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1.
Pang Y  Wang H  Li Z  Piao J  Piao J  Chi Y  Jin J  Liu Q  Li W 《Avian diseases》2012,56(2):347-353
In order to generate Salmonella enterica serovar Enteritidis fimbriae antigens (rSEF21), the intact region encoding SEF21 was amplified from Salmonella Enteritidis by PCR and subcloned into a prokaryotic expression vector pET-28a(+) to yield pET-28a(+)-SEF21. The rSEF21 protein was highly expressed and purified by nickel affinity chromatography. Liposomeassociated rSEF21 was prepared for oral immunization to seek protective efficacy for intestinal infection with Salmonella Enteritidis. Evidence of IgA and IgG responses were found in the intestinal tracts and in the sera of a group of chickens immunized. Two weeks after the booster immunization, the chickens were challenged orally with 2 x 10(6) colony-forming units of live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 4 wk after challenge. The numbers of bacteria in the intestinal contents (cecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Therefore, oral immunization with liposome-associated rSEF21 elicits both systemic and mucosal antibody responses, leading to a reduction in bacterial colonization in the intestinal tract and excretion of Salmonella Enteritidis in the feces.  相似文献   

2.
Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.  相似文献   

3.
FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.  相似文献   

4.
Long-term feed withdrawal has been shown to increase ileocecal intestinal colonization and fecal shedding of Salmonella enterica serovar Enteritidis in challenged hens. Less information is available regarding effects of fasting on crop colonization. Two trials were conducted to compare effects of 14-day feed withdrawal vs. full feed on crop colonization in hens challenged with Salmonella Enteritidis. The levels of Salmonella Enteritidis in the crops of fasted hens were significantly higher than in nonfasted hens on days 3 and 10 and days 3, 9, and 16 postinfection (PI) in trials 1 and 2, respectively. Fecal shedding of Salmonella Enteritidis was significantly increased in the fasted hens on day 10 PI in trial 1. Analysis of crop IgA anti-Salmonella Enteritidis lipopolysaccharide levels in crop lavage samples of hens in trial 1 revealed a humoral response PI in both treatment groups with no significant differences, although peak response for fasted hens occurred 1 wk later. Histologic evaluation of hematoxylin and eosin-stained crop sections from trial 1 birds revealed mild to moderate heterophilic infiltration within the crop lamina propria (LP) or LP and epithelium of nonfasted infected hens at 24 and 96 hr PI. In comparison, heterophils in crops of fasted hens infected at this time point were sparse, indicating a possible diminished heterophil response in the fasted birds. Multifocal areas of tissue inflammation, as indicated by marked heterophil infiltration, with necrosis and sloughing of epithelium, were observed in crops from fasted hens at day 11 PI (14th day of feed withdrawal) but not in the fed groups. This severe heterophilic inflammation was observed in both challenged and nonchallenged fasted hens, suggesting that some factor other than Salmonella Enteritidis was responsible. These results indicate that feed withdrawal can have a dramatic effect on the integrity of the crop and its ultimate response to infection.  相似文献   

5.
Distribution of maternally transmitted Salmonella antibodies and their protective effects were studied in the progeny of broiler breeder birds which had been vaccinated with live S. Typhimurium and inactivated S. Enteritidis vaccines. Vaccination resulted in a significant increase of the antibody concentration in yolk of hatching eggs and in serum and jejunum of the progeny of immunized breeder birds. Higher antibody titres for isotypes IgG and IgA were still seen on day 21 of age. Antibody production of isotypes IgA and IgM by the chickens themselves was found between 14 and 21 days of age. Two challenge models (10(2) cfu/bird on day 1 of age and a seeder bird model, respectively) were used to evaluate the efficacy of maternal antibodies against challenge with S. Enteritidis. Using both models numbers of challenge organisms were lower in the caeca of the progeny of immunized parent birds between day 7 and day 21 of age (maximum about 1.5 log10 units) compared with control chicks. The results indicate the efficacy of maternally transferred antibodies but it remains the question of their practical relevance. The effects of acquired maternal antibodies on an active immunization of the progeny of immunized breeder birds with live Salmonella vaccines are discussed.  相似文献   

6.
This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 108 colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 106 CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine’s value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars.  相似文献   

7.
8.
A series of experiments was undertaken to investigate the infection dynamics of various doses of S. typhimurium in day-old and 14-day-old broiler chickens kept in isolators. The infections were followed quantitatively in ceca and ileum by enumerating the colony forming units (cfu) of the challenge strain. It was found that the inoculation of 10(7) cfu of S. typhimurium to day-old chickens established stable cecal infection in all the animals for 35 days. For 14-day-old chickens, stable and lasting infections were seen with inoculation of 10(9) cfu. Lower doses yielded more variable results, and the bacteria were rapidly eliminated from most birds, especially in 14-day-old inoculated chickens. Salmonella was found in spleen and liver 2-3 days postinoculation. Salmonella was cleared from both organs or reduced to very low numbers within 3 weeks.  相似文献   

9.
The adjuvant effect of chicken interferon-gamma (ChIFN-gamma) was examined for protecting chickens against intestinal colonization of Salmonella Enteritidis (SE) following oral exposure. Ten 7-week-old chickens per group were immunized with inactivated SE twice with or without co-administration of ChIFN-gamma intramuscularly, and all chickens were challenged with SE. Sera collected from immunized groups with or without ChIFN-gamma, and from unimmunized group were measured for SE antibody by agglutination test. The levels of antibodies were raised by 1 week post-immunization and did not show any difference between groups with and without ChIFN-gamma. No antibodies were detected in unimmunized group before challenge. Fecal samples from each group were cultured at 1, 4, 7, and 13 days post-challenge to determine the incidence of intestinal colonization and the numbers of SE shed into the environment. Co-administration of ChIFN-gamma, significantly reduced the incidence of intestinal colonization (P<0.05). At 13 days post-challenge, the bacterial counts of SE in organs were also reduced in ChIFN-gamma administered group. These data suggest co-administration of ChIFN-gamma with SE antigen enhances protection against SE challenge without acceleration of antibody production.  相似文献   

10.
Eighteen chickens were immunized subcutaneously with purified type 1 fimbriae from Salmonella enterica serotype Enteritidis at 18 and 21 weeks of age. Evidence of IgG and IgA responses was found in the eggs and in the sera of the immunized hens. Three weeks later, immunized and non-immunized chickens (n=18) were challenged intravenously with 2x10(7) live Salmonella enterica serotype Enteritidis. There was no significant difference in the numbers of eggs laid by immunized and non-immunized birds. The percentage of Salmonella contaminated eggs was significantly higher in the non-immunized group than in the immunized group due to a higher percentage of contamination of the externally disinfected egg shells. There were no statistical differences in the percentages of contaminated yolks and egg whites between control and immunized birds. No differences in the number of colonizing bacteria could be found in the spleen nor in the liver between the immunized and the control groups throughout the experiment. Salmonella was cleared from the ovary of the immunized birds in the second week p.i., in contrast to the control birds where Salmonella was isolated till the third week after infection. Oviducts were significantly more infected in the control group than in the immunized group. Salmonella was cleared from the oviducts at 3 weeks p.i. in the immunized hens but not in the control hens. In conclusion, we demonstrated that the immunization of laying hens with type 1 fimbriae reduced the number of contaminated eggs and reduced the colonization of the reproductive organs.  相似文献   

11.
Four-day-old chickens infected orally with a spectinomycin-resistant (Spcr) mutant of a highly invasive avian Salmonella typhimurium strain excreted salmonellae in the feces for at least 10 weeks. When these chickens were reinfected at this time with a nalidixic acid-resistant (Nalr) mutant of the same strain, they excreted this mutant in significantly smaller numbers (P less than 0.01) than did a previously uninfected control group. The Nalr mutant had a shorter survival rate in the tissues of the immunized chickens than in tissues of the control birds. The Spcr mutant stimulated strong IgG, IgA, and IgM responses in serum, small-intestinal contents, and bile. These were detected by enzyme-linked immunosorbent assay (ELISA) against antigens of crude whole bacterial cell protein sonicate, lipopolysaccharide, flagella, and outer-membrane proteins. There was some evidence of an anamnestic response with IgA in bile following reinfection with the Salmonella. The peak response of antibody-producing cells from the spleens of infected chickens, assayed by solid-phase ELISA, occurred at 3 weeks postinoculation. A strong delayed hypersensitivity reaction, detected by foot-pad swelling after inoculation with either whole-cell or outer-membrane proteins, was observed between 2 and 5 weeks after infection with the Spcr mutant. The data indicate that outer-membrane proteins are major immunogens for both humoral and cell-mediated arms of the immune system.  相似文献   

12.
Administration of live Salmonella strains to day-old chicks provides profound protection against superinfection with a related strain within a matter of hours by a colonisation-inhibition mechanism, which is primarily a bacterial physiological process. Although currently available, commercial, live attenuated Salmonella vaccines induce protection by adaptive immunity, none of them is able to induce protection against Salmonella organisms by colonisation-inhibition and, therefore, they are unable to protect newly-hatched birds immediately after oral vaccination. In this study, mutants of Salmonella Typhimurium and Enteritidis with deletions in phoP and rpoS, either alone or in combination with ompC, were characterised and tested for their level of attenuation and their ability to inhibit the intestinal colonisation of the isogenic parent strains in chickens. Mutants with deletions only in rpoS demonstrated an unaffected potential to inhibit the intestinal colonisation of the challenge strain but were still fully virulent for the chickens. Mutants with deletions in phoP, either alone or in combination with rpoS, resulted in a high level of attenuation, unimpaired ability to colonise the gut and a nearly unaffected potential to inhibit the challenge strain from caecal colonisation. Mutants with an additional deletion in ompC revealed a reduced capacity of intestinal colonisation-inhibition when compared to the control strains and both the single rpoS and the phoP deletion mutants. Mutations in phoP- or phoP-regulated genes may therefore be used for the development of live attenuated Salmonella vaccines possessing these novel characteristics.  相似文献   

13.
Chickens were immunized orally with 10(9)cfu of the temperature-sensitive (T(s)) mutant E/1/3 of Salmonella enteritidis at 1, 2, 3 and 7 days of age. The animals were challenged with wild-type strains of Salmonella of different serotypes 7 or 14 days following immunization. Chickens receiving multiple oral doses of the vaccine strain showed no signs of disease. Immunized animals shed the vaccine strain for at least 2 weeks after the last inoculation; on the other hand, colonization by the attenuated mutant of internal organs such as spleen and liver was limited. Early exposure of the immunized animals to the virulent bacteria resulted in a reduced cecal colonization by the pathogen. Visceral invasion by the wild-type strain of S. enteritidis or S. gallinarum was drastically diminished in birds challenged 14 days after immunization. Significant differences in the number of these Salmonella were found in the cecal contents, spleen and liver of immunized birds compared with the control animals. In addition, cecal colonization by the virulent strain was reduced in birds challenged with S. typhimurium. These results demonstrate that immunization of newly hatched chickens with live attenuated T(s) mutant E/1/3 of S. enteritidis is safe and reduces Salmonella shedding.  相似文献   

14.
肠炎沙门氏菌SEFA基因表达和间接ELISA检测方法的初步建立   总被引:2,自引:0,他引:2  
为了给临床检测肠炎沙门氏菌的感染和血清学调查提供一种切实可行的方法,本研究根据肠炎沙门氏菌SEF14菌毛操纵子亚单位sefA基因序列设计一对引物,利用PCR技术从国内标准株CMCC(B)50336中扩增sefA基因,并按预定的阅读框插入表达载体pET22b+中,获得重组质粒pET-sefA,限制性内切酶结合琼脂糖凝胶电泳分析和序列测定结果表明,该序列大小为498bp,与已发表的sefA结构编码序列完全一致。重组质粒pETsefA转化大肠杆菌E.coli BL21(DE3)并能获得高效诱导表达,通过对菌体裂解上清液SDS-PAGE和western blot分析鉴定,该重组菌可以表达大小为15.2ku的可溶性重组蛋白rSEFA。纯化的rSEFA免疫小鼠所得高免血清以及标准株CMCC(B)50336感染小鼠后所获阳性血清,均能识别标准株肠炎沙门氏菌SEF14菌毛蛋白以及纯化的重组蛋白rSEFA,结果表明明体外表达的rSEFA蛋白有较好的免疫原性和反应原性。而基于rSEFA介导的间接ELISA有较好的特异性,对肠炎沙门氏菌特异性抗体检测有潜在的应用前景。  相似文献   

15.
Salmonella enterica subspecies enterica infection remains a serious problem in a wide range of animals and in man. Poultry-derived food is the main source of human infection with the non-host-adapted serovars while fowl typhoid and pullorum disease are important diseases of poultry. We have assessed cecal colonization and immune responses of newly hatched and older chickens to Salmonella serotypes Enteritidis, Infantis, Gallinarum and Pullorum. S. Enteritidis and S. Infantis colonized the ceca more efficiently than S. Gallinarum and S. Pullorum. Salmonella infection was also associated with increased staining for B-lymphocytes and macrophages in the cecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds stimulated the expression of CXCLi1 and CXCLi2 chemokines in the cecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the cecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes.  相似文献   

16.
We have recently reported that Salmonella enterica serovar Enteritidis (S. Enteritidis) strains circulating in Uruguay, are unevenly distributed among different genetic subtypes, with a predominant genotype that is a common contaminant of poultry-derived food and that accounts for the vast majority of human cases of food-borne disease. Herein, we describe the construction of a genetically-defined aroC derivative (LVR02) of a local strain of S. Enteritidis belonging to the major genetic type. We demonstrated the attenuation and the immunogenicity of that strain in a mouse model, and evaluated it as a vaccine for commercial layer chickens. LVR02 proved to be stable, attenuated, innocuous, immunogenic and to induce protective immunity against a S. Enteritidis challenge when used for oral vaccination. A single oral dose of LVR02 administered to newly hatched chickens induced protection against oral challenge with the parental virulent strain, preventing systemic and persistent intestinal infection and significantly reducing the shedding of the challenge strain in birds' feces. A second vaccine dose at 15 days post-hatching boosted the immunogenicity of the vaccine, and strengthened the protection achieved with a single dose. This strain may represent the basis of a live vaccine to be included in national control programs to reduce circulation of this pathogen in the country.  相似文献   

17.
The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.  相似文献   

18.
根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计1对引物,通过RT-PCR扩增出鹅源NDV分离株JS5F基因(约1700bp),测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1-F。pVAX1-F经脂质体转染COS-7细胞,间接免疫荧光试验检测出F基因在COS-7细胞中的表达产物。将pVAX1-F转化减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pVAX1-F)。重组菌以109CFU/只的剂量2次免疫BALB/c小鼠,免疫小鼠可以检测到特异性针对NDVF蛋白的血清抗体和小肠粘膜抗体应答,SL7207(pVAX1-F)免疫组抗体水平显著高于SL7207(pVAX1)组(P<0.05)。将SL7207(pVAX1-F)以109CFU/只剂量口服免疫1日龄雏鸡,免疫保护试验结果显示,SL7207(pVAX1-F)免疫组对鸡具有良好的保护率(77.27%),与空白对照组和SL7207(pVAX1)空载体组之间存在显著性差异(P<0.05)。结果表明,该运送DNA疫苗的减毒沙门氏菌系统在体内能成功释放所携带的质粒,并能刺激机体产生免疫应答,可对NDV强毒攻击提供良好的免疫保护作用,提示该疫苗候选株对新城疫的控制有重要应用前景。  相似文献   

19.
The purpose of this work was to develop a live, attenuated vaccine strain to protect chickens against colonization by group C Salmonella. We constructed two candidate vaccines: a deltacya deltacrp derivative and a deltaphoP derivative of Salmonella hadar. White Leghorn chickens were vaccinated at day of age and at 2 wk with one of the two strains. A nonvaccinated group served as a control. At 4 wk of age, all birds were challenged with wild-type S. hadar and necropsied 6 days later. Numbers of S. hadar in the ceca were determined. Enzyme-linked immunosorbent assay-derived serum immunoglobulin G responses against S. hadar lipopolysaccharide indicated that both strains induced a serum antibody response. The average optical density450 for birds vaccinated with the deltaphoP or deltacya deltacrp derivatives was 0.456 and 0.881, respectively. Although the deltacya deltacrp derivative induced higher levels of serum antibody, it did not provide an immune response protective against colonization by S. hadar. Conversely, birds vaccinated with the deltaphoP strain showed significant protection against S. hadar challenge. Seventy percent of the nonvaccinates, 60% of the deltacya deltacrp vaccinates, and 15% of deltaphoP vaccinates were positive for S. hadar in tissues. In a second experiment, birds were vaccinated with either the deltaphoP strain or buffer and challenged with a 10-fold higher dose than in the first experiment. After challenge, all of the birds in both groups were colonized. The geometric mean number of cecal S. hadar isolated from the control group was 1.0 x 10(6) colony-forming units (CFU)/g, and from the vaccinated group, this value was 32 CFU/g, indicating a four to five log reduction in colonization by the challenge strain.  相似文献   

20.
Probiotics are currently employed for control of pathogens and enhancement of immune response in chickens. In this study, we investigated the underlying immunological mechanisms of the action of probiotics against colonization of the chicken intestine by Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella serovar Typhimurium). Birds received probiotics by oral gavage on day 1 of age and, subsequently, received Salmonella serovar Typhimurium on day 2 of age. Cecal tonsils were removed on days 1, 3 and 5 post-infection (p.i.), RNA was extracted and subjected to real-time quantitative RT-PCR for measurement of interleukin (IL)-6, IL-10, IL-12 and interferon (IFN)-gamma gene expression. There was no significant difference in IL-6 and IL-10 gene expression in cecal tonsils of chickens belonging to various treatment groups. Salmonella serovar Typhimurium infection resulted in a significant increase in IL-12 expression in cecal tonsils on days 1 and 5p.i. However, when chickens were treated with probiotics prior to experimental infection with Salmonella, the level of IL-12 expression was similar to that observed in uninfected control chickens. Treatment of birds with probiotics resulted in a significant decrease in IFN-gamma gene expression in cecal tonsils of chickens infected with Salmonella compared to the Salmonella-infected birds not treated with probiotics. These findings reveal that repression of IL-12 and IFN-gamma expression is associated with probiotic-mediated reduction in intestinal colonization with Salmonella serovar Typhimurium.  相似文献   

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