共查询到20条相似文献，搜索用时 31 毫秒
1.
The aroA gene from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 was isolated and sequenced. The gene complemented the aroA mutation in Escherichia coli AB2829. A kanamycin resistance cassette was inserted into the aroA gene and the mutant gene was reintroduced into A. pleuropneumoniae by allelic replacement. Intratracheal infection of susceptible pigs with A. pleuropneumoniae aroA caused no signs of respiratory disease or lung lesions in any of the animals at a dose 10(4) times the dose reliably known to induce acute pleuropneumonia; all animals infected with the unaltered control strain developed acute disease. The aroA mutant was rapidly eliminated from the lungs and tonsil of infected animals. The mutant may represent a safely attenuated strain for use in live bacterial vaccination or the delivery of antigen by the intranasal route. However, the residence time of the mutant in the respiratory tract of the pig may be too short for it to be useful in generating a protective immune response. 相似文献
2.
Chen AY Fry SR Forbes-Faulkner J Daggard GE Mukkur TK 《Veterinary microbiology》2006,114(3-4):252-259
The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-gamma level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-gamma was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-gamma production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced. 相似文献
3.
嗜水气单胞菌不同分离株生化特性及胞外蛋白酶的检测 总被引：2，自引：0，他引：2
嗜水气单胞菌可引起多种宿主患病,该菌致病性与其产生的多种毒力因子特别是胞外蛋白酶有关。本研究检测了26株嗜水气单胞菌不同分离株的生化指标,采用脱脂乳平板法检测了胞外蛋白酶的产生情况,并利用PCR技术检测了2种胞外蛋白酶基因ahp-A(编码丝氨酸胞外蛋白酶)和eprCAI(编码温敏胞外蛋白酶)。结果表明,大部分菌株生化指标表现为蔗糖(+)、阿拉伯糖(+)、葡萄糖(+)、甘露醇(+)、鸟氨酸脱羧酶(-)、肌醇(-)、七叶苷(+);脱脂乳平板法检测发现,91.7%(22/24)的致病菌株均能产生胞外蛋白酶,而2株无毒株则不能产生,对2个胞外蛋白酶基因的检测也得到相似的结果。本研究将为嗜水气单胞菌的流行病学调查和致病机制的深入研究奠定基础。 相似文献
4.
The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related. 相似文献
5.
Aeromonas hydrophila is a broad-host-range pathogen and its pathogenesis is multifactorial. A regulatory mechanism known as quorum sensing has been found to be involved in the regulation of virulence in many bacteria. In A. hydrophila the ahyR gene encodes LuxR-type response regulator. Here we describe the inactivation of the ahyR gene of A. hydrophila J-1 by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila J-1 by means of the suicide plasmid pJP5603. Cytotoxic effects on EPC cells assay and LD(50) determinations in fish demonstrated that the ahyR mutant was highly attenuated relative to the wild-type strain. Compared with the parent strain, some characteristics, such as biochemical characters and outer membrane protein profiles, had changed. Some main virulent determinants could not be detected, including proteases, amylase, Dnase, hemolysin and S layer. This article confirmed the important function of AhyR in the pathogenesis of A. hydrophila J-1. 相似文献
6.
Previously we described the development of an attenuated Pasteurella multocida mutant that expresses only the N-terminal truncated fragment of P. multocida toxin (N-PMT) and its protective effects in a mouse model. This paper details our evaluation of the vaccine potential of this mutant strain in pigs. Pigs vaccinated with the mutant showed significantly higher rates of antibody induction and lower nasal conchal (turbinate) scores for atrophic rhinitis than controls, which suggests that this mutant strain may be a good candidate for a live attenuated vaccine. 相似文献
7.
8.
Combination of competitive exclusion and immunization with an attenuated live Salmonella vaccine strain in chickens 总被引：3，自引：0，他引：3
To use the advantages of both the competitive exclusion (CE) technique and immunization with a live Salmonella vaccine, the combination of these methods was studied. Specific-pathogen-free chickens were pretreated by combined or single administration of a CE culture and a commercial live Salmonella typhimurium vaccine on days 1 and 2 of life and challenged with Salmonella typhimurium on day 3 to study the exclusion effect by both the CE preparation and the Salmonella vaccine. The exclusion effect by the CE culture combined with the immunologic effect by the live vaccine was studied after challenge of the birds on day 43 of age. The number of challenge organisms in ceca was used to evaluate the efficacy of the pretreatment. The protective exclusion effect of the CE culture was substantial in very young chicks and still detectable in 6-wk-old birds. The attenuated Salmonella typhimurium vaccine produced only an initially occurring exclusion effect. Because the exclusion effect of the CE culture was considerably stronger than the exclusion effect of the attenuated Salmonella typhimurium vaccine, the combination of both did not result in an additive protective effect. In order to exploit the exclusion potential between Salmonella strains and to attain an additive exclusion effect by a CE culture and a vaccine strain, live Salmonella vaccines are needed that are sufficiently attenuated without affecting genes essential for colonization exclusion of other Salmonella organisms. In 6-wk-old birds, the exclusion effect by the CE culture combined with the immunologic effect by the live Salmonella vaccine resulted in a degree of protection considerably beyond that generated by the exclusive use of the two methods. The administration of the live Salmonella vaccine strain prior to or simultaneously with the CE culture revealed the best protective effect because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of an exclusion effect in very young chicks and the development of a strong immune response affording protection in older birds. 相似文献
9.
In vitro and in vivo efficacy of a live attenuated Salmonella Typhimurium vaccine at preventing intestinal colonization in chicks 
下载免费PDF全文
下载免费PDF全文 Alexander J. Howard Kapil K. Chousalkar Andrea R. McWhorter 《Zoonoses and public health》2018,65(6):736-741
Vaccination of chicks with Salmonella (S .) Typhimurium aroA deletion mutants has previously been shown to inhibit intestinal colonization of wild‐type S. Typhimurium strains. In Australia, Bioproperties VaxSafe? STM1 strain is the only licensed and commercially available S . Typhimurium vaccine. This vaccine is a live attenuated aroA deletion mutant. Currently, it is recommended that the first dose of the STM1 vaccine is administered through coarse spray. It is unclear whether this mode of administration effectively permits intestinal colonization. Furthermore, it is not known whether the STM1 strain prevents or inhibits Salmonella colonization of chicks following this first dose. This study investigated both in vitro and in vivo colonization parameters. Invasiveness was assessed using an in vitro invasion assay into sections of ileum and caecum collected from day‐old chicks. The S. Typhimurium definitive types (DT) 9 and 44 exhibited the greatest invasion into both intestinal segments. STM1 was invasive but was significantly less so than both isolates of S. Typhimurium. In dual and triple infections, no competitive microbial interactions between STM1 and wild‐type Salmonella were observed. In vivo colonization inhibition was also tested. Vaccinated and nonvaccinated day‐old chicks were challenged with S. Typhimurium DT9. Both STM1 and S. Typhimurium DT9 were found in spleen, liver, ileum, caecum and caecal contents from day 2 postinfection. No significant exclusion effect was observed in vaccinated and challenged chicks. 相似文献
10.
重组转移载体pBSKA通过电转化导入亲本菌胸膜肺炎放线杆菌血清7型（APP-7）WF83株,电转化后的产物涂布于TSB／Kan平板,2d获得突变株。卡那霉素抗性实验证实突变株有卡那霉素抗性;NAD依赖性实验证实突变株依赖NAD生长;PCR鉴定证实了卡那霉素抗性基因置换了apxlIC基因,并证实突变株中无pBSKA质粒的存在;溶血活性实验证实突变株完全失去了溶血活性;细胞毒性实验证实突变株的细胞毒性完全丧失;对小鼠的安全性实验证实突变株的毒力显著减弱,突变株对小鼠是安全的;遗传稳定性实验证实,突变株在体外连续传30代和在体内传10代均不会发生卡那霉素抗性的丢失。结果表明实验成功构建了基因缺失减毒株,为进一步以此突变株作为基因工程弱毒活疫苗株开展研究奠定了一定的基础。 相似文献
11.
猪丹毒ＱＬ２５１弱毒株在马丁肉汤、血液琼脂斜面、小白鼠及猪丹毒敏感猪体内连续传代后，测其安全性和免疫原性。试验证明，ＱＬ２５１无论是在培养基上，还是在动物体内传代后都是稳定的，仍然保持了原有的特性。 相似文献
12.
Deletion of the M063 gene from myxoma virus produces a virus that is unable to replicate in rabbit cells in vitro or in live rabbits but can be propagated in non-rabbit cell lines. A targeted M063 deletion mutant was constructed in the attenuated Uriarra strain of myxoma virus and the ability of this virus to act as a safe, non-transmissible vaccine against myxomatosis was tested in outbred laboratory rabbits. Immunization with the M063 deletion vaccine provided good short-term protection against lethal challenge with virulent myxoma virus. Long-term protection was similar to reported results with heterologous live virus, with some rabbits protected but others succumbing to challenge. Replication-deficient poxvirus vaccines, like the Modified Vaccinia Virus Ankara (MVA) in man and the myxoma virus vaccine described here in rabbits, are very attractive from a safety perspective. Seasonal boosting would be predicted to provide long-term protection. Targeted host-range gene deletions could have potential for rapid development of poxvirus vaccines in general. 相似文献
13.
Najdenski H Golkocheva-Markova E Kussovski V Vesselinova A Garbom S Wolf-Watz H 《Zoonoses and public health》2009,56(4):157-168
Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain. 相似文献
14.
A promising approach to reduce the impact of influenza is the use of an attenuated life virus as a vaccine. Using reverse genetics, a mutant of strain A/WSN/33 with a modified cleavage site within its haemagglutinin was generated which depends on proteolytic activation by elastase. Unlike the wild-type requiring trypsin, this mutant is strictly dependent on elastase. Both viruses grow equally well in cell culture in the presence of the respective protease. In contrast to the lethal wild-type, the mutant is entirely attenuated in mice at a virus dose of 10(6) pfu. At a dose of 10(5) pfu it induced complete protection against lethal challenge. This approach allows the conversion of any epidemic strain into a genetically homologous attenuated virus. 相似文献
15.
Characterization of virulent and attenuated strains of pseudorabies virus for thymidine kinase activity, virulence and restriction patterns. 总被引：1，自引：1，他引：0 下载免费PDF全文
下载免费PDF全文 A pseudorabies virus mutant lacking thymidine kinase activity (TK-) was isolated and characterized. The mutant replicated as well in cell culture as the parental TK+ strain, was not temperature sensitive at 38.5 degrees C, and did not revert to TK+. Two pseudorabies virus field isolates and three commercial modified live virus vaccine strains were compared for TK activity and virulence for the mouse; all strains expressed TK: Km values for thymidine of the viral TKs ranged from 2.9 to 3.9 microns; the commercial modified live virus vaccine strains were reduced in virulence for the mouse two to ten-fold. The TK- mutant was avirulent for the mouse. Restriction enzyme analysis of the pseudorabies virus DNA from the strains under study revealed that two of the modified live virus vaccine strains are closely related and that all three modified live virus vaccine strains lack the largest PstI fragment characteristic of the other strains included in the study. 相似文献
16.
Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine. 相似文献
17.
Aeromonas hydrophila is a Gram-negative opportunistic pathogen that causes disease in a wide range of hosts due to its multifactorial virulence. Here we describe the application of transposon insertion mutagenesis approach to obtain an exoenzyme mutant of A. hydrophila strain J-1. Immunization of swordtail fish (Xiphophorus helleri Heckel) with the highly attenuated mutant provided protection (survival of 27 out of 35 fish, compared to survival of only 13 out of 35 control fish) in the fish given the highest immunization dose (10(7) CFU) against intraperitoneal challenge with the wild J-1 strain. Immunization with doses of 10(5) or 10(3) did not provide significant protection. 相似文献
18.
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
19.
兔支气管败血波氏杆菌PRN基因缺失突变株的构建及特性研究 总被引：1，自引：1，他引：0
利用自杀性质粒构建兔支气管败血波氏杆菌百日咳黏附素(PRN)缺失突变株以研究PRN在支气管败血波氏杆菌(Bb)致病机理中的作用,同时为支气管败血波氏杆菌病减毒活疫苗的研究提供理论依据.PCR扩增出PRN1(PRN上游基因)和PRN2(PRN下游基因)2个目的基因片段,运用基因重组技术将庆大霉素抗性基因(GM)连接到PRN1和PRN2之间,将连接好的基因片段克隆到pMEG-375自杀性载体中,构建自杀性载体pMEG375-PRN1-GM-PRN2,将其转化到宿主菌SM-10中,通过宿主菌SM-10与受体菌Bb固相滤膜交配,自杀性载体转移到受体菌,根据同源重组原理,抗性筛选得到基因缺失突变株,命名为Bb(△PRN).对突变株Bb(△PRN)与野生株WT进行了遗传稳定性、生长特性、溶血特性、细胞黏附特性、毒力、免疫保护性等比较研究.结果表明:Bb(△PRN)具有遗传稳定性;与野生株相比,突变株生长速度较慢,毒力有所下降,溶血活性及对Hep-2细胞的黏附能力没有明显变化;小鼠免疫原性试验结果显示,突变株免疫小鼠后可以产生强有力的免疫力,能够抵抗野生株的攻击.Bb(△PRN)突变株构建成功并具有良好的免疫原性,为支气管败血波氏杆菌病减毒活疫苗的研究奠定了基础. 相似文献
20.
Growth of Babesia bovis parasites in stationary and suspension cultures and their use in experimental vaccination of cattle 总被引：1，自引：0，他引：1
A combination of stationary culture and suspension culture was used to produce litre quantities of Babesia bovis parasites suitable for use as live vaccine. The Australian vaccine strain of B bovis, Ka, was maintained continuously in microaerophilus stationary phase (MASP) cultures, and for a short period in batch and flow-through spinner flask cultures. Although continuous culturing was not achieved in spinner flasks, the production of litre quantities of heavily parasitised erythrocytes was achieved more simply than by using MASP cultures. Ka strain parasites were maintained continuously in MASP culture for 174 days without altering their virulence or immunogenicity when compared to calf-derived parasites. Cultured parasites also survived storage at 4 degrees C for six days in basal medium, adding to their potential usefulness as a live vaccine in field situations. 相似文献