Orally administered attenuated Salmonella enteritidis reduces chicken cecal carriage of virulent Salmonella challenge organisms

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.     

Evaluation of the efficacy of an oil-emulsion bacterin for protecting chickens against Salmonella enteritidis.

To assess the potential protective efficacy of a Salmonella enteritidis bacterin, an acetone-killed oil-emulsion vaccine was prepared from a phage type 13a S. enteritidis strain and administered subcutaneously to hens in two experiments. Hens were housed individually, and every other hen was vaccinated (at 23 weeks of age in one experiment and at 45 weeks in the other). A second (booster) bacterin injection was administered 6 weeks later in both experiments. Three weeks after the second vaccination, all hens were challenged with an oral dose of approximately 10(9) cells of a heterologous (phage type 14b) S. enteritidis strain. In both trials, S. enteritidis was isolated from fewer internal organs (spleens, ovaries, and oviducts) and pools of egg contents from vaccinated hens than from unvaccinated control hens. Vaccination did not, however, affect the percentage of hens that shed S. enteritidis in feces in either experiment.     

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.     

Virulence of Salmonella enteritidis phagetypes 4, 8 and 13 and other Salmonella spp. for day-old chicks, hens and mice.

Virulence of three Canadian poultry strains of Salmonella enteritidis, namely phagetypes (PT) 4, 8 and 13, and one Salmonella heidelberg strain was assessed in orally and intraperitoneally inoculated one-day old chickens and compared to the virulence of a human S. enteritidis PT 4 strain from the United Kingdom (UK). The two PT 4 strains were also compared in orally inoculated adult laying hens. In addition, orally inoculated Balb/c mice were used to evaluate virulence of the above strains and two strains of Salmonella typhimurium containing different plasmids. In orally inoculated one-day old chickens, the UK S. enteritidis PT 4 strain was more virulent than the Canadian PT 4 strain. The UK PT 4 strain was also more virulent and invasive in adult laying hens than the Canadian PT 4 strain. The S. enteritidis PT 8 strain and one S. typhimurium strain isolated from a chicken hatchery were the most virulent for orally inoculated Balb/c mice. This strain of S. typhimurium contained the 60 megadalton plasmid associated with virulence for Balb/c mice which was not present in the S. typhimurium strain isolated from a pig with septicemic disease.     

Use of a live attenuated Salmonella typhimurium vaccine to protect hens against Salmonella enteritidis infection while undergoing molt

Previous studies demonstrated that Salmonella enteritidis infections in hens undergoing molt via feed withdrawal were more severe than in full fed hens. We conducted two trials to determine if immunizing specific-pathogen-free, Salmonella-culture-negative hens via aerosol exposure to MeganVacl, a commercially available attenuated Salmonella typhimurium vaccine, would reduce transmission of S. enteritidis from infected hens to uninfected but contact-exposed hens during a molt. In trial 1, one group of hens received two aerosol doses of vaccine 2 wk apart whereas a second group of hens remained nonvaccinated. In trial 2, the vaccinated group received only one dose of vaccine. Two weeks after the final immunization, feed was removed from all the hens, and on day 4, the center hen in rows of 11 hens received a dose of 3 x 10(5) (trial 1) or 1.3 x 10(6) (trial 2). Transmission to the unchallenged hens was followed 3, 10, 17, and 24 days later. Vaccination reduced the horizontal spread of S. enteritidis in vaccinated hens compared with their nonvaccinated counterparts, with vaccinated hens shedding significantly less S. enteritidis on day 10 postchallenge in trial 1 and on days 3, 10, 17, and 24 in trial 2. Recovery of S. enteritidis from ovaries was significantly reduced in the vaccinated hens in trial 1 and from livers/spleens, ovaries, and cecum in trial 2. These studies indicate that immunization of hens with a live S. typhimurium vaccine could help reduce S. enteritidis problems during a molt situation.     

Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.     

Investigation of the efficacy of a genetically-stabile live Salmonella typhimurium vaccine for use in swine

Hybrid swine (Landrace x Pietrain) aged 3-4 weeks were immunized twice at an interval of 3 weeks solely by the oral route and by the oral/parenteral route to evaluate the efficacy of a live S. Typhimurium vaccine. In each experiment a control group was run without vaccination. The animals were challenged at the age of 8-10 weeks by oral test infection with a labelled S. Typhimurium DT 104 strain. An ELISA was used to establish the presence of antibodies to S. Typhimurium in serum samples, coupled with clinical investigation. The presence of the challenge strain in the ileal and caecal mucosa and in the ileocolic lymph nodes was investigated quantitatively using the Koch plating method to determine the degree of colonization of those organs at the time of slaughter. The clinical course of disease was used to assess the success of vaccination. However, it was not possible to trigger, in a reproducible manner, clinical signs of disease in unvaccinated animals through infection. The vaccinated animals had a significantly lower (p < 0.05) colonization of the ileal and caecal mucosa than the unvaccinated animals. This was also seen to a lesser degree for the ileocolic lymph nodes.     

减毒鸡沙门氏菌97A疫苗株安全性和免疫效力试验

本试验将减毒鸡沙门氏菌97A疫苗株分别以不同剂量经口服和肌肉注射接种10日龄AA肉鸡，结果表明，97A对10日龄雏鸡有良好安全性。将97A分别以10     

Resistance to fecal shedding of salmonellae in pigs and chickens vaccinated with an aromatic-dependent mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the effectiveness of an aromatic-dependent mutant of Salmonella typhimurium as a parenteral vaccine for prevention of fecal shedding of Salmonella spp. Pigs and chickens were vaccinated IM, with 1 x 10(9) and 1 x 10(8) organisms, respectively, followed by a second identical vaccination 2 weeks later. Salmonella organisms were not detected by analysis of fecal or cloacal swab specimens from any animal after vaccination. Deleterious side effects were not noticed after vaccination. Pigs were challenge-inoculated PO with 1 x 10(12) virulent S typhimurium 1 week after the second vaccination. Chickens were challenge-inoculated PO with 3 x 10(8) organisms of either S enteritidis or the virulent parent strain of S typhimurium 3 weeks after the second vaccination. Vaccinated pigs shed Salmonella spp significantly less frequently than did nonvaccinated pigs. Vaccinated chickens challenge-inoculated with either S enteritidis or S typhimurium also shed Salmonella less frequently than the corresponding nonvaccinated control birds; however, the difference was not significant.     

A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimurium challenge in chickens

A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enteritidis vaccine was used to vaccinate chicks at 1 day and again at 4 weeks of age, with challenge by a high and a low dose of S. Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S. Typhimurium. In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls. Vaccination reduced colonisation of internal organs after challenge by contact seeder birds. However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent. In conclusion, the data indicate that the vaccine should be a useful tool in the control of S. Typhimurium infection in chickens.     

Adjuvanted Outer Membrane Protein Vaccine Protects Poultry against Infection with Salmonella enteritidis

The immunogenicity of a sonicated extract (SE) and of outer membrane proteins (OMP) of Salmonella enteritidis was tested in birds of about 8 weeks of age. The dose, route of vaccination and the adjuvant used varied in different groups of birds. Two vaccine doses with or without adjuvant were given parenterally or orally 3 weeks apart. OMP vaccines gave significantly higher antibody titres than SE vaccines, as indicated by ELISA. The vaccines adjuvanted with oil produced higher antibody titres than those without any adjuvant. A dose of 1 mg of vaccine produced higher antibody titres than 0.5 mg of vaccine. Adjuvanted vaccine given subcutaneously elicited higher antibody responses than oral vaccines given without adjuvant. The birds were challenged with virulent S. enteritidis organisms at the end of the second week after a booster dose. None of the birds given 1 mg of OMP vaccine subcutaneously shed the organisms when tested by culturing cloacal swabs, although a few birds vaccinated with 0.5 mg of OMP vaccine did so. In general, adjuvanted OMP vaccines gave better protection than SE vaccines.     

Aromatic-dependent Salmonella dublin as a parenteral modified live vaccine for calves

A virulent Salmonella dublin isolate was made histidine-requiring (his-) to allow recognition. The his- derivative, SL1367 (still calf-virulent), was then given by transduction and mutation, a transposon-generated non-reverting aromatic biosynthesis (aro) defect; this defect caused loss of virulence for the mouse. The his- aro- derivative strain, SL1438, was effective as a live vaccine in mice. Twenty male Holstein calves were divided into 4 groups. Groups I, II, and III were vaccinated IM at 2 weeks and at 3 weeks of age with aromatic-dependent (aro-) S dublin strain SL1438. Groups I and III received freshly prepared vaccine and group II received lyophilized vaccine. Serious adverse reactions to the vaccination were not seen. After vaccination, the mean maximum increase in rectal temperature was 1.8 C in group I and III calves and 0.6 C in group II calves. Fewer group II calves developed diarrhea (1 of 5) or positive blood cultures (0 of 5) after vaccination compared with group I and III calves (6 of 10 and 5 of 10, respectively). Postvaccination diarrhea was mild and of short duration. Group IV was comprised of 5 nonvaccinated calves. At 5 weeks of age, all calves were challenge exposed orally. Group I, II, and IV calves were challenge exposed with 10(11) virulent S dublin SL1367. Group III was challenge exposed with 10(11) virulent S typhimurium UCD 108-11. Subsequently, fever and diarrhea (lasting 1 to 3 days), but no deaths, were observed in the vaccinated calves. Four of the 5 nonvaccinated (group IV) calves died (P less than 0.001) within 8 days after challenge exposure. Aro- S dublin SL 1438 did not cause serious adverse effects and provided protection against oral challenge exposure with either virulent S dublin or S typhimurium.     

Salmonella enterica serovar enteritidis burden in broiler breeder chicks genetically associated with vaccine antibody response

The relationship between antibody response to Salmonella enteritidis vaccine and internal organ burden of S. enteritidis is not fully understood. The genetic relationship, therefore, between postchallenge S. enteritidis burden and antibody response to S. enteritidis vaccine was determined in broiler breeder chicks. Sibling chicks from a broiler breeder male line were either inoculated with a pathogenic S. enteritidis or vaccinated with a commercial S. enteritidis vaccine. Spleen, liver, cecal wall, and cecal content samples from S. enteritidis-challenged chicks (n = 120) were cultured for enumeration of bacteria. Unchallenged chicks (n = 314) were vaccinated at 11 days of age, and serum samples were taken at 10 days postvaccination. Antibody response to vaccination and number of S. enteritidis in cecal content cultures were negatively correlated (-0.772), demonstrating that genetic potential for greater antibody response to S. enteritidis vaccine is associated with lesser S. enteritidis bacterial burden in cecal content of broiler breeder chicks. The findings suggest that genetic selection for vaccine antibody responsiveness can lower bacterial burden in the gut lumenal content and, thus, potentially reduce contamination of poultry products at processing.     

Comparison of different attenuation strategies in development of a Salmonella hadar vaccine

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.     

Safety and efficacy of the cell-associated vaccine prepared by an attenuated infectious laryngotracheitis virus.

The safety and efficacy of the cell-associated (C-A) vaccine prepared by chicken embryo fibroblast (CEF) cells infected with the tissue-culture-modified strain of infectious laryngotracheitis (ILT) virus were studied in chickens. Over seventy percent of chickens inoculated with the C-A vaccine by the subcutaneous (S.C.) or intramuscular (I.M.) route at 1 day of age was protected against challenge with a virulent strain of ILT virus without any clinical signs. Chickens vaccinated with the C-A vaccine at 1 day of age acquired immunity within 6 days after vaccination, and the protection rate maintained more than 60% until 10 weeks post-vaccination. The C-A vaccine was invariably effective for chickens at various age. There was no evidence that the development of immunity was hindered by further vaccination with Newcastle disease and infectious bronchitis combined live vaccine. In addition, the C-A vaccine was safe when chickens were inoculated with 10 doses. In the field trials of the C-A vaccine, no adverse reaction was observed, and over 65% of vaccinated chickens was protected against the challenge of the virulent ILT virus at 8 weeks after vaccination.     

Feasibility of using proteins from Salmonella gallinarum vs. 9R live vaccine for the prevention of fowl typhoid in chickens

Proteins from a field strain of Salmonella gallinarum MSG1 were compared with 9R live vaccine strain for their protection against experimental fowl typhoid in chickens. Proteins from S. gallinarum gave better protection than the 9R live vaccine as measured by clearance of challenge organism from internal organs. Proteins given twice with an adjuvant at 200 micrograms/100 g body weight resulted in 95% protection, compared with 60% protection with 9R given orally. The 9R live vaccine produced more hepatic and splenic lesions and, when administered orally as a single dose, was the least protective (60%). In the group vaccinated subcutaneously with a single dose of 9R without an adjuvant, both the challenge strain and the 9R vaccine strain were isolated from the ovaries of some birds. All chickens vaccinated with 9R strain or with proteins developed antibodies detectable by microagglutination test, and in some vaccinated groups as many as 100% of the birds developed antibody levels detected by seroagglutination.     

Vaccination of calves against Salmonella dublin with aromatic-dependent Salmonella typhimurium

Ten Holstein calves were divided into 2 groups. Five calves served as nonvaccinated controls, and 5 calves were vaccinated IM at 2 and 3 weeks of age with 10(9) aromatic-dependent (aro-) Salmonella typhimurium strain SL1479 containing O antigens 1, 4, 12. Serious adverse reactions to vaccination were not observed in the calves. Mean maximum rectal temperature increase in the vaccinated calves was 1.5 C. One calf had diarrhea and depressed appetite for 1 day after vaccination. At 5 weeks of age, all calves were challenge exposed orally with 1.5 X 10(11) virulent S dublin strain SL1367 (O antigens 9,12). After challenge-exposure inoculum was given, 1 of 5 vaccinated calves died and 4 of the 5 nonvaccinated calves died (P less than 0.05). Thus, some cross serotype protection against S dublin was induced by parenteral vaccination of calves with aro- S typhimurium strain SL1479, although protection was not complete.     

Induction of humoral immune response and protective immunity in chickens against Salmonella enteritidis after a single dose of killed bacterium-loaded microspheres.

Formalin-inactivated Salmonella enteritidis phage type 4 strain 119/95 (SE) was encapsulated in biodegradable poly (DL-lactide co-glycolic acid) PLGA; (65:35) microspheres by a modified water-in-oil-in-water (w/o/w) double-emulsion solvent extraction/evaporation technique. These SE-loaded microspheres (SE-MS) were porous and spherical in shape with diameters of 0.4-10 microm and 20-80 microm in two preparations. SE-MS were subsequently used to vaccinate specific-pathogen-free chickens in a single dose in order to investigate the potency of a single-dose vaccination in inducing immune responses and protective immunity. In Experiment 1, 4-wk-old chickens that were vaccinated intramuscularly with 20-80-microm SE-MS generated long lasting (over 6 mo) and persistently high serum anti-SE immunoglobulin (Ig)G antibody response. In Experiment 2, 2-wk-old chickens were vaccinated orally with 0.4-10-microm or intramuscularly with 20-80-microm SE-MS and challenged with 10(9) colony-forming units of homologous SE strain at 6 wk postvaccination. When challenged intramuscularly, one each of the orally vaccinated (n = 10) and the intramuscularly vaccinated birds (n = 10) showed clinical signs and death, whereas all of the nonvaccinated control birds (n = 12) were sick and 11 of them were killed. When challenge was via oral route, 26.1% of cloacal swabs and 24.0% of organs (liver, spleen, and cecum) collected from orally vaccinated birds (n = 35) were positive for SE, comparable to 27.9% of feces and 18.7% of organs from the intramuscularly vaccinated birds (n = 35). These figures were significantly lower than those for nonvaccinated birds (n = 30) from which 59.3% of feces and 44.0% of organs tested SE positive (P < 0.05). The humoral immune response was also determined after vaccination with a single dose. The intramuscular vaccination elicited higher serum IgG response than oral administration, but the latter elicited a significant intestinal mucosal IgA antibody response. This is the first evidence that chickens vaccinated with killed SE-loaded PLGA microspheres, intramuscularly and orally in a single dose, developed systematic and local immune responses, thereby conferring protective immunity.     

Laboratory assessment of protection given by an experimental Salmonella enteritidis PT4 inactivated, adjuvant vaccine

An experimental inactivated Salmonella enteritidis PT4 vaccine, containing 10(11) cfu/ml and 50 per cent oil adjuvant, was used in laboratory vaccination trials in chickens. A single subcutaneous vaccination at three weeks or two vaccinations at three and six weeks old provided good protection against challenge with 10(9) cfu or 10(8) cfu of virulent organisms administered intramuscularly or intravenously at five and eight weeks old, respectively. The degree of protection was assessed according to the severity of the clinical signs, the mortality, the post mortem lesions and the recovery of the challenge organisms from post mortem material.     

Vaccination of calves with a diaminopimelic acid mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the safety and efficacy of a diaminopimelic acid mutant of Salmonella typhimurium as a vaccine for calves. Transposon techniques were used to create a stable nonreverting diaminopimelic acid mutant of a virulent S. typhimurium strain. Calves were vaccinated at weekly intervals with the diaminopimelic acid mutant given as an oral dose of 10(10) organisms, followed by two subcutaneous doses of 10(9) organisms. The calves tolerated vaccination well and the vaccine strain was eliminated from the feces within four days. Of five vaccinated calves, three survived challenge with 5 X 10(9) organisms of the parent strain whereas all five unvaccinated calves that were challenged died. The surviving calves eliminated the challenge organism from the feces within three weeks.