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.     

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.     

Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups

This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 10⁸ colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 10⁶ CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine’s value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars.     

Risk factors of Salmonella infection in laying hens in Menoua Division,Western region of Cameroon (Central Africa)

Salmonella infections in poultry farms are overlooked in many African countries; yet these infections are mostly zoonotic with impact on both poultry industry and public health. Considering the impact of Salmonella in laying hens, and the role of laying hens as a source of Salmonella outbreak in human, knowledge of the status of Salmonella on laying hen farms as well as the factors influencing the presence of Salmonella is important. In a cross sectional study, cloacal swabs were collected from 270 commercial laying hens on 27 farms located in Menoua Division. These samples were cultured on standard media. A questionnaire was used to collect information on animals, farms and farmer’s characteristics. The prevalence of Salmonella was 93.34%; three zoonotic isolates namely S. Enteritidis (75.90%), S. Paratyphi (11.90%), and S. Typhimurium (5.60%) were identified. The location of farms was significantly associated with presence of Salmonella, and the risk of infection was 10-fold higher in Nkong-ni than Santchou (p < 0.05). Other potential risk factors such as flock size, age of the farm (infrastructure), or water source were not associated with Salmonella infection. The prophylactic measures against avian diseases in the country must include measures against Salmonella to protect poultry industry and public health.     

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.     

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.     

Temporal changes in the expression of avian β-defensins in the chicken vagina during sexual maturation and Salmonella infection

Avian β-defensins (AvβDs) constitute a family of antimicrobial peptides that are critical to innate immunity in chickens, providing protection against microbial pathogens including Salmonella Enteritidis (SE). As apart from the digestive tract another main route of SE colonization in birds is via infection of the oviduct and specifically of the vagina, the aim of this study was to investigate the expression of the complete family of AvβDs, in the chicken vagina in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the vaginal AvβDs expression. Expression analysis revealed that 11 members of the AvβD family were expressed in the chicken vagina. Quantitative real-time PCR analysis revealed that the mRNA abundance of five AvβDs was up regulated and of one AvβD was down regulated with respect to sexual maturation. In addition SE infection resulted in a significant induction of AvβD5, 7, 10, 11, 12 and 14 in the vagina of sexually mature birds, and in a significant induction of AvβD5 and 11 in the vagina of aged birds. These findings provide strong evidence to suggest that an AvβD-mediated immune response mechanism exists in the chicken vagina providing protection against bacterial pathogens including Salmonella species.     

Salmonella Infantis and Salmonella Enteritidis specific bacteriophages isolated form poultry faeces as a complementary tool for cleaning and disinfection against Salmonella

Salmonellosis represents an important public health concern. Several authors point out the inefficiency of the cleaning and disinfection protocols to remove the bacteria from the field. For this reason, innovative techniques, as bacteriophages, could be implemented to control the bacteria. The main objectives of this study were to assess the effect of bacteriophages against Salmonella Infantis and Salmonella Enteritidis on farm surfaces, and to evaluate bacteriophage procedure application as sanitiser against Salmonella in field conditions. Thus, most prevalent serovars in poultry production were selected (Salmonella Infantis and Salmonella Enteritidis) to contaminate farm facilities. Then, two specific bacteriophages isolated from poultry faeces were applied against them. Results showed Salmonella Infantis and Salmonella Enteritidis decreased of 4.55 log₁₀CFU/mL and 3.85 log₁₀CFU/mL, respectively; the maximum reduction in Salmonella was the 5^th day, after 10⁸ PFU/mL and 10³ PFU/mL bacteriophage application. These results highlight bacteriophages as a promising tool together with cleaning and disinfection.     

Evaluation of the colonization capabilities of Salmonella Enteritidis in Quails using an RT-PCR approach

We used a real-time PCR assay and indirect fluorescent antibody (IFA) assay to detect genomic DNA of Salmonella Enteritidis in the internal organs of quails after an oral challenge. The results showed that S. Enteritidis was detected in all the samples at different time points. This study will assist a future understanding of the pathogenesis of S. Enteritidis.     

Wild Bonelli’s eagles (Aquila fasciata) as carrier of antimicrobial resistant Salmonella and Campylobacter in Eastern Spain

Wild birds have repeatedly been found to be involved in the dissemination of enteric bacterial pathogens in the environment. The aim of this study was to determine the occurrence of Salmonella and Campylobacter as well as the antimicrobial resistance in wild Bonelli’s eagles nestlings in Eastern Spain. In addition, we compared the efficiency of two sampling methods (fresh faecal samples from nest and cloacal swabs from nestlings) for detection of both bacteria. A total of 28 nests with 45 nestlings were analysed. In the nest, Salmonella occurrence was 61 ± 9.2%, while Campylobacter occurrence was 11 ± 5.8% (p < 0.05). In the nestlings, Salmonella occurrence was 36 ± 7.1%, while Campylobacter occurrence was 11 ± 4.7% (p < 0.05). Eight Salmonella serovars were identified, and the most frequently isolated were S. Enteritidis, S. Typhimurium, S. Houston, and S. Cerro. Only one Campylobacter species was identified (C. jejuni). Regarding antimicrobial resistance, the Salmonella strains isolated were found to be most frequently resistant to ampicillin and to tigecycline; however, the sole Campylobacter strain recovered was multidrug resistant. In conclusion, this study demonstrated that wild Bonelli’s eagles nestlings are greater carriers of Salmonella than of Campylobacter. Both Salmonella and Campylobacter isolates exhibited antimicrobial resistance. In addition, faecal samples from nests were most reliable for Salmonella detection, while cloacal swab from nestlings were most reliable for Campylobacter detection.     

Use of blends of organic acids and oregano extracts in feed and water of broiler chickens to controlSalmonella Enteritidis persistence in the crop and ceca of experimentally infected birds

The aim of the present evaluation was to assess the efficacy of blends of organic acids and oregano extracts supplied from d 1 to 21 and from d 35 to 42 in feed, drinking water, or both, in controllingSalmonella Enteritidis persistence in the crop and ceca of broiler chicken. A total of 105 one-day-old male broiler breeder chicks were randomly distributed into 5 different treatments according to the supply of referred blends andSalmonella Enteritidis challenge.Salmonella Enteritidis was inoculated directly in the crop of birds at the 15 d of age. Treatments were unsupplemented unchallenged, unsupplemented challenged, supplemented through water and challenged, supplemented through feed and challenged, and supplemented through feed and water and challenged. Use of the additives in feed and water and in water alone efficiently controlledSalmonella shedding and reduced cecal persistence. Immune mechanisms involved are proposed.     

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.     

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.     

Effect of a mannanoligosaccharide-supplemented diet on intestinal mucosa T lymphocyte populations in chickens challenged withSalmonella Enteritidis

Salmonellosis is one of the most common worldwide zoonosis, and the Enteritidis and Typhimurium serotypes are most prevalent. In this study, we evaluated the effects of a diet supplemented with mannanoligosaccharides (MOS) on CD3+, CD4+, CD8+, and goblet cell counts in the intestines of broilers vaccinated and challenged withSalmonella Enteritidis. The evaluation was based on randomized experiments including 45 chickens divided into 3 experimental groups. Group 1 received a nonsupplemented control diet. Group 2 received a diet supplemented with 1 kg MOS/t from d 1 to 21 and 0.5 kg MOS/t from d 22 to 56. Group 3 received a diet supplemented with 2 kg MOS/t from d 1 to 21 and 1 kg MOS/t from d 22 to 56. Chickens fed the lower level of MOS demonstrated reduced fecal shedding ofSalmonella 14 d after being challenged (P < 0.05). Both groups of MOS-supplemented chickens showed an increase in CD4+ and CD8+ cell counts in the ileal and cecal mucosa after being challenged withSalmonella Enteritidis (SE). Supplementation with MOS increased the specific T lymphocyte infiltration in the intestinal mucosa of chickens vaccinated and challenged withSalmonella Enteritidis.     

Adhesion of Salmonella enterica serotype Enteritidis isolates to chicken isthmal glandular secretions

The ability of Salmonella enterica serotype Enteritidis isolates to adhere to immobilized secretions of the isthmus of the laying hen was determined in an ELISA-type assay. One-third of the 56 isolates tested in the logarithmic growth phase, adhered to the isthmal secretions. Using a binding assay of the isolates to thin paraffin sections of the oviduct, we demonstrated that the receptor of the adhesion was localized inside the tubular gland cells of the isthmus. The adhesion to immobilized isthmal secretions as well as to the paraffin sections was blocked by the addition of mannose. A fimD mutant of S. Enteritidis, lacking type 1 fimbriae, did not adhere, confirming that the adhesion was mediated by type 1 fimbriae. Mannosylated glycoproteins were demonstrated in the isthmus glandular cells using confocal laser scanning microscopy by FITC-labelled Lens culinaris lectins. It is hypothesized that the binding of S. Enteritidis to isthmal secretions could play a role in the contamination of eggs through incorporation of the bacteria in the shell membranes.     

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.     

Estimation of the Rate of Egg Contamination from Salmonella‐Infected Chickens

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent causes for human gastroenteritis and is by far the predominant Salmonella serovar among human cases, followed by Salmonella Typhimurium. Contaminated eggs produced by infected laying hens are thought to be the main source of human infection with S. Enteritidis throughout the world. Although previous studies have looked at the proportion of infected eggs from infected flocks, there is still uncertainty over the rate at which infected birds produce contaminated eggs. The aim of this study was to estimate the rate at which infected birds produce contaminated egg shells and egg contents. Data were collected from two studies, consisting of 15 and 20 flocks, respectively. Faecal and environmental sampling and testing of ovaries/caeca from laying hens were carried out in parallel with (i) for the first study, testing 300 individual eggs, contents and shells together and (ii) for the second study, testing 4000 eggs in pools of six, with shells and contents tested separately. Bayesian methods were used to estimate the within‐flock prevalence of infection from the faecal and hen post‐mortem data, and this was related to the proportion of positive eggs. Results indicated a linear relationship between the rate of contamination of egg contents and the prevalence of infected chickens, but a nonlinear (quadratic) relationship between infection prevalence and the rate of egg shell contamination, with egg shell contamination occurring at a much higher rate than that of egg contents. There was also a significant difference in the rate of egg contamination between serovars, with S. Enteritidis causing a higher rate of contamination of egg contents and a lower rate of contamination of egg shells compared to non‐S. Enteritidis serovars. These results will be useful for risk assessments of human exposure to Salmonella‐contaminated eggs.