRSV was detected in these samples with higher detection rate than CSFV and JEV.Among 174 clinical samples,PRRSV-positive percentage was 30.4,CSFV-positive percentage was 4.6,JEV-positive percentage was 1.7,co-infection of CSFV and PRRSV percentage was 1.7.In order to confirm the reliability of the multiplex PCR,we selected PCR products of CSFV-positive JEV-postive and PRRSV-positive respectively that detected clinical samples by multiplex PCR.After sequences analysis and BLAST,it is showed that homologies between PCR products of CSFV,PRRSV,JEV and target genes sequences were 92%,90% and 89%,respectively.Those results proved that multiplex RT-PCR have Applied value in practices.