Sherryll Layton

Evaluation of Salmonella-Vectored Campylobacter Peptide Epitopes for Reduction of Campylobacter jejuni in Broiler Chickens

ABSTRACT Campylobacter is a leading cause of bacterial gastroenteritis in humans and is often linked to contaminated poultry products. Live Salmonella vectors expressing three linear peptide epitopes from Campylobacter proteins Cj0113 (Omp18/CjaD), Cj0982c (CjaA), and Cj0420 (ACE393) were administered to chicks by oral gavage on the day of hatch, and the chicks were challenged with Campylobacter jejuni on day 21. All three candidate vaccines produced consistent humoral immune responses with high levels of serum IgG and mucosal secretory IgA (sIgA), with the best response from the Cj0113 peptide-expressing vector. Campylobacter challenge following vaccination of three candidate vaccine groups decreased Campylobacter recovery from the ileum compared to that for controls on day 32. The Cj0113 peptide-expressing vector reduced Campylobacter to below detectable levels. The Salmonella-vectored Cj0113 subunit vaccine appears to be an excellent candidate for further evaluation as a tool for the reduction of Campylobacter in poultry for improved food safety.

Vaccination of chickens with recombinant Salmonella expressing M2e and CD154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge.

Avian influenza (AI) is a significant public health concern and serious economic threat to the commercial poultry industry worldwide. Previous research demonstrates that antibodies against M2e confer protection against influenza challenge. Using the Red recombinase system in combination with overlapping extension PCR, we recently developed several novel attenuated Salmonella Enteritidis strains that express a protective M2e epitope in combination with a potential immune-enhancing CD154 peptide sequence on the Salmonella outer membrane protein lamB. Commercial Leghorn chicks were orally immunized (immunization dose: 10(6) to 10(8) cfu/chick) with saline (negative control) or one of the recombinant Salmonella strains [DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, DeltaaroA-DeltahtrA M2e(4)-CD154] on day of hatch and 21 d posthatch. These candidate vaccine strains were evaluated for their ability to invade, colonize, and persist in tissues and elicit an M2e-specific antibody response. The vaccine candidate strain DeltaaroA M2e-CD154 exhibited significantly greater organ invasion in the liver and spleen at d 7 (P > 0.05); however, no marked differences in colonization of the cecal tonsils were observed. Vaccinated chickens exhibited significantly increased M2e-specific IgG responses, which were further enhanced by simultaneous expression of CD154 (P < 0.05). Virus neutralization assays gave neutralizing indices of 6.6, 6.3, and 6.3 for DeltaaroA M2e-CD154, DeltahtrA M2e-CD154, and DeltaaroA-DeltahtrA M2e(4)-CD154 seven days post booster immunization, respectively, indicating effective neutralization of AI by serum IgG of vaccinated chickens. In a subsequent direct challenge study, specific-pathogen-free Leghorn chicks immunized with DeltaaroA-DeltahtrA M2e(4)-CD154 offered significant protection against direct challenge with low pathogenic AI H7N2, but not highly pathogenic H5N1 AI. Taken together, these data suggest that these Salmonella-vectored vaccines expressing M2e in association with CD154 are effective at protecting chickens against low pathogenic AI.

Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.

BackgroundA variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome.ResultsThe final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis.ConclusionWe have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.

Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens

Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative cells or spores is advisable.

Development and evaluation of candidate recombinant Salmonella-vectored Salmonella vaccines

Attenuated Salmonella Enteriditis (ΔSE) recombinant vaccine vectors incorporating a Salmonella flagellar filament protein (fliC) subunit, a putative cell-mediated epitope, for expression of the lamB gene (encoding a maltose outer membrane porin), with or without co-expression of a putative immune-enhancing CD154 oligopeptide, were developed and compared with wild-type Salmonella Enteriditis (experiments 1 and 2) or the attenuated ΔSE empty vector (experiment 3) as initial vaccine candidates against Salmonella infection. A total of 3 experiments were performed to assess the infection and clearance rate of each of these constructs. Each construct or Salmonella Enteriditis was orally administered to broiler chicks at day of hatch by oral gavage (~10(8) cfu/chick). In experiments 1 to 3, liver-spleen and cecal tonsils were removed aseptically for recovery of wild-type Salmonella Enteriditis or ΔSE mutants. These experiments suggested that cell surface expression of fliC alone markedly increased the clearance rate of the vector at or before 21d postvaccination in all 3 experiments. In a fourth experiment, broilers were vaccinated with one of the vaccine constructs or the ΔSE empty vector and then challenged with wild-type Salmonella Typhimurium. At 19 d posthatch, 16 d postinfection, neither candidate protected against challenge significantly better than the ΔSE empty vector, although there was significantly less Salmonella recovered from vaccinated chickens as compared with nonvaccinated controls. These experiments indicate that these experimental vaccines did not protect against heterologous challenge or enhance clearance after Salmonella Typhimurium challenge; as such, their value as vaccines is limited. The increased clearance of the candidate vaccines, particularly the vector expressing fliC alone, may have value in that the fliC epitope may decrease the clearance time of other recombinant vectored Salmonella vaccines.

Evaluation of recombinant Salmonella expressing the flagellar protein fliC for persistence and enhanced antibody response in commercial turkeys

Salmonella enterica serovar Enteritidis (SE) is one of the most common causes of human foodborne illness in the United States. Previous research indicates that antibodies against the fliC protein can provide protection against Salmonella challenge in mice. To generate a vaccine that effectively protects poultry against multiple Salmonella serotypes, novel attenuated strains of SE were developed to express a fliC peptide sequence on the outer membrane protein lamB in association with an M2e (marker) epitope. In 3 separate trials, poults were immunized with 10(7) to 10(8) cfu/poult of the appropriate recombinant Salmonella strains (ΔSE-M2e or ΔSE-M2e-fliC) via oral gavage on the day of hatch and again on d 21 posthatch. Liver, spleen, and cecal tonsils were aseptically removed on d 7, 14, 21, 28, 35, and 42 posthatch for detection of Salmonella, and blood samples were obtained at these same time points for determination of an M2e-specific antibody response. In all 3 trials, the ΔSE-M2e-fliC strain exhibited significantly less invasion of the liver and spleen at d 7 and 14 when compared with ΔSE-M2e or SE phage type 13A (P < 0.05). Similarly, colonization of the cecal tonsils was decreased in the poults immunized with the ΔSE-M2e-fliC strain. By d 21, the ΔSE-M2e-fliC strain exhibited a significantly higher M2e-specific antibody response when compared with the negative control and SE phage type 13A groups (P < 0.05). However, no significant differences in M2e-specific antibody responses were observed between the ΔSE candidate vaccine strains throughout the study. Overall, these data suggest that oral live attenuated Salmonella-vectored vaccines expressing a fliC peptide sequence are able to elicit a humoral immune response in commercial poults and may contribute to a reduction in Salmonella organ invasion and colonization.

Evaluation of effects of EarlyBird associated with FloraMax-B11 on Salmonella Enteritidis, intestinal morphology, and performance of broiler chickens

A posthatch fasting period of 24 to 72 h is a common and inevitable practice in commercial poultry production. This delay in start of feed intake has been reported to negatively affect yolk utilization, gastrointestinal development, slaughter weight, breast meat yield, performance, and to also depress immunological development, making the birds more susceptible to infection from pathogens such as Salmonella. Furthermore, public concerns regarding the considerable human rates of illness reported and the emergence of antibiotic-resistant strains of Salmonella have doubled the challenge on the poultry industry to find alternative means of Salmonella control. In the present study, we evaluated the effects of a combination of early feeding with probiotic supplementation on morphological development of mucosa, control of Salmonella, and overall performance in broiler chickens. We used a blend of a commercially available perinatal supplement, EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR), and a successful probiotic supplement, FloraMax-B11 (FM; Pacific Vet Group USA Inc.), to evaluate the effects on gut morphology, Salmonella intestinal colonization, and horizontal transmission, along with its effects on BW and related performance in broiler chickens under simulated commercial hatching management and shipping conditions. Morphometric analysis showed increased villus height, villus width, villus to crypt ratio, and villus surface area index in chickens treated with EB + FM groups. Significant reductions in Salmonella recovery, incidence, and horizontal transmission were also observed among the same groups, suggesting beneficial effects of early feeding and competitive exclusion by probiotic bacteria. Improved gut morphology and Salmonella exclusion was very well supported by BW data with significantly lower early BW loss and overall BW gains in birds treated with EB + FM mixture. The results of this study demonstrated that the combination of EB and FM improved gut morphology, reduced the amount of Salmonella that could be recovered, as well as improved BW when compared with controls and each product individually. These data address both animal welfare and food safety concerns faced by the poultry industry.

Evaluation of recombinant Salmonella expressing CD154 for persistence and enhanced antibody response in commercial turkeys

Foodborne illness due to Salmonella is a worldwide public health concern and epidemiological evidence has identified poultry and poultry products as a significant source of human Salmonella infection. To discover an effective vaccine that protects poultry against multiple Salmonella serotypes, several novel attenuated Salmonella Enteritidis strains (DeltaSE) were developed to express variations of a potential immune-enhancing CD154 peptide sequence on the outer membrane protein lamB in association with a M2e (marker) epitope. The 3 CD154 peptide sequences evaluated in this study correspond to those naturally occurring in turkeys, humans, and chickens. In 3 separate trials, poults were immunized with 10(7) to 10(8) cfu/poult of the appropriate recombinant Salmonella strains (DeltaSE-M2e, DeltaSE-M2e-T/CD154, DeltaSE-M2e-H/CD154, DeltaSE-M2e-C/CD154) via oral gavage on day of hatch and again on 21 d posthatch. Liver, spleen, and cecal tonsils were aseptically removed on d 7, 14, 21, 28, 35, and 42 posthatch for detection of Salmonella and blood samples were obtained at these same time points for determination of an M2e-specific antibody response. In all 3 trials, DeltaSE strains exhibited significantly less invasion of the liver and spleen at d 7 when compared with Salmonella Enteritidis phage type 13A (P < 0.05). In 2 of the 3 trials, the DeltaSE strains expressing a CD154 peptide sequence further decreased invasion of the liver and spleen. Similarly, colonization of the cecal tonsils was also decreased in the poults immunized with the DeltaSE strains. However, there were no differences in colonization or invasion due to the amino acid sequence of the CD154 insert in all 3 trials. By d 21, the DeltaSE strains exhibited a significantly higher M2e-specific antibody response when compared with the negative control and SE13A groups (P < 0.05). However, no significant differences in M2e-specific antibody responses were observed between any of the DeltaSE candidate vaccine strains expressing CD154 throughout the study. Overall, these data suggest that oral live attenuated Salmonella-vectored vaccines expressing a foreign peptide sequence are able to elicit a humoral immune response in commercial poults and may contribute to a reduction in Salmonella organ invasion and colonization.

Glycerol supplementation enhances the protective effect of dietary FloraMax-B11 against Salmonella Enteritidis colonization in neonate broiler chickens

Two independent trials were conducted in the present study to evaluate the effect of 5% glycerol supplementation combined with dietary FloraMax-B11 (FM) against Salmonella Enteritidis colonization in neonate broiler chickens. In each trial, 60 chicks were randomly assigned into 4 groups. Group 1 received a control diet. Group 2 received a control diet supplemented with 5% glycerol. Group 3 received a control diet supplemented with FM, and group 4 received a control diet supplemented with 5% glycerol and FM. At placement, chickens were challenged with Salmonella Enteritidis at 10(4) cfu/bird. In each trial, 12 chicks were humanely killed 72 h postchallenge, respectively, for Salmonella Enteritidis colonization. Supplementation of 5% glycerol or FM by themselves, showed no significant effect on Salmonella Enteritidis recovery or incidence when compared with control nontreated chickens in both trials. However, no detectable Salmonella Enteritidis was observed in the chickens that received the supplementation of 5% glycerol combined with FM in both trials. Further studies are in progress in older birds to substantiate these findings.