Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.     

The effect of exposure to a delta cya/delta crp mutant of Salmonella typhimurium on the subsequent colonization of swine by the wild-type parent strain.

This study characterizes the clinical response and colonization pattern of caesarean-derived, colostrum-deprived swine exposed to a delta cya/delta crp mutant (chi 4233) of S. typhimurium and challenged with the wild-type parent strain. chi 4233 was mildly virulent in swine and induced transient fever and soft stools. Chi 4233 colonized the ileum, cecum, liver, spleen, tonsils, and mandibular and ileocolic lymph nodes of swine in a manner similar to the parental wild-type, but the numbers of S. typhimurium (chi 4233) in the ileum were 100- to 1000-fold less than those of pigs exposed to the parental wild-type. Pigs exposed to chi 4233 21 days before parental wild-type challenge demonstrated a milder clinical response to challenge than did pigs that did not receive chi 4233. The wild-type populations in the ilea of chi 4233-exposed pigs after challenge were 100- to 10,000-fold less than those in pigs not receiving chi 4233. The liver, spleen, and ileocolic lymph nodes were cleared of wild-type S. typhimurium more quickly after challenge in chi 4233-exposed pigs. The populations of chi 4233 in the ilea of exposed pigs after wild-type challenge were also less than would have been expected in unchallenged pigs. Thus, exposure of swine to a delta cya/delta crp mutant of S. typhimurium modulated the subsequent response to parental wild-type challenge and reduced carrier populations of wild-type S. typhimurium in infected swine.     

Increased tracheal colonization in chickens without impairing pathogenic properties of avian pathogenic Escherichia coli MT78 with a fimH deletion

Several studies suggest that the expression of F1 fimbriae could be involved in the virulence of Escherichia coli for chickens. F1 fimbriae display multivalent properties such as adhesion to epithelia or interaction with the immune system that imply specific interactions between the adhesin FimH and different cell receptors. We constructed a delta fimH mutant of the avian pathogenic E. coli MT78 and evaluated its in vivo colonization and pathogenicity, as compared to that of the parent strain. The generated mutant PA68 was unable to adhere in vitro to chicken epithelial pharyngeal or tracheal cells; mutant bacteria were mostly afimbriated although a minority of them displayed altered piliation phenotypes. Two inoculation routes were used to compare the ability of MT78 and PA68 to colonize the respiratory tract and to induce colibacillosis in chickens. In the first model, 2-wk-old axenic chickens were inoculated intratracheally with one or both E. coli strains, after primary infection with infectious bronchitis virus. In the second model, 3-wk-old specific-pathogen-free chickens were inoculated via the caudal thoracic air sac. After intratracheal inoculation, the delta fimH mutant was found to be a better colonizer than MT78 in the trachea of inoculated chickens. Furthermore, when both strains were inoculated simultaneously, the delta fimH mutant constituted 98% of the bacterial population in the trachea at day 7 postinoculation. Irrespective to the inoculation route, MT78 and PA68 showed similar abilities to induce macroscopic lesions in chickens, to provoke bacteremia, and to colonize the internal organs. However, 4 days after intra-air sac inoculation, bacterial counts of the mutant were lower in the spleen and liver than those of MT78. Our results show that FimH is not required for colonization of the trachea of axenic chickens by E. coli and that it is not a major determinant of bacterial pathogenicity. On the contrary, the lack of expression of FimH seems to favor the in vivo colonization of the trachea of chickens by E. coli.     

In vitro adhesion of an avian pathogenic Escherichia coli O78 strain to surfaces of the chicken intestinal tract and to ileal mucus

The role of fimbria in adherence of an avian pathogenic Escherichia coli (APEC) O78 strain 789 to chicken intestine was studied. Bacterial adhesion to tissue sections representing the regions within the chicken intestinal tract was determined by using immunohistochemical methods. E. coli 789 grown to express the type 1 fimbria adhered efficiently to the crop epithelium, to the lamina propria of intestinal villi, and to the apical surfaces of both the mature as well as the crypt-located enterocytes in intestinal villi, whereas no adhesion to mucus-producing goblet cells was detected. The adhesion was inhibited by mannoside and the role of type 1 fimbriae in the observed adhesion was confirmed with a recombinant strain expressing type 1 fimbriae genes cloned from E. coli and Salmonella enterica. E. coli 789 strain grown to favor AC/I fimbriae expression as well as the recombinant E. coli strain expressing the fac genes adhered to goblet cells but only poorly to the other epithelial sites. E. coli strain 789 as well as S. enterica serovar Typhimurium IR715 and S. enterica serovar Enteriditis TN2 strains were able to multiply in ileal mucus medium. The type 1 fimbria expressing bacteria adhered to the ileal mucus, whereas the AC/I fimbriated strains showed poor adherence to the mucus. The adhesion of E. coli 789 onto the crop epithelium and the follicle associated epithelium of the chicken ileum was efficiently inhibited by an adhesive strain ST1 of Lactobacillus crispatus isolated from chicken, whereas poor inhibition of E. coli adherence was observed with the weakly adhesive L. crispatus strain 134mi. The type 1 fimbriae may be important in colonization of the chicken intestine by APEC and Salmonella.     

Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli.

An avirulent, wild-type avian Escherichia coli (E. coli Av) was electrotransformed with a plasmid coding for the production of microcin 24 (pGOB18) and was designated E. coli AvGOB18. The transformant inhibited the growth of seven serotypes of Salmonella commonly associated with colonization and contamination of poultry products and seven strains of E. coli O157:H7 in the in vitro colicin/microcin assay. The transformant did not inhibit the replication of multiple isolates of Listeria monocytogenes or Campylobacter jejuni in similar assays. The transformant is nonconjugative, indicating that the plasmid would not be transmitted to other intestinal microflora in the environment. The transformant also survived in sterile tap and deionized water incubated at 25 C and 37 C in the laboratory for 30 days and was recovered from drinkers and birds in in vivo floor pen studies. In in vivo studies, E. coli AvGOB18 did not colonize the intestinal tract of broiler chicks when given as a single or multiple dose and did not reduce the Salmonella load in the broilers. But Salmonella typhimurium was reduced significantly in the intestinal tracts of broiler chickens when E. coli AvGOB18 was administered continually in the water supply.     

Attenuation of an avian pathogenic Escherichia coli strain due to a mutation in the rpsL gene

The diseases caused by pathogenic Escherichia coli constitute a major economic loss to the poultry industry. The development of a live oral E. coli vaccine to prevent or reduce diseases in poultry had been the objective of our work. Four spontaneous streptomycin-dependent (str-dependent) mutants were generated from a virulent avian strain that contains a mutation in the fur region of the chromosome. Genetic analysis of the mutants indicated that the str-dependent phenotype was due to a base change of C --> T at base 272 in the rpsL gene. The mutants were tested for attenuation using the day-old chick model. Day-old birds, in groups of 20, were either challenged with 10(6) colony-forming units (CFU) of the str-dependent mutant, the parent strain (containing the fur mutation), or the wild-type strain without the fur mutation. The parent strain and the wild-type strain were highly virulent, and 80% or more of the birds died. None of the birds challenged with the str-dependent mutants died, indicating attenuation of the mutants. The protective effect of the mutant as a live vaccine against the challenge with 10(6) CFU of the wild-type strain EC317 was investigated. Vaccination by both aerosol (day 1) and oral (days 14 and 28) routes using 10(8) CFU of the str-dependent mutant (EC1598) had no effect on the occurrence of cellulitis in the birds. Two vaccinations given as aerosol on day 1 and given orally on day 14 also had no significant effect on the occurrence of systemic lesions. Three immunizations on days 1, 14, and 28 resulted in a significant reduction in the number of birds with systemic lesions. Antibody titers prior to challenge were not predictive of outcome of challenge.     

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.     

Isolation and characterization of type 1 fimbriae from a chicken pathogenic Escherichia coli serotype O78.

Type 1 fimbriae from chicken pathogenic Escherichia coli strain PDI-386 (serotype O78) was purified and characterized. Because of the acid-induced autoagglutination (T. Sekizaki, Y. Nakasato, and I. Nonomura, J. Vet. Med. Sci. 54, 493-499, 1992), the fimbriae could be easily purified by repeating acid sedimentation, washing, and dissolving in buffer (pH 8.0). In electron microscopy, the purified fimbriae showed a filament of 8 nm in diameter and 10 microns in average length. The molecular mass of the protein subunit of the purified fimbriae estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 19,000 daltons. The amino acid composition and its NH2-terminal sequence were similar to the previously described one of the Klebsiella pneumoniae type 1 fimbriae. Moreover, there was an immunological relatedness between them. These results indicated that a molecular diversity found between the fimbriae of E. coli and that of K. pneumoniae has already been existed among chicken pathogenic E. coli strains.     

Population of Salmonella serovar typhimurium in the cecum of gnotobiotic chickens with Escherichia coli and intestinal bacteria.

To test the interaction between various species of bacteria and Salmonella serovar typhimurium (S. typhimurium), the population of S. typhimurium was measured in the cecum of gnotobiotic chickens in the presence of Escherichia coli (E. coli) and one of the four intestinal bacteria; Lactobacillus acidophilus. Clostridium perfringens, Bifidobacterium thermophilum and Bacteroides vulgatus. Competitive exclusion of S. typhimurium by di-flora chicken was not demonstrated. But the population of S. typhimurium was temporarily suppressed in di-flora chickens with E. coli and L. acidophilus. In penta-flora chickens with E. coli and these four intestinal bacteria, the population of S. typhimurium was suppressed for only 2 days. In normalized chickens, the population of S. typhimurium was markedly suppressed.     

Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.     

Escherichia coli ghosts or live E. coli expressing the ferri-siderophore receptors FepA, FhuE, IroN and IutA do not protect broiler chickens against avian pathogenic E. coli (APEC)

The aim of this study was to investigate if immunization with the ferri-siderophore receptors FepA, FhuE, IroN and IutA could protect chickens against avian pathogenic Escherichia coli (APEC) infection. The antigens were administered as recombinant proteins in the outer membrane (OM) of E. coli strain BL21 Star DE3. In a first immunization experiment, live E. coli expressing all 4 recombinant ferri-siderophore receptors (BL21(L)) were given intranasally. In a second immunization experiment, a mixture of E. coli ghosts containing recombinant FepA and IutA and ghosts containing recombinant FhuE and IroN was evaluated. For both experiments non-recombinant counterparts of the tentative vaccines were administered as placebo. At the time of challenge, the IgG antibody response for BL21(L) and a mixture of E. coli ghosts containing recombinant FepA and IutA and ghosts containing recombinant FhuE and IroN was significantly higher in all immunized groups as compared to the negative control groups (LB or PBS) confirming successful immunization. Although neither of the tentative vaccines could prevent lesions and mortality upon APEC infection, immunization with bacterial ghosts resulted in a decrease in mortality from 50% (PBS) to 31% (non-recombinant ghosts) or 20% (recombinant ghosts) and these differences were not found to be significant.     

Weaning of piglets. Effects of an exposure to a pathogenic strain of Escherichia coli

The influence of weaning on day 32 and a simultaneous challenge with a pathogenic strain of Escherichia coli was studied in eight piglets. Another nine weaned but non-infected piglets were used as controls. The distribution of peripheral blood mononuclear cells (PBMC) into subpopulations, as well as their response when stimulated in vitro by pokeweed mitogen, changed in a similar manner during post-weaning in both groups. In contrast, superior responses were recorded for PBMC collected from the challenged pigs when stimulated in vitro with concanavalin A and with a heat-inactivated extract of the E. coli strain used for infection, respectively. Despite a successful colonization of the challenge strain, no clinical signs of disease were recorded. Nor did the daily weight gain or the number of E. coli, enterococci, or Clostridium perfringens excreted per gram of faeces differ between the groups. However, the weaning induced a marked decrease in the diversity of coliforms in individual piglets, which announced a reduced colonization resistance of that flora. Also, a decreased homogeneity between coliform floras of different piglets was observed following weaning. The decreased homogeneity indicated that different strains of E. coli were predominant in different animals, which may in turn facilitate the spread of pathogenic strains. The enteric changes were more pronounced and lasted longer in infected animals. Still, the influence of a sole pathogenic strain of E. coli was not enough to induce post-weaning diarrhoea.     

鸡粪中沙门氏菌和大肠杆菌O78多重PCR检测方法的建立

为建立同时快速检测沙门氏菌和大肠杆菌O78的方法,本研究根据沙门氏菌inv A基因和大肠杆菌O78 O-抗原特异基因序列设计引物,并在细菌16S r DNA区设计内参引物,通过各项参数调整优化,建立了能够直接从样品中同时检测沙门氏菌和大肠杆菌O78的多重PCR检测方法。结果显示:该方法可以同时扩增出O781 113 bp和沙门氏菌821 bp的特异片段以及细菌16S r DNA 527 bp的通用片段,而对于其他13种肠道致病菌只能扩增出527 bp的细菌同源片段,具有较强的特异性。该体系检测沙门氏菌和大肠杆菌O78的最低检出限分别为10~3cfu/m L和10~2 cfu/m L。增菌5 h,鸡粪模拟污染菌样品中沙门氏菌和大肠杆菌O78的检测灵敏度分别为10~3 cfu/m L和10~2 cfu/m L。本研究建立的检测方法能够在8 h内完成鸡粪样品中两种靶标菌的快速检测,对于沙门氏菌和大肠杆菌O78的鉴别诊断和快速检测具有重大意义。     

牦牛致病性大肠杆菌多价蜂胶灭活苗的研制

牦牛大肠杆菌病是严重影响牦牛生产的细菌性传染病,该病既是一种条件性疾病,又是其他诸多疾病的致病因素,因而是继主要的病毒性传染病得到控制以后,引起牦牛生产重大经济损失的重要传染病。抗生素对该病的防治效果良好,但其易产生耐药性,且常呈多重耐药,同时对肠道的微生态具有不良影响,因此对牦牛大肠杆菌病疫苗的研制日益受到广泛的重视。     

R factor types found in Salmonella typhimurium and Escherichia coli isolated from calves in a confined environment.

Typing of R factors by genetic properties was done with Salmonella typhimurium and Escherichia coli isolated from calves on a feedlot where epizootics of clinical or subclinical calf salmonellosis had repeatedly occurred during 5 years. Forty-nine R factors from S typhimurium were fi- (no fertility inhibition) and spp- (no restriction against phage lambda vir). Twenty-three (46.9%) of them belonged to compatibility group Ialpha and the remainder were nontypable. Fourteen R factors from E coli belonged to different genetic types: fi+ (11=78.6%) and fi- (3=21.4%); spp+ (1=7.1%) and spp- (13=92.9%); compatibility groups FII (5=35.7%), N (1=7.1%), and nontypable (8=57.2%). In contrast to the R factors of S typhimurium, 9 (64.3%) of the 14 R factors of E coli carried resistance against aminobenzyl penicillin with or without kanamycin resistance. The compatibility groups of R factors of S typhimurium seemed to be useful as a subsidiary epizootiologic marker in this feedlot.     

Feedstuffs as a vehicle of cattle exposure to Escherichia coli O157:H7 and Salmonella enterica

Feed has been reported as a vehicle for transmission of Salmonella enterica in cattle and several lines of evidence suggest that feed can be a vehicle for transmitting Escherichia coli O157:H7 as well. To show whether microbial contamination of feeds could contribute to the populations of S. enterica and E. coli O157:H7 on a farm, we compared isolates from feed samples to bovine fecal isolates from the same farm using pulsed-field gel electrophoresis (PFGE). Four of 2365 component feed samples (0.2%) and 1 of 226 feed mill samples (0.4%) were positive for E. coli O157:H7. Twenty of 2405 (0.8%) component feed samples and none of 226 feed mill samples were positive for Salmonella. PFGE profiles from E. coli O157:H7 isolated from a component feed sample closely resembled that from a fecal isolate collected later from the same farm, and a similar observation was made of a Salmonella Tyhpimurium isolate from component feed on another farm. There were indistinguishable PFGE profiles from component feed Salmonella Tyhpimurium DT104 isolates and fecal isolates from the same farm. These results provide evidence for a role of cattle feed in transmission of E. coli O157:H7; S. enterica; cattle-bacteria.     

Generation of Salmonella ghost cells expressing fimbrial antigens of enterotoxigenic Escherichia coli and evaluation of their antigenicity in a murine model

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.     

Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens

In order to develop a novel, safe and immunogenic fowl typhoid (FT) vaccine candidate, a Salmonella Gallinarum ghost with controlled expression of the bacteriophage PhiX174 lysis gene E was constructed using pMMP99 plasmid in this study. The formation of the Salmonella Gallinarum ghost with tunnel formation and loss of cytoplasmic contents was observed by scanning electron microscopy and transmission electron microscopy. No viable cells were detectable 24 h after the induction of gene E expression by an increase in temperature from 37 °C to 42 °C. The safety and protective efficacy of the Salmonella Gallinarum ghost vaccine was tested in chickens that were divided into four groups: group A (non-immunized control), group B (orally immunized), group C (subcutaneously immunized) and group D (intramuscularly immunized). The birds were immunized at day 7 of age. None of the immunized animals showed any adverse reactions such as abnormal behavior, mortality, or signs of FT such as anorexia, depression, or diarrhea. These birds were subsequently challenged with a virulent Salmonella Gallinarum strain at 3 weeks post-immunization (wpi). Significant protection against the virulent challenge was observed in all immunized groups based on mortality and post-mortem lesions compared to the non-immunized control group. In addition, immunization with the Salmonella Gallinarum ghosts induced significantly high systemic IgG response in all immunized groups. Among the groups, orally-vaccinated group B showed significantly higher levels of secreted IgA. A potent antigen-specific lymphocyte activation response along with significantly increased percentages of CD4⁺ and CD8⁺ T lymphocytes found in all immunized groups clearly indicate the induction of cellular immune responses. Overall, these findings suggest that the newly constructed Salmonella Gallinarum ghost appears to be a safe, highly immunogenic, and efficient non-living bacterial vaccine candidate that protects against FT.     

Development of a novel live vaccine delivering enterotoxigenic Escherichia coli fimbrial antigens to prevent post-weaning diarrhea in piglets

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.     

Oral immunisation of mice with a recombinant rabies virus vaccine incorporating the heat-labile enterotoxin B subunit of Escherichia coli in an attenuated Salmonella strain

To investigate effective new rabies vaccines, a fusion protein consisting of the rabies virus (RV) glycoprotein and the heat-labile enterotoxin B subunit of Escherichia coli (LTB) was successfully constructed and delivered in a live attenuated Salmonella strain LH430. Mice were immunised with LH430 carrying pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. The antibody titres of mice immunised with oral LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTB were significantly higher than those of pVAX1-G-immunised mice. The results of the challenge with the rabies virus standard strain (CVS-11) showed that the LH430 strain carrying the G-LTB gene induced immunity and elevated IL-2 levels in immunised mice (⁷P < 0.01), whereas LH430 carrying pVAX1-G did not contribute to protection. These results show that LH430 carrying recombinant G-LTB could provide overall immunity against challenge with CVS-11 and should be considered to be a potential rabies vaccine.