Passive protection against Salmonella enterica serovar Enteritidis infection from maternally derived antibodies of hens vaccinated with a ghost vaccine

This study evaluated maternal immunity against Salmonella enterica serovar Enteritidis acquired through the egg yolk. Two-hundred 19-week-old specific pathogen free (SPF) broiler breeders which were randomly divided into two groups of equal size were injected with S. Enteritidis ghosts (5 × 10⁹ colony forming units in 0.1 ml per hen) and phosphate-buffered saline (PBS, 0.01 mol⋅l⁻¹, pH 7.4) twice, respectively, with an interval of 2 weeks. An indirect enzyme-linked immunosorbent assay (ELISA) was applied to detect specific antibodies against S. Enteritidis. S. Enteritidis-specific antibody levels in the vaccinated group increased over time and were significantly higher than those of the control group on days 28 (P < 0.001) and 35 (P < 0.001) post-vaccination. Ten 7-day-old chicks from hens that were vaccinated with a S. Enteritidis ghost vaccine were challenged at 14 days of age with 5 × 10⁹ CFU of S. Enteritidis DH091 (homologous to the vaccine strain), 8/10 (80%) chicks from vaccinated hens survived, whereas 3/10 (30%) chicks from unvaccinated hens survived. The chicks acquired high levels of serum antibodies against S. Enteritidis. These results reveal that maternal antibodies in chicks acquired from vaccinated hens through eggs can confer a significant protection against S. Enteritidis infection.     

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 ± 0.09 and 0.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 ± 0.21 and 1.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.     

Analysis of Salmonella enterica serotype Enteritidis isolated from human and chickens by repetitive sequence-PCR fingerprinting, antibiotic resistance and plasmid profiles

A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.     

Molecular typing of Salmonella enterica serovar Enteritidis isolates from food-producing animals in Japan by multilocus variable-number tandem repeat analysis: evidence of clonal dissemination and replacement

Background

Salmonella enterica serovar Enteritidis is a zoonotic pathogen. Human infections are associated with contaminated eggs and egg products. In Japan, since 1989, the incidence of food-borne disease caused by S. Enteritidis has increased and a pandemic has occurred; however, little is known about changes that occurred before and after this pandemic event in the dominant lineage of isolates from food-producing animals. This study aimed to determine the S. Enteritidis lineages in Japan over the last few decades by using multilocus variable-number tandem repeat analysis (MLVA).

Findings

MLVA was used to analyse 79 S. Enteritidis isolates collected from chickens (n = 63), cattle (n = 12), pigs (n = 2), and goats (n = 2) during 1975–2009. The S. Enteritidis isolates showed 14 different MLVA allele combinations, which were classified into two major clusters (A and C) and a minor cluster (B). All the 62 isolates in cluster A were isolated after 1988, whereas 13 of the 17 isolates belonging to cluster B and C were isolated before 1989.

Conclusions

The MLVA results showed that cluster C was predominant before 1989, and isolates in cluster A disseminated since 1989 and replaced the previous dominant clone, suggesting that isolates of cluster A originated from imported S. Enteritidis infection.     

Introduction: Reducing Salmonella Enteritidis contamination of shell eggs

Salmonella Enteritidis is a leading food-borne pathogen in the United States, with many outbreaks in humans traced back to shell eggs. As a result, the implementation of effective strategies for reducing Salmonella Enteritidis in commercial layer flocks has become a critical public health and economic objective. In this paper, we share the findings of 2 multistate USDA-National Integrated Food Safety Initiative grant teams and their work aimed at Salmonella Enteritidis reduction in shell eggs. One project, led by K. Venkitanarayanan, is using plant-derived antimicrobial molecules as dietary supplements to reduce Salmonella Enteritidis colonization of the digestive and reproductive tract of chickens. The same molecules are being evaluated for their effect on Salmonella Enteritidis in egg wash solutions. The project led by S. Kariyawasam used on-farm investigation and novel bacterial typing methods to study Salmonella Enteritidis transmission in diverse layer environments to update and optimize Egg Quality Assurance Programs that will significantly reduce Salmonella Enteritidis contamination of shell eggs. The current US Food and Drug Administration Egg Safety Rule and Egg Quality Assurance Programs are based on critical control points and best management practices developed from studies of large flocks (>50,000 hens) conducted in the 1990s, indicating that opportunities exist to improve preharvest programs to reduce Salmonella Enteritidis contamination. This paper will share the findings of these 2 projects.     

Differences in immune responses of pigs vaccinated with Salmonella Typhimurium and S. Choleraesuis strains and challenged with S. Choleraesuis

S. Choleraesuis (Choleraesuis) and S. Typhimurium (Typhimurium) cause salmonellosis in pigs and humans. The effects of vaccine strains pSV-less Typhimurium OU5048 and Choleraesuis OU7266 and SPI-2-mutant Choleraesuis SC2284 on the immune responses of pigs against Typhimurium, Choleraesuis, and S. Enteritidis (Enteritidis) with or without the virulence plasmid (pSV) were determined. After oral vaccination of three vaccine groups and challenge with Choleraesuis CN36, the level of Salmonella-specific IgG in sera and the bactericidal effects and superoxide generation of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) against the above strains were determined using ELISA and NBT assay, respectively. Among three vaccine strains tested, OU7266 stimulated the highest Salmonella-specific IgG levels. Complement inactivation increased IgG concentration, while E. coli absorption reduced IgG levels. The pSV-containing strains were less resistant to serum killing than the pSV-less strains, and Enteritidis exhibited the lowest resistance to serum killing. Serovars tested, vaccine strains, and timeline periods postvaccination and challenge were important factors affecting superoxide production. The two Choleraesuis vaccine strains stimulated greater levels of superoxide from PMNs and PBMCs than the Typhimurium strains. The PMNs and PBMCs in challenged and vaccinated pigs reduced more superoxide than those in challenged hosts. In vaccinated hosts, pSV-less Salmonella strains triggered lower levels of PMN/PBMC-generated superoxide upon challenge than strains with pSV against Enteritidis and Choleraesuis. Overall, Choleraesuis OU7266 may be better than the other vaccine strains in generating the greatest IgG levels, serum bactericidal activity and superoxide levels. The pSV likely influences the immune responses.     

The population structure of Salmonella enterica Enteritidis in Iran analyzed by multiple-locus variable-number tandem repeat analysis

Salmonella enterica Enteritidis is the most frequent etiological agent of salmonellosis in humans and poultry. To understand the genetic diversity of S. Enteritidis in Iran, we examined 69 chicken isolates from 18 broiler farms and six non-epidemic human isolates from six geographically distant provinces by multi-locus variable-number tandem repeat analysis (MLVA). Among SE2, SE3, SE5, SE7, SE8, SENTR4, and SENTR7, only SE5 with four and SENTR7 with two alleles, respectively, proved variable giving estimates of locus genetic diversity of 0.58 and 0. In all, six closely related MLVA profiles were identified among which three were commonly represented by human and chicken isolates. This population homogeneity contrasts with the high diversity at these loci reported elsewhere and is likely a consequence of a single clone of S. Enteritidis distributed across Iran.     

Poultry as a possible source of non-typhoidal Salmonella enterica serovars in humans in Bangladesh

We investigated Salmonella enterica isolates from human clinical cases of gastroenteritis to determine the distribution of non-typhoidal Salmonella serovars in the human population, and compared them to isolates originating from poultry by serotyping, phage typing, plasmid profiling, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) to evaluate the potential role of poultry in human non-typhoidal salmonellosis in Bangladesh. Nine different serovars were identified among the human isolates of which Salmonella Paratyphi B var Java (S. Java), S. Kentucky, S. Enteritidis, S. Virchow and S. Weltevreden also were commonly isolated from poultry. The poultry isolates belonging to S. Java, S. Kentucky and S. Enteritidis were indistinguishable from human isolates or genetically closely related, based on PFGE profiles and MLST. S. Kentucky clone ST198 and S. Java clone ST43 both well-known cause of human infections were also isolated from poultry.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Integrated farm management to prevent Salmonella Enteritidis contamination of eggs

Salmonella Enteritidis in contaminated eggs is a public health hazard that may cause hospitalization or death in the elderly, infants, and individuals with impaired immune systems. Prevention of Salmonella Enteritidis infection of laying hens is an essential first step in reducing Salmonella Enteritidis outbreaks in humans. Multiple interventions at several stages during egg production can combine to reduce numbers of infected chickens and keep egg contamination to low levels. Every effort should be made to exclude Salmonella Enteritidis from egg production premises by implementing effective biosecurity measures, stocking the farm with Salmonella Enteritidis-free replacement pullets, controlling rodent and insect vectors, and denying wild birds and pets access to chicken houses. Diligent cleaning and disinfection of chicken houses before introduction of a new flock will minimize environmental exposure and indirect horizontal transmission of multiple pathogens, including Salmonella Enteritidis. Increased resistance of chickens to intestinal colonization by Salmonella Enteritidis can be attained by the use of probiotics, prebiotics, and synbiotics. Laying hens should be immunized with live and killed vaccines to stimulate mucosal and systemic immunity and reduce the prevalence of Salmonella Enteritidis-contaminated eggs. Shell eggs should be refrigerated as soon as possible after laying to keep Salmonella Enteritidis cells at low levels in any contaminated eggs. Comprehensive Salmonella Enteritidis-control programs have proven to be successful in reducing the incidence of Salmonella Enteritidis infections in both egg-laying flocks and humans.     

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Efficacy of plant-derived antimicrobials for reducing egg-borne transmission of Salmonella Enteritidis

Egg-borne transmission of Salmonella Enteritidis is a significant public health concern in the United States and worldwide. This review discusses the potential of plant-derived antimicrobials for reducing egg-borne transmission of Salmonella Enteritidis in layer chickens. In addition to documenting the research conducted on plant-derived antimicrobials, a general overview of other pre- and postharvest approaches to control Salmonella Enteritidis in layers and on shell eggs is presented.     

On-farm risk factors for Salmonella Enteritidis contamination

Forty layer farms from 2 states participated in a study to examine the risk factors and incidence of Salmonella Enteritidis from multiple samples, including environmental drag swabs from the bird areas, feed, water, flies, rodents, live rodent traps, and environmental swabs from areas occupied by other livestock. Twenty-four of these farms had between 3,000 and 31,000 bird flocks (medium-sized flocks) and 16 had less than 3,000 birds (small-sized flocks). All were housed in cage-free production systems. Twenty-two farms included outside pasture areas for the birds. Most of the participants had just come under the FDA Egg Rule and had not yet tested their flocks (flocks under 3,000 birds are exempt) for Salmonella Enteritidis. Many, however, obtained their pullets from commercial Salmonella Enteritidis-clean breeder sources hatched in National Poultry Improvement Plan hatcheries. Vaccination against Salmonella Enteritidis was performed on 21 of the 40 farms (combination of live and killed vaccines). Salmonella Enteritidis was detected on 7 out of the 40 farms, primarily in rodents, their feces, or from swabs taken inside live traps. Of these 7 Salmonella Enteritidis-positive farms, 3 farms that had vaccinated their pullets with live Salmonella Typhimurium vaccine and killed-Salmonella Enteritidis vaccine; no Salmonella Enteritidis was isolated from the environmental drag swabs taken from the bird area or from the eggs on these farms. However, on the farms that had not vaccinated for Salmonella Enteritidis, the organism was isolated from 4 environmental drag swabs and 3 egg pools. The last 4 farms had flocks under 3,000 birds. No Salmonella Enteritidis was isolated from any of the samples of feed, flies, water, or swabs taken from other livestock areas. Based on the initial findings in this study, we suggest the 2 most important risk factors for Salmonella Enteritidis contamination inside the bird area and in the eggs in these small- and medium-sized flocks are the presence of infected rodents and the absence of an Salmonella Enteritidis vaccination program.     

Antibacterial Effect of Trans-Cinnamaldehyde on Salmonella Enteritidis and Campylobacter jejuni in Chicken Drinking Water,

Salmonella Enteritidis and Campylobacter jejuni are 2 major foodborne pathogens in the United States, estimated to cause more than 3 million cases of human illness annually. Chickens are the natural hosts of these bacteria, and their drinking water can be a source of S. Enteritidis and C. jejuni, contributing to the colonization of birds. In this study, trans-cinnamaldehyde (TC), a natural, generally recognized as safe ingredient in cinnamon oil was evaluated for its efficacy to inactivate S. Enteritidis and C. jejuni in the drinking water of chickens. Well water containing 0, 0.016, 0.03, and 0.06% TC was inoculated with a 5-strain mixture of S. Enteritidis or C. jejuni (˜6 log₁₀ cells/mL). Water samples containing 1% chicken feces or feed were also included. The samples were incubated at 12.5 or 25°C for 7 d and analyzed for bacterial populations on d 0, 1, 3, 5, and 7. Duplicate samples of treatments and control were included, and the study was replicated 3 times. Trans-cinnamaldehyde at 0.06% inactivated Salmonella completely after 24 h in water with 1% feces at both temperatures. In water containing 1% feed, TC (0.06%) reduced S. Enteritidis to undetectable levels after 3 d at 12.5°C or 7 d at 25°C. Presence of feed or feces in water reduced the antibacterial effect (P < 0.001) of TC. The effect of TC on C. jejuni was more pronounced than that on S. Enteritidis. The TC at 0.06% completely inactivated the pathogen after 1 d of incubation at both temperatures. The presence of feces or feed did not have any effect (P > 0.001) on the antibacterial property of TC on C. jejuni. Results indicate that TC is effective in killing S. Enteritidis and C. jejuni in chicken drinking water and may decrease the likelihood that water will contribute to colonization of chickens by these pathogens.     

Comparison of ITS Profiling,REP‐ and ERIC‐PCR of Salmonella Enteritidis Isolates from Poland

Thirty‐one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP‐PCR, ERIC‐PCR and ITS profiling (PCR‐ribotyping). Analysis of DNA banding patterns generated by REP‐PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC‐PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC‐PCR fingerprints. REP‐ and ERIC‐PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S‐23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south‐west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP‐ and ERIC‐PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.     

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (10⁹ CFU – 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10⁸ CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.     

Natural antimicrobials for control of Salmonella Enteritidis in feed and in vitro model of the chicken digestive process

This study evaluated the antimicrobial effect of essential oils (EO) and organic acids (OA) against Salmonella Enteritidis in chicken feed and during an in vitro model that mimics the chicken digestive process. The minimal inhibitory concentration (MIC) of allyl isothiocyanate (AITC), carvacrol (CV), propionic acid (PROP) and caproic acid (CAP) were individually determined. Then, based on the MICs of each compound, combinations of EOs and/or OAs were tested to evaluate their synergic antimicrobial effect. The synergic effect of AITC and CAP was the most efficient against the bacterial strain tested. Commercial feed was inoculated with a 5‐strain cocktail of S. Enteritidis and treated with different doses of AITC + CAP to evaluate their effect on the growth/survival of the pathogen. In addition, the simulated digestion model was used to access the antimicrobial effect of AITC + CAP added to the feed towards S. Enteritidis and Lactobacillus plantarum. Synergistic effect was found between AITC (0.065 mM) and CAP (17.5 mM) against S. Enteritidis in chicken feed, where S. Enteritidis was reduced to undetectable levels (<1.00 log CFU/g). AITC (1.95 mM) + CAP (45 mM) also decreased (p < 0.05) the population of S. Enteritidis in the simulated digestion, while the growth of L. plantarum was not affected. Therefore, the addition of AITC + CAP in feed might be a potential natural antimicrobial able to prevent economic losses caused for Salmonella in chicken.     

Blocking the expression of syntaxin 4 interferes with initial phagocytosis of Brucella melitensis in macrophages

Brucella melitensis is the Brucella species most frequently associated with brucellosis in humans. It is also the causative agent of the disease in goats and other ruminants. Although significant aspects of the pathogenesis of infection by this intracellular pathogen have been clarified, several events during invasion of host cells remain to be elucidated. In this study, infections of human macrophages from the THP-1 monocyte cell line were conducted with B. melitensis Bm133 wild-type strain and a strain of Salmonella serovar Enteritidis as a control. A multiplicity of infection of 100 was used in trials focused on defining the relative expression of syntaxin 4 (STX4), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, in the early events of phagocytosis (at 15, 30, 45, and 60 min). Immunoblot assays were also done to visualize expression of the protein in cells infected with either bacterial strain. The expression of STX4 was not significantly different in cells infected with B. melitensis strain Bm133 compared to that observed in cells infected with S. Enteritidis. When the expression of STX4 mRNA was inhibited with short or small interfering, or silencing, RNA in the THP-1 cells, the survival of B. melitensis was significantly reduced at time 0, when gentamicin treatment of cultures was begun (after 1 h of phagocytosis), and also at 2 h and 12 h after infection.