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1.
In order to construct a novel vaccine candidate for preventing post-weaning diarrhea in swine, the individual genes for Escherichia coli K88ab, K88ac, FedA, and FedF fimbriae were inserted into a secretion plasmid pBP244 containing asd, lepB, secA, and secB. These were transformed into Salmonella Typhimurium Δlon ΔcpxR Δasd. Secretion of the individual recombinant fimbrial antigens was confirmed by immunoblot analysis. Groups 1 and 2 mice received a single oral dose of the vaccine mixture and S. Typhimurium carrying pBP244 only as a control, respectively. In groups 3 and 4, mice were primed and boosted with the vaccine mixture and S. Typhimurium carrying pBP244 only as a control, respectively. In general, all immunized mice had significantly increased serum immunoglobulin (Ig)G (P < 0.05) and intestinal secretory IgA against the individual fimbrial antigens compared with those mice in the control group. In the IgG2a and IgG1 titer assay, only IgG2a titer was increased in group 1, while both IgG2a and IgG1 titers were increased in group 3. Furthermore, the vaccine strains were not detected in the excreted feces of any immunized mice. Thus, the vaccine candidate can be highly immunogenic and be safe to the environment.  相似文献   

2.
The efficacy of a novel, live delivery vaccine was examined for protection against post-weaning diarrhea in pigs. An expression/secretion plasmid harboring genes encoding enterotoxigenic Escherichia coli K88ab, K88ac, FedA and FedF fimbriae was constructed and harbored in an attenuated Salmonella, which was used as the vaccine candidate. Groups A (n=3) and B (n=3) sows were orally immunized with the candidate vaccine and PBS as a control, respectively, at 8 and 11 weeks of pregnancy. All group piglets were challenged with two challenge strains at 5-week-old. All immunized sows had significantly increased IgG and IgA levels in both serum and colostrum to individual adhesins compared to the control (p ≤ 0.05). Immune response in Group A piglets were significantly increased (p ≤ 0.05). Furthermore, no clinical signs were observed in Group A piglets after the challenge and no challenge strains were detected in rectal swabs, while diarrhea was observed in 47.8% control piglets and challenge strains were isolated from all the diarrheic piglets. These results show that immune response of sucking piglets can maintain at higher levels through the milk of the immunized sows and vaccination of sows with the candidate may protect colibacillosis in weaned piglets.  相似文献   

3.
Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-γ, IL-4, TNF-α, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection.  相似文献   

4.
We have previously developed a novel attenuated Salmonella Typhimurium (S. Typhimurium) ΔcpxR Δlon vaccine. This study was carried out to examine whether this vaccine could effectively protect growing piglets against Salmonella infection. Attenuated S. Typhimurium secreting the B subunit of Escherichia coli heat-labile enterotoxin was also used as a mucosal adjuvant. Pregnant sows in groups A and B were primed and boosted with the vaccine and mucosal adjuvant, whereas sows in groups C, D and E received PBS. Piglets in groups A and C were intramuscularly primed with formalin-inactivated vaccine and orally boosted with live vaccine, while piglets in groups B, D and E received PBS. Piglets in groups A, B, C, and D were challenged with a wild type virulent S. Typhimurium at the 11th weeks of age. Colostrum sIgA and IgG titers in vaccinated groups A and B sows were approximately 50 and 40 times higher than those of non-vaccinated groups C, D and E sows (P < 0.001). Serum IgG titers of group A piglets were also significantly higher than those of groups D and E piglets during the study (P < 0.001). Furthermore, no clinical signs were observed in group A piglets during the entire experimental period after the challenge, while diarrhea was observed in many of the piglets in groups B, C, and D. No Salmonella was isolated from fecal samples of the groups A and C piglets on day 14 after challenge, whereas the challenge strain was isolated from several piglets in groups B and D. These results indicate that vaccination of the piglets with the vaccine and mucosal adjuvant in addition to vaccination of their sows induced effective protection against Salmonella infections in the growing piglets.  相似文献   

5.
本研究旨在构建基于减毒鼠伤寒沙门菌的平衡致死系统。将打靶基因片段Cat基因重组入鼠伤寒沙门菌LH430株asd基因位置,之后将温敏型质粒pCP20转入菌体中用以消除Cat基因片段,通过PCR技术两步法筛选出缺失株Δasd LH430;将asd+的原核表达质粒pYA3493转入Δasd LH430,经PCR及测序鉴定表明鼠伤寒沙门菌平衡致死系统Δasd LH430（pYA3493）构建成功。对构建的基因缺失株研究表明,该缺失株失去了在二氨基庚二酸（DAP）阴性环境中生存的能力;糖发酵试验表明,缺失菌株在利用碳源的能力方面与亲本菌株保持一致;9种生化试验结果显示,缺失菌株遗传了亲本菌株的生化特性;遗传稳定性试验表明,缺失菌株能够稳定遗传缺失的423 bp的基因片段;将绿色荧光基因转入所构建的平衡致死系统,成功构建携带绿色荧光基因的重组菌Δasd LH430（pYA-gfp）,对其研究表明该重组菌能够有效表达绿色荧光蛋白。本研究成功构建了鼠伤寒沙门菌平衡致死系统Δasd LH430（pYA3493）,能够有效表达所携带的基因,该系统可作为疫苗活载体来携带外源基因。  相似文献   

6.
为研制仔猪副伤寒—猪水肿病新型二联疫苗,从pMDSLT-Ⅱ中扩增去信号肽后的SLT-ⅡvB基因,将2拷贝SLT-ⅡvB与pYA3493连接。阳性质粒pYA2B电转化C500crp-/asd-缺失株,进行SDS-PAGE电泳和Western blotting,重组菌C500crp-/asd-(pYA2B)口服免疫小鼠,腹腔攻10 LD50的猪霍乱沙门氏菌强毒C78-1和2 LD50的猪水肿大肠杆菌强毒ED3株,计算攻毒保护率。结果显示,2 SLT-ⅡvB在C500crp-/asd-缺失株中获得了表达。C500crp-/asd-(pYA2B)免疫组对ED3的攻击能提供60%保护,而对照组不能提供任何保护。2组对C78-1攻击均提供100%保护,为研制仔猪副伤寒—猪水肿病二联疫苗及进一步提高免疫效果提供依据。  相似文献   

7.
We evaluated the protective efficacy of 94-kb virulence plasmid-cured, and phoP- or aroA-deficient strains of Salmonella enterica serovar Typhimurium (ΔphoP or ΔaroA S. Typhimurium) as oral vaccine candidates in BALB/c mice. Two weeks after the completion of 3 oral immunizations with 1 × 108 colony-forming units (CFU) of virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium at 10-day intervals, S. Typhimurium lipopolysaccharide (LPS)-specific mucosal secretory immunoglobulin A (s-IgA) antibody titers were detected in the cecal homogenate, bile and lung lavage fluid, but not in the intestinal lavage fluid. In addition, the increases in S. Typhimurium LPS-specific immunoglobulin G (IgG) and IgA antibody titers in the serum were also observed 2 weeks after completing 3 oral immunizations with virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium. The series of 3 oral immunizations protected the mice against an oral challenge with 5 × 108 CFU of the virulent strain of S. Typhimurium, suggesting that both the virulence plasmid-cured, and ΔphoP and ΔaroA S. Typhimurium strains are promising candidates for safe and effective live S. Typhimurium vaccines.  相似文献   

8.
Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.  相似文献   

9.
This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross‐bred buffaloes. A total of 134 cross‐bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 1010 CFU/ml of the C501 (pVAX‐asd‐IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX‐asd‐IS) elicited significant immune responses, and anti‐inhibin IgG antibody titres in group T1 were significantly higher (p < .01) than groups T3 and C. After the second GnRH treatment, the growth speed of the dominant follicles in group T1 was significantly faster (p < .05) than groups T3 and C. The number and diameter of large follicles (≥10 mm) as well as ovulatory follicles in group T1 were the greatest in all groups, resulting in a greater conception rate in buffaloes with positive anti‐inhibin antibodies. These results demonstrate that immunization with the C501 (pVAX‐asd‐IS) vaccine, coupled with the Ovsynch protocol, could be used as an alternative approach to improve reproductive performance in cross‐bred buffaloes.  相似文献   

10.
小鼠对猪囊虫抗原基因TS76的免疫应答   总被引：3，自引：0，他引：3  
将猪囊虫抗原基因 TS76的真核表达型质粒 VTS76单独或与 p UC18联合肌肉免疫注射于 BAL B/ c小鼠 ,以MTT比色法检测小鼠脾淋巴细胞 Con A刺激的增殖反应及 IL - 2的诱生活性 ,EL ISA方法检测免疫小鼠 Ig G总量和特异性抗体水平 ,常规法检测外周血免疫细胞数量的动态变化。结果发现 ,VTS76免疫小鼠各项细胞和体液免疫应答反应指数均比空白对照组小鼠显著提高 ;联合免疫组小鼠的细胞免疫应答和体液免疫应答反应指数均比 VTS76小鼠提高。由此可见 ,以 VTS76免疫小鼠可诱导其特异性细胞和体液免疫反应 ,而 p UC18又明显提高了 VTS76免疫小鼠的免疫应答水平 ,具有显著的免疫增强作用  相似文献   

11.
In this study, the Actinobacillus pleuropneumoniae antigens ApxIA, ApxIIA, ApxIIIA and OmpA were expressed in an attenuated strain of Salmonella (?lon?cpxR?asd) for prevention of porcine pleuropneumonia. In order to evaluate the immunization strategy of the construct, a total 60 BALB/c mice were equally divided into four groups (n?=?15). Group A mice were intranasally immunized only at 6-weeks-of-age, while group B mice were intransally primed and boosted at 6- and 9-weeks-of-age, respectively, and group C mice were intransally primed at 6-weeks-of-age and subsequently boosted twice at 9- and 12-weeks-of-age. Group D mice were used as a control, which were inoculated with sterile PBS. Groups A, B, and C showed significantly higher serum IgG and fecal IgA immune responses than those of the control group. After virulent challenge with a wild type A. pleuropneumoniae, the immunized groups A, B and C showed 33.3 %, 13.3 % and 26.7 % mortality as the control group showed 60 % mortality. These results showed that the protection against porcine pleuropneumonia using the construct can be optimized by a double intranasal vaccination.  相似文献   

12.
To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×10(7)CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×10(9)CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish.  相似文献   

13.
In seeking to develop a safe fowl typhoid (FT) vaccine, a novel candidate lacking cpxR, lon, and asd Salmonella Gallinarum (SG) genes was constructed with the plasmid-containing araC::P(araBAD)::asd system. A balanced-lethal host-vector system based on the essential bacterial gene for aspartate beta-semialdehyde dehydrogenase (asd) was used to construct the SG mutant strain. A plasmid (p15A ori) with an araC::P(araBAD)::asd cassette was introduced into an auxotrophic mutant to prevent ex vivo survival. The safety, immunity, and protective properties of the SG mutant were evaluated. Inoculation of the mutant at 10(6) colony-forming units (CFU) did not result in recovery in feces and internal organs, whereas inoculation at 10(8) and 10(10) CFU resulted in moderate bacterial recovery from feces and organs. Birds immunized with the mutant were challenged with a virulent SG strain at day 14 postimmunization; significantly reduced mortality and induced plasma immunoglobulin (Ig)G and mucosal IgA responses were noted. Cellular immune responses as evaluated by a peripheral lymphocyte proliferation assay were also significantly induced. The balanced-lethal host-vector system for construction of SG mutants is an effective and improved approach for safe vaccine construction against FT.  相似文献   

14.
Previous findings, viz. that mice can be successfully immunized against infection with Salmonella dublin with either live or inactivated vaccine, were confirmed. Immunity lasted for at least 12 weeks in mice which had been immunized with inactivated alum-precipitated vaccine. The immunogenicity of inactivated vaccine gradually decreased on storage at 4 degrees C, but this was only detectable if a single injection was used for immunization: 2 injections virtually eliminated this phenomenon. The immunogenicity of live vaccine in mice was not enhanced by levamizole or the simultaneous injection of inactivated organisms. Both live and inactivated vaccines provided immunity in calves. A single injection of lyophilized vaccine, prepared from live rough Salmonella dublin strain (HB 1/17),protected 3 out of 6 calves, while 2 injections of a formalin-inactivated, alum-precipated vaccine, containing 1% packed cells of S. dublin strain 2652 V, protected 5 out of 6 calves against intraduodenal challenge with 2 x 10(9), S. dublin strain 2652 V. Two calves which had been immunized with an inactivated oil adjuvant vaccine were also solidly immune to this challenge. Serum antibody response in calves was poor when measured by the tube agglutination and the haemagglutination tests. Similarly, the sera had only marginal protective values when tested by means of a passive protection test in mice. Antibody titres alone are not a valid measure therefore, for the immune status of immunized animals.  相似文献   

15.
将SPF级Balb/c小鼠随机分为免疫组和对照组,免疫组小鼠以pLA-F41/L.casei重组干酪乳杆菌滴鼻免疫,对照组用pLA/L.casei干酪乳杆菌滴鼻免疫,应用间接ELISA测定血清中特异性IgG抗体水平和鼻咽部冲洗液、肠道冲洗液、阴道冲洗液及粪便中ETEC F41特异性SIgA抗体水平.分离并计数NALT、NC及PP、IEL淋巴细胞数目,利用免疫细胞化学法检测CD4+、CD8+T细胞亚群水平,观察重组干酪乳杆菌滴鼻免疫小鼠诱导的鼻相关淋巴组织(NALT)和肠相关淋巴组织(GALT)黏膜部位的免疫应答.结果表明重组干酪乳杆菌滴鼻免疫小鼠可有效诱导NALT和GALT黏膜部位免疫应答.  相似文献   

16.
为了研究猪水肿病(ED)大肠杆菌SLT-Ⅱe基因突变菌株作为口服疫苗的免疫效果,本实验用已构建的猪ED大肠杆菌SLT-Ⅱe基因突变菌株口服免疫BALB/c小鼠,检测其血清中的IgG抗体及粪便和肠黏液中的slgA抗体水平,并进行淋巴细胞增殖检测及攻毒保护实验.结果表明该基因突变菌株具有良好的免疫性,能诱导小鼠体内产生IgG和sIgA抗体,并且能引起T淋巴细胞增殖反应.攻毒保护实验结果显示,口服免疫突变菌株能对小鼠提供良好的保护,保护率为75%(15/20).本研究结果证明,该大肠杆菌基因突变菌株在小鼠体内能激发体液免疫和细胞免疫反应,可作为猪ED口服疫苗的候选菌株.  相似文献   

17.
无乳链球菌是导致奶牛乳腺炎的重要病原菌,本研究评价了SIP(surface immunogenic protein)亚单位疫苗对小鼠无乳链球菌乳腺炎的免疫保护效果。制备无乳链球菌SIP亚单位疫苗和灭活疫苗,对小鼠进行免疫,并设PBS阴性对照。免疫前后采血测定IgG及IgG亚类的抗体滴度。免疫小鼠分娩后第4天,进行无乳链球菌乳腺攻毒试验。24h后扑杀攻毒鼠,进行乳腺内CFU的测定,制作并观察乳腺组织病理切片。结果显示,SIP亚单位疫苗免疫组IgG及IgG亚类抗体滴度显著高于灭活疫苗组(P0.01)。免疫哺乳小鼠乳腺攻毒后,SIP亚单位疫苗免疫组小鼠乳腺CFU显著低于灭活疫苗组及对照组(P0.001)。乳腺病理切片显示SIP亚单位疫苗组乳腺组织结构较对照组完整,且中性粒细胞浸润程度最小。本研究表明,无乳链球菌SIP亚单位疫苗有望作为奶牛无乳链球菌乳腺炎的候选亚单位疫苗。  相似文献   

18.
This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.  相似文献   

19.
Nine lambs, approximately 9 months of age were allocated to three groups (A, B, C), with three animals in each. Sheep in Groups A and B were trickle-infected with doses of 1000 third-stage larvae (L3) of Ostertagia leptospicularis (five times per week) over periods of 7.5 and 10.5 weeks, respectively, and were subsequently treated with fenbendazole (7.5 mg/kg). Approximately 3 weeks after anthelmintic treatment, all sheep were challenged with a single dose of 100,000 L3, whereas sheep of Group C received the same dose as a primary infection. Sheep of Groups A and B were almost completely refractory against the challenge infection, as indicated by negative faecal egg counts and adult worm burdens. A relatively high infection level was present in the sheep of Group C. The results indicate that a comparatively short immunization period of 7.5 weeks is sufficient to protect lambs against subsequent larval challenge. During immunization, the pepsinogen-, gastrin- and IgA-responses were similar in the individual sheep. In contrast to parasite-specific IgG1 and IgG2 levels, IgA decreased rapidly after cessation of trickle infection and parallel anthelmintic treatment, and may therefore indicate current exposure to parasite antigen. After challenge, the majority of the immunized sheep exhibited immediate and short-term responses of pepsinogen, gastrin and IgA in the serum. The time course and the level of each of these responses were very similar in the individual sheep, suggesting that the release of pepsinogen, gastrin and IgA into the circulation was influenced by related mechanisms.  相似文献   

20.
Salmonella Typhimurium ghost cells expressing K88ab, K88ac, K99, and FasA fimbriae of enterotoxigenic Escherichia coli (ETEC) in their envelopes were constructed. The genes encoding the fimbriae were individually cloned into an expression plasmid, pMMP81, carrying the asd gene, which was subsequently electroporated into the Δasd S. Typhimurium mutant. Plasmid pJHLP99, carrying the phiX174 lysis gene E, was also subsequently electroporated into the Salmonella mutant. The presence of the individual fimbriae on the ghost cells was examined by Western blot analysis. Forty BALB/c mice were equally divided into 2 groups of 20 mice each. Group A mice were intramuscularly vaccinated with a mixture of the 4 ghost cells expressing the individual fimbriae. The group B mice were inoculated with sterile phosphate-buffered saline as a control. The antigen-specific serum IgG concentrations were significantly higher in group A than in group B from week 2 until week 6 after inoculation. In addition, the antigen-specific IgA concentrations in fecal samples were significantly higher in group A than in group B at week 2 after inoculation. A large difference between the groups in the number of antigen-specific IgA-secreting cells in the small intestine was observed by immunohistochemical study. Also, the splenic lymphocyte proliferative responses were significantly greater in group A than in the control mice. These results suggest that vaccination with our Salmonella ghost cells can induce both humoral and cell-mediated immune responses and that the increased number of antigen-specific IgA-secreting cells in the small intestine may be correlated with the elevated fecal IgA immune response.  相似文献   

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