Effect of vaccination of hens with an avirulent strain of Salmonella typhimurium on immunity of progeny challenged with wild-Type Salmonella strains

The avirulent Salmonella typhimurium chi3985 was used to vaccinate white leghorn chickens at 16 and 18 weeks of age, and the effect of maternal antibody on Salmonella colonization of progeny of vaccinated hens was assessed with S. typhimurium F98 or chi3985. Progeny of hens that had been vaccinated at 1 and 3 or 2 and 4 weeks of age with chi3985 were used to determine the effect of maternal immunity on vaccine efficacy. Vaccination of hens induced long-lasting Salmonella-specific antibodies which were transferred into eggs and were detected as immunoglobulin G (IgG) in the egg yolk. Maternal antibody was detected in the progeny of vaccinated birds as IgG and IgA in serum and intestinal fluid, respectively. The titer of maternally transmitted IgG or IgA was highest in the first week of life of the progeny and declined with age. Maternal antibodies prevented colonization of the chicks by S. typhimurium chi3985 and reduced colonization by S. typhimurium F98. Overall, chicks from vaccinated hens had significantly higher antibody responses than did the progeny of nonvaccinated hens after oral infection with Salmonella strains. Maternal antibody reduced the efficacy of vaccination of progeny with chi3985 at 1 and 3 weeks of age. But vaccination at 2 and 4 weeks of age induced excellent protection against challenge with S. typhimurium F98 or S. enteritidis 27A PT 8 in birds from vaccinated hens and in specific-pathogen-free chickens. Vaccination of chickens at 2 and 4 weeks of age has been shown to protect the birds against challenge with homologous and heterologous Salmonella serotypes. A combination of vaccination of adult animals and use of the progeny of vaccinated birds will enhance effective control of Salmonella infections in the poultry industry. This will complement the present control of Salmonella-associated food poisoning caused by Salmonella enteritidis in eggs because the avirulent S. typhimurium vaccine strain chi3985 induced excellent protection against S. enteritidis in chickens.     

Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract

Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine. Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions. A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium. Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions. Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella. Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine.     

Comparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen

We compared the abilities of different Salmonella enterica var. Typhimurium (S. typhimurium) strains harboring mutations in the genes aroA, aroAD, purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system. Plasmid pTETtac4, encoding C fragment, was transferred into the various S. typhimurium mutants, and the levels of antigen expression were found to be equivalent. After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer's patches and spleens of mice. Of all strains compared, the delta purA mutant colonized and persisted in the Peyer's patches at the lowest level, whereas the delta htrA mutant colonized and persisted in the spleen at the lowest level. The level of specific antibody elicited by the different strains against either S. typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants' ability to colonize the spleen. The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S. typhimurium mutants. The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S. typhimurium delta purA. Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen. Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S. typhimurium mutants tested induced a Th1-type immune response. Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization. With the exception of the S. typhimurium delta purA mutant, all strains elicited a protective immune response. These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S. typhimurium strain.     

Expression of the serine rich Entamoeba histolytica protein (SREHP) in the avirulent vaccine strain Salmonella typhi TY2 chi 4297 (delta cya delta crp delta asd): safety and immunogenicity in mice

Infection by the intestinal protozoan parasite Entamoeba histolytica remains a significant threat to health in much of the world. Here we describe the successful expression of the serine rich Entamoeba histolytica protein (SREHP), a protective antigen of ameba, in an attenuated vaccine strain Salmonella typhi TY2 chi 4297 (delta cya delta crp delta asd). The attenuation of S. typhi TY2 chi 4297 was not altered by expression of the SREHP-maltose binding protein (MBP) fusion protein and mice parenterally vaccinated with S. typhi TY2 chi 4297 expressing SREHP-MBP developed serum anti-amebic and anti-LPS antibodies. S. typhi TY2 chi 4297 expressing SREHP-MBP represents a prototype combination vaccine designed to prevent both amebiasis and typhoid fever.     

Evaluation of bacterins containing three predominant phage types of Salmonella enteritidis for prevention of infection in egg-laying chickens

Six Salmonella enteritidis bacterin formulations differing in adjuvant content and whole-cell inactivation procedures were evaluated in egg-laying chickens. Chickens given S enteritidis bacterins containing modified Freund's incomplete adjuvant had greater humoral immune responses to S enteritidis than did birds given other bacterin formulations (P < 0.05). Better protection against infection by S enteritidis phage types 8, 13a, and 23 was obtained in birds vaccinated with bacterin 5. Bacterin 5 contained S enteritidis cells inactivated by 20% acetone and modified Freund's incomplete adjuvant.     

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.     

Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B

Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma. The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice. UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain. Evaluation of protection against H. pylori was performed in Balb/c mice by oral immunization with a single dose of the vaccine strain. Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H. pylori wild type strain and, six weeks later, mice were sacrificed to determine H. pylori infection by detection of urease activity from the antral region of the mouse stomachs. In several independent experiments, we observed 100% infection with H. pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S. typhimurium expressing recombinant UreA and UreB. Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays. These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H. pylori colonization. Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H. pylori infection. In addition, this model will aid to elucidate the effective protection mechanisms against H. pylori in the gastric mucosa.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

Metabolites produced during the peroxisomal beta-oxidation of linoleate and arachidonate move to microsomes for conversion back to linoleate

The present work compared the accumulation of intraperitoneal heterophils in day-of-hatch chicks following treatment with Salmonella enteritidis-immune lymphokine (ILK) and challenge with various strains of Salmonella enteritidis (SE). Day-of-hatch chicks received ILK by intraperitoneal injection and were challenged 1 hr later by intraperitoneal inoculation with one of the following SE strains: a wild-type, SE 890034-3; a delta cya-12 delta cyp-11 avirulent vaccine strain, chi 4357; and an invasion-deficient strain, InvA::kan, chi 4420. Four hours after challenge heterophils were recovered from the peritoneal cavity by lavage. The concentration of heterophils in the recovered lavage fluid was determined. Heterophil concentrations increased in response to challenge with each SE strain but there was a lower response to the invasion-deficient strain. The difference was statistically significant. This diminished heterophil response to challenge with invasion-deficient salmonellae supports existing evidence that the initial defensive reaction occurs at the earliest stages of the Salmonella-host interaction.     

Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Multiple fimbrial adhesins are required for full virulence of Salmonella typhimurium in mice

Adhesion is an important initial step during bacterial colonization of the intestinal mucosa. However, mutations in the Salmonella typhimurium fimbrial operons lpf, pef, or fim only moderately alter mouse virulence. The respective adhesins may thus play only a minor role during infection or S. typhimurium may encode alternative virulence factors that can functionally compensate for their loss. To address this question, we constructed mutations in all four known fimbrial operons of S. typhimurium: fim, lpf, pef, and agf. A mutation in the agfB gene resulted in a threefold increase in the oral 50% lethal dose (LD50) of S. typhimurium for mice. In contrast, an S. typhimurium strain carrying mutations in all four fimbrial operons (quadruple mutant) had a 26-fold increased oral LD50. The quadruple mutant, but not the agfB mutant, was recovered in reduced numbers from murine fecal pellets, suggesting that a reduced ability to colonize the intestinal lumen contributed to its attenuation. These data are evidence for a synergistic action of fimbrial operons during colonization of the mouse intestine and the development of murine typhoid fever.     

Synergism of lactate and succinate as metabolites utilized by Veillonella to inhibit the growth of Salmonella typhimurium and Salmonella enteritidis in vitro

The inhibition of salmonellae growth by a Veillonella bacterium isolated from the cecal contents of adult chickens was examined. The Veillonella isolate was grown on an agar medium supplemented with 175 mumol of lactate or succinate/ml. Either 0, 100, 125, 150, or 175 mumol of succinate/ml was added to the lactate medium; either 0, 100, 125, 150, or 175 mumol of lactate/ml was added to the succinate medium; and the pH of all media was adjusted to 6.0. Agar overlays of Veillonella cultures grown on the media were inoculated with Salmonella typhimurium or S. enteritidis. The largest zones of inhibition of salmonellae growth were produced by Veillonella cultures grown on medium supplemented with 175 mumol/ml of both lactate and succinate. The widths of the zones of inhibition decreased as the concentration of lactate was reduced in the succinate medium and as the concentration of succinate was reduced in the lactate medium. Analyses of lactate broth and succinate broth inoculated with Veillonella indicated that inhibition of salmonellae growth on the agar media was related to the production of volatile fatty acids by Veillonella, the presence of residual succinate in the media, and the final pH of the media.     

Delivery of epitopes by the Salmonella type III secretion system for vaccine development

Avirulent strains of Salmonella typhimurium are being considered as antigen delivery vectors. During its intracellular stage in the host, S. typhimurium resides within a membrane-bound compartment and is not an efficient inducer of class I-restricted immune responses. Viral epitopes were successfully delivered to the host-cell cytosol by using the type III protein secretion system of S. typhimurium. This resulted in class I-restricted immune responses that protected vaccinated animals against lethal infection. This approach may allow the efficient use of S. typhimurium as an antigen delivery system to control infections by pathogens that require this type of immune response for protection.     

Resistance against Salmonella enteritidis cecal colonization in Leghorn chicks by vent lip application of cecal bacteria culture

Leghorn chicks were treated with cultures of cecal bacteria from adult chickens by crop gavage, upper body spray, or vent lip application on the day of hatch. The chicks were challenged orally with 10(4) Salmonella enteritidis (SE) at 3 d of age and evaluated for SE cecal colonization at 10 d of age. The concentration of volatile fatty acids (VFA) in the cecal contents was determined on the day after culture treatment and at 10 d of age. Compared with controls, SE colonization was significantly decreased in each of the treatment groups. Vent lip application of a single .05-mL drop of cecal bacteria culture resulted in resistance against SE challenge comparable to crop gavage or spray treatment with .5 mL of culture. Resistance to SE challenge was directly associated with the concentrations of total VFA and propionic acid in the cecal contents of the treated chicks on the day after culture treatment. The results indicated that cecal bacteria from adult chickens that increase SE colonization resistance may rapidly become established in the ceca of newly hatched chicks following contact with the vent lips.     

Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization

An avirulent, streptomycin-resistant Salmonella typhimurium strain, SL5319, and its lipopolysaccharide (LPS)-deficient mutant strain, SL5325, differ in their ability to colonize the large intestines of streptomycin-treated mice. When fed to mice independently, the strains colonize equally well, but when fed together, the LPS-deficient mutant is outcompeted by the wild-type strain during establishment in the gut (J.J. Nevola, B.A.D. Stocker, D.C. Laux, and P.S. Cohen, Infect. Immun. 50:152-159, 1985). In the present study, the spatial distribution in the intestinal mucosal layer of the two strains was visualized by specific hybridization to bacterial rRNA in histological sections of mouse colon and cecum. The first day after infection, 9.8% of the smooth SL5319 cells observed in mucus were found to be associated with the mouse epithelial cells, but three days after infection, the corresponding fraction of adhering bacteria was reduced to 2.1%. The LPS-deficient S. typhimurium strain was confined to the part of the mucosal layer closest to the colonic lumen and was not observed to adhere to the epithelium either at day 1 or 3 after infection. Quantitative determinations of the distance from the S. typhimurium cells to the epithelial wall confirmed that the average distance for the rough S. typhimurium SL5325 was much larger than for its smooth counterpart, S. typhimurium SL5319. Quantification of the hybridization signal from bacteria isolated from the cecal mucus revealed that the two strains had the same ribosome concentration, indicating that they have the same potential for growth in the intestinal environment. On the basis of these observations, we suggest that the better colonization ability of the strain carrying wild-type LPS is due to the better abilities to penetrate the intestinal mucosal layer and to subsequently bind to the epithelial cells in vivo.     

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.     

Induction of the phoE promoter upon invasion of Salmonella typhimurium into eukaryotic cells

Live attenuated Salmonella typhimurium strains expressing foreign antigens can be used for vaccination purposes. Due to deleterious effects of constitutive, high-level expression of the heterologous antigens, there is often strong selection pressure against plasmids encoding these antigens, resulting in rapid segregation in vivo. In vivo-inducible promoters may be a good alternative for constitutive promoters. The outer membrane protein PhoE of Escherichia coli is being used as a carrier for foreign antigenic determinants. Here we studied whether its expression from a plasmid is induced in S. typhimurium upon invasion of eukaryotic cells. This appeared to be the case. Furthermore, a S. typhimurium phoE mutant was constructed and the effects of the mutation on invasion, intracellular survival and virulence were studied. Survival in HEp-2 cells or in the macrophage-like cell line J744 was not, or only slightly, affected. Furthermore, the mutant appeared to be as virulent for mice as the wild-type strain.     

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.     

Yeast treatment to reduce Salmonella and Campylobacter populations associated with broiler chickens subjected to transport stress

The prevalence of Campylobacter and Salmonella on retail poultry carcasses remains a significant public health concern. The stresses associated with transporting poultry prior to slaughter have been shown to increase pathogen populations both in the intestinal tract and on the carcass exterior. The yeast, Saccharomyces boulardii, was evaluated for its ability to reduce populations of Salmonella and Campylobacter in broiler chickens subjected to transport stress. Chicks, inoculated with individual strains of Salmonella and Campylobacter were held for 6 wk and then divided into two groups with half of the chickens receiving 10% dried yeast in the feed for 60 h. The birds were then caged and transported to simulate commercial conditions. After euthanatizing the birds, the ceca were aseptically removed and analyzed for Salmonella and Campylobacter. With no yeast treatment, transport stress caused the Salmonella colonization frequency to increase more than fivefold, from 3.3 to 16.7%. Yeast treatment significantly reduced the frequency of Salmonella colonization to lower than prestress levels, as no Salmonella were recovered from the ceca of these birds (P < 0.05). Similar results were obtained from birds challenged with a mixture of Salmonella and Campylobacter strains. Before transport, 53.3% of these chickens were positive for Salmonella. Transport stress increased the colonization rate to 67.5% in control birds, whereas the colonization of yeast-treated chickens decreased to 40% (P < 0.05). Frequency of Campylobacter isolation from the ceca was not affected by treatment, but Campylobacter populations present in the ceca were significantly reduced in the mixed strain trial (P < 0.05).