Identification of possible chicken intestinal mucosa receptors for SEF21-fimbriated Salmonella enterica serovar Enteritidis

In order to test whether glycosphingolipids (GSLs) on the chicken intestinal mucosa serve as a receptor for Salmonella enterica serovar Enteritidis with fimbriae, we analyzed neutral GSLs and gangliosides from chicken intestinal mucosa and investigated the binding of bacteria to neutral GSLs and gangliosides. Four kinds of neutral GSLs, designated as N-1 to N-4 and four kinds of gangliosides, named G-1 to G-4, were identified on high-performance thin-layer chromatography (HPTLC) plates. In TLC immunostaining tests, fimbriated S. Enteritidis bound only to glucosylceramide (GlcCer) standard, N-1, GM3 standard and G-1, but neither to N-2, N-3, N-4, nor to G-2, G-3 and G-4. Further, the bacterial binding to N-1 and G-1 was completely inhibited by preincubation of bacteria with anti-S. Enteritidis fimbriae (SEF) 21 antibody, but not by anti-SEF14 antibody. These results suggest that both GlcCer (N-1) and ganglioside GM3 (G-1) on the epithelial cell surfaces of chicken intestine act as receptors for fimbriated S. Enteritidis.     

Analysis of neutral glycosphingolipids from Trypanosoma brucei

Neutral glycosphingolipids (GSLs) were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC), TLC/secondary ion mass spectrometry (TLC/SIMS), and liposome immune lysis assay (LILA). Three species of neutral GSLs, designated as N-1, -2, and -3 were separated on TLC. N-1 GSL migrated very close to glucosylceramide (GlcCer) and N-2 GSL showed the same mobility as lactosylceramide (LacCer). On the other hand, the mobility of N-3 GSL on the TLC plate was slower than globotetraosylceramide (Gb4). In order to characterize the molecular species of neutral GSLs from T. brucei, N-1, -2 and -3 GSLs were analyzed by TLC/SIMS. The TLC/SIMS analysis of N-1 of the parasites revealed a series of (M–H)⁻ ions from m/z 698 to 825 representing the molecular mass range of ceramide monohexoside (CMH) (GlcCer or galactosylceramide). On the other hand, the TLC/SIMS spectra of N-2 GSL revealed a series of (M–H)⁻ ions from m/z 944–987 indicating the molecular mass range of LacCer. In the TLC/SIMS analysis of N-3 GSL, however, the characteristic molecular ions that can elucidate the structure of N-3 GSL were not obtained. In order to confirm the results obtained from TLC/SIMS, N-1, -2, and -3, GSLs were tested by LILA with specific antibodies against GlcCer, LacCer, and Gb4, respectively. N-1 GSL had reactivity to anti-GlcCer antibody and N-2 GSL reacted with the antibody against LacCer. However, N-3 GSL was not recognized by anti-Gb4 antibody. Using anti-GlcCer and anti-LacCer antibodies, furthermore, we studied the expression of GlcCer and LacCer in T. brucei parasites. Both GlcCer and LacCer were detected on the cell surface of T. brucei.     

Binding of Vibrio anguillarum to neutral glycosphingolipids from intestinal mucosa of rainbow trout (Oncorhynchus mykiss)

To test whether glycosphingolipids (GSLs) on the intestinal mucosa of rainbow trout (Oncorhynchus mykiss) serve as a binding receptor for Vibrio anguillarum, we analyzed neutral GSLs from rainbow trout intestinal mucosa and investigated the binding of bacteria to neutral GSLs. Two kinds of neutral GSLs, designated N-1 and N-2, were identified on high-performance thin-layer chromatography (TLC) plates. In TLC immunostaining tests, V. anguillarum bound only to galactosylceramide (GalCer), lactosylceramide and N-1 having the same TLC mobility as GalCer, but neither to glucosylceramide nor to N-2. These results suggest that N-1 is GalCer (Gal beta 1-1Cer) and also that N-1 (GalCer) on rainbow trout intestinal mucosa act as a receptor for V. anguillarum.     

Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens

A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.     

Entry and survival of Salmonella enterica serotype Enteritidis PT4 in chicken macrophage and lymphocyte cell lines

Various leukocytes are involved in the reaction to counter Salmonella infection in chicken. The various leukocyte types react differently after an infection, since some clear the infection while others may cause dissemination of Salmonella throughout the chicken. Therefore, we investigated in vitro the entry and survival of Salmonella enterica serotype Enteritidis in chicken cell lines of various cell types, including two macrophage cell lines, HD11 and MQ-NCSU (NCSU), two B-cell lines LSCC-1104-X5 (1104) and LSCC-RP9 (RP9), and a T-cell line MDCC-MSB-1 (MSB-1). The macrophages were able to internalize high numbers of S. Enteritidis. In contrast and as expected, cells of the T-cell line MSB-1 and the B-cell line RP9 internalized bacteria at a much lower level. After S. Enteritidis entered the macrophages, the number of intracellular S. Enteritidis decreased over time, so that after 48h no more than 20% of the bacteria, which had entered, survived intracellularly. In contrast to macrophages, the number of S. Enteritidis in cells of the T-cell line MSB-1 and the B-cell line RP9 increased rapidly within 12h post-inoculation. Thereafter the number of intracellular S. Enteritidis decreased only slowly. In conclusion, all three different cell types were able to control and to start clearing S. Enteritidis, although macrophages were far more effective compared to T- and B-cells. However, none of the cell lines were able to clear S. Enteritidis fully within 48h. These results suggest that the three cell types play an important but different role in the dissemination and elimination of S. Enteritidis throughout the animal.     

Preliminary evaluation of the use of the sefA fimbrial gene to elicit immune response against Salmonella enterica serotype Enteritidis in chickens

In the last 2 decades, the prevalence of Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) has dramatically increased worldwide, becoming the leading cause of food-borne illnesses and an important public health issue. Many studies have suggested the role of the SEF14 fimbrial protein in the adhesion of Salmonella Enteritidis to the host. In the present study, the sefA gene, which encodes the main subunit of the SEF14 fimbrial protein, was cloned into a temperature-sensitive expression vector and transformed into a nonpathogenic, avirulent strain of Escherichia coli. The recombinant strain was used as a vaccine to elicit specific immune response against the SefA protein of Salmonella Enteritidis in 1-day-old chickens. The recombinant strain was reisolated from the intestines of treated birds for up to 21 days posttreatment, demonstrating its ability to colonize the intestinal tracts of 1-day-old chickens. In addition, immunoglobulin A (IgA) against the SefA protein was detected in intestinal secretions from treated birds at 7 days posttreatment and in bile samples from 14 to 21 days posttreatment by enzyme-linked immunosorbent assay. Nontreated birds did not show any evidence of intestinal colonization by the recombinant strain or anti-SefA IgA response in their bile or intestinal secretions. Preliminary evaluation of the recombinant strain showed a potential use of this strain to elicit protection against Salmonella Enteritidis infection in chickens. Further experiments are needed to study the ability of the recombinant strain to protect birds against Salmonella Enteritidis colonization.     

Immune response to liposome-associated recombinant SEF21 following oral immunization in chickens

In order to generate Salmonella enterica serovar Enteritidis fimbriae antigens (rSEF21), the intact region encoding SEF21 was amplified from Salmonella Enteritidis by PCR and subcloned into a prokaryotic expression vector pET-28a(+) to yield pET-28a(+)-SEF21. The rSEF21 protein was highly expressed and purified by nickel affinity chromatography. Liposomeassociated rSEF21 was prepared for oral immunization to seek protective efficacy for intestinal infection with Salmonella Enteritidis. Evidence of IgA and IgG responses were found in the intestinal tracts and in the sera of a group of chickens immunized. Two weeks after the booster immunization, the chickens were challenged orally with 2 x 10(6) colony-forming units of live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 4 wk after challenge. The numbers of bacteria in the intestinal contents (cecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Therefore, oral immunization with liposome-associated rSEF21 elicits both systemic and mucosal antibody responses, leading to a reduction in bacterial colonization in the intestinal tract and excretion of Salmonella Enteritidis in the feces.     

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.     

Expression of Putative Escherichia coli Heat-labile Enterotoxin (LT) Receptors on Intestinal Brush Borders from Pigs of Different Ages

Many strains of enterotoxigenic Escherichia coli (ETEC) that cause diarrhoea in young piglets secrete a heat-labile enterotoxin (LTp) that binds to specific glycoconjugates on porcine intestinal epithelial cells. Binding of LTp to an appropriate glycoconjugate facilitates the uptake and trafficking of the toxin into the cell, where it stimulates intracellular changes that promote fluid secretion and diarrhoea. The objective of the current study was to identify the LTp-binding glycoconjugates on porcine intestinal epithelial cells, the natural target cells for LTp. We found that LTp binds, in an age-correlated manner, to an acidic glycosphingolipid (GSL) that co-migrated with GM1 on thin-layer chromatography (TLC), a small acidic GSL that appears to be a sulphatide, a neutral GSL that co-migrated with neolactotetraglycosylceramide (nLc4) on TLC, and two glycoproteins (36 and 205 kDa). Of these potential LTp receptors, the GM1-co-migrating GSL was detected most intensely in young animals, while the other four LTp-binding glycoconjugates were detected most intensely in older pigs (54 weeks). Since ETEC primarily cause disease in young piglets, the GM1-co-migrating GSL is the most likely candidate for a functional LTp receptor.     

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.     

An epidemiological analysis of Salmonella enteritidis contamination in a rat-infested chicken layer farm, an egg processing facility, and liquid egg samples by pulsed-field gel electrophoresis

In order to determine the epidemiological link between the Salmonella Enteritidis contamination in a rat-infested chicken layer farm, an attached egg processing facility and liquid egg samples, several S. Enteritidis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. A total of 33 S. Enteritidis strains were isolated from a total of 4,081 samples. Similar pulsed-field patterns were generated by S. Enteritidis isolates from liquid eggs, rats and effluent water. Additionally, only two phage types were detected among the S. Enteritidis isolates, PT 1b and PT 6. These results suggest that S. Enteritidis isolates from rats, egg processing facility, and liquid eggs are genetically related. Furthermore, S. Enteritidis infection in rats in layer farms poses a serious public health concern and should be included in future epidemiological studies.     

The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nalr) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nalr and PCP separately there were no significant differences in the numbers of S1400/94 Nalr and PCP bacteria in tissues sampled on days 1, 2, and 5. By day 42 after challenge S1400/94 Nalr bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nalr. In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nalr. In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays an important role in poultry infections although this role is less important in the newly hatched chick.     

Comparison of real-time PCR with conventional PCR and culture to assess the efficacy of a live attenuated Salmonella enterica serovar Typhimurium vaccine against Salmonella enterica serovar Enteritidis in commercial leghorn chicks vaccinated under field and laboratory conditions

The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.     

Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing mannose-sensitive FimH adhesin from Salmonella enterica serovar Enteritidis

To investigate the role of non-hemagglutinating type 1 fimbriae in the pathogenesis of Salmonella Gallinarum, the isogenic mutant elaborating type 1 fimbriae with mannose-sensitive (MS) variant of the FimH adhesin from Salmonella Enteritidis and the mutant strain with no FimH expression were constructed. Their binding to chicken leukocytes in vitro and invasiveness in 1-day-old chicks were studied. Our results demonstrated that S. Gallinarum type 1 fimbriae with an endogenous variant of the FimH adhesin mediated mannose-resistant (MR) binding to avian leukocytes and did not bind to human epithelial cells. However, after allelic replacement of the FimH, mutated fimbriae with S. Enteritidis variant of the FimH adhesin bound to both cell types in a mannose-dependent manner. In chick model, S. Gallinarum expressing wild-type FimH variant colonized cecal tonsils and bursa of Fabricius more effectively and invaded the spleen and liver in greater numbers than S. Gallinarum fimH knockout strain or mutant expressing MS FimH variant from S. Enteritidis. The invasive potential of the latter was greatly reduced in chicks since no viable bacteria expressing MS variant of the adhesin could be recovered from intestinal lymphoid tissues or liver over a 6 days course of infection. Together, these results demonstrate that the S. Gallinarum type 1 fimbriae with the endogenous MR variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of these adhesive structures in the virulence of S. Gallinarum.     

Influence of enrofloxacin and chloramphenicol on the level of IgY in serum and egg yolk after immunostimulation of hens with Salmonella enteritidis antigens

In the chicken, maternal antibodies are transferred into the egg and subsequently transported into the developing embryo. IgG (called IgY) is the primary immunoglobulin isotype of the egg yolk. Their level in serum depends on the correct function of immunological system in laying hens. Many factors have a direct or indirect influence on antibody level in fowl. One of them is a commonly used antibiotic, but its influence on avian immune system is still unknown. The objective of the study was to determine the effect of enrofloxacin and chloramphenicol on the level of IgY antibody in serum and egg yolk after immunostimulation of hens with living cells of Salmonella enterica subsp. enterica serovar Enteritidis and lipopolisaccharide. Forty adult egg-laying Arbor Acres and Isa 215 hens (32 and 50 weeks old) from the reproductive flocks and 1640 of their eggs were used for the investigation. No clinical symptoms of any diseases were observed in birds during the entire breeding period. Additionally the birds were checked as free from Salmonella spp. in the beginning of the experiment. The birds were divided into 6 experimental and 2 control groups (5 birds in one group). The hens in the experimental groups were immunized with S. Enteritidis antigens: living bacteria and lipopolisaccharide and treated with enrofloxacin or chloramphenicol. Antibiotics were administered in drinking water for 10 days (from 3rd to 13th day of experiment). To indicate anti-S. Enteritidis, antibodies in sera and egg yolk were used indirectly on ELISA based on lipopolisaccharide from S. Enteritidis. As conjugate these were applied anti-chicken IgY with horseradish peroxidase and ABTS with H2O2 as obtained. Additionally, to detect antibody in serum, a rapid slide test was used with Pullognost and Enterognost standard antigens made in the laboratory. The study revealed that both antibiotics tested decreased the level of specific IgY in laying hens immunized with living bacteria and lipopolisaccharide. It seems that antibiotics have a suppressive effect on the immunological system. The strongest immunosuppressive effect was exerted by chloramphenicol.     

Simplified preparation of a specific S. enteritidis antigen for ELISA and other immunological techniques

This study was conducted to prepare a specific S. enteritidis antigen (FG-Antigen) for the serological detection of S. enteritidis infections in chicken flocks. This antigen (FG-Antigen) consistent mainly of the flagellar fraction H:g and partly of the fimbrial fraction SEF14 from a S. enteritidis-phage type 4 strain. The initial steps followed in the preparation of this antigen were conducted based on a previously described procedure, which involved the application of heat at 60 degrees C. The purification process (filtration and concentration) enabled the exclusion of the cross-reaction causing LPS antigens from the preparation and allowed the retention of S. enteritidis-specific antigens composed of fimbria and H:g fractions. As a result, no cross-reaction with S. typhimurium nor with S. gallinarum was exhibited by the prepared FG-antigen. To characterize and determine its specificity, the following laboratory tests were conducted: indirect ELISA, immunoblotting and a SEF14 agglutination test. In these examinations, rabbit and chicken reference sera as well as chicken field sera and absorbed hyperimmune sera against H:g-carrying serovars were used.     

Characterization of Salmonella spp. isolated from an integrated broiler chicken operation in Korea

The purpose of this study was to investigate the biological and genetic characterization of persistent Salmonella isolates in an integrated broiler chicken operation, in an attempt to elucidate the source of contamination. From the breeder farm, the hatchery, the broiler farm and the chicken slaughter house of an integrated broiler chicken operation, a total of 6 serotypes were observed. Although S. Heidelberg was not detected in the broiler farm, it was consistently found in the breeder farm, the hatchery and the chicken slaughter house. Also, S. Enteritidis and S. Senftenberg were found in the hatchery and the chicken slaughter house, and the hatchery and the broiler farm, respectively. S. Gallinarum and S. Blockley were found only in the broiler farm, and S. Virchow was only recovered in the chicken slaughter house. Isolated S. Heidelberg, S. Enteritidis and S. Senftenberg strains were divided into 3, 5 and 7 types, respectively, on the basis of all properties. Especially, S. Senftenberg isolates, divided into four types by their antimicrobial resistance patterns, were all obviously the XbaI PFGE pattern. Also, four S. Enteritidis isolates resistant to nalidixic acid showed a difference in phage type and PFGE pattern. Such a different pattern was shown despite Salmonella isolates originating from an integrated broiler operation, suggesting that further epidemiological studies on many integrated chicken companies in Korea are needed.     

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.     

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.