Étude de l’efficacité de la vaccination à Salmonella Enteritidis chez la poule pondeuse et de la protection contre l’infection

Les infections à Salmonella Enteritidis chez les humains sont associées à la consommation d’œufs ou d’ovoproduits contaminés. La vaccination est un outil utilisé pour diminuer les risques d’infection à SE chez la volaille, mais avec des résultats variables. Au Canada deux bactérines, MBL SE4C et Layermune, sont couramment utilisées pour lutter contre SE. Cependant, leur efficacité n’a pas été complètement déterminée chez les poules pondeuses plus âgées. Par ailleurs, la capacité de ces vaccins à prévenir la transmission verticale et horizontale n’a pas encore été étudiée. L’objectif principal de cette étude était d’évaluer l’effet des deux bactérines sur la réponse immunitaire chez les poules pondeuses, de vérifier la protection conférée par ces vaccins contre l’infection expérimentale à SE, et d’identifier des protéines immunogènes afin de développer un vaccin sous-unitaire. Les oiseaux ont été vaccinés avec deux protocoles d’immunisation en cours d’élevage (soit à 12 et 18, ou à 16 semaines d’âge). Le groupe contrôle a été injecté avec la solution saline. Les oiseaux ont été inoculés per os avec 2 x 109 CFU de la souche SE lysotype 4 à 55 ou à 65 semaines d’âge. Les anticorps (IgG et IgA) ont été mesurés à différents temps avec un ELISA maison en utilisant l’antigène entier de SE. La phagocytose, flambée oxydative, les populations des splénocytes B et T ont été analysées en utilisant la cytométrie en flux. Les signes cliniques, l’excrétion fécale, la contamination des jaunes d’œufs et l’invasion des salmonelles dans les organes ont été étudiés pour évaluer l’efficacité de protection. La transmission horizontale a aussi été étudiée en évaluant l’infection à SE chez les oiseaux mis en contact avec les oiseaux inoculés. Les protéines immunogènes ont été identifiées par SDS-PAGE et Western blot à l’aide d’antisérums prélevés suite à la vaccination et/ou à l’infection expérimentale/naturelle, puis caractérisées par la spectrométrie de masse. Le protocole de vaccination avec deux immunisations a généré un niveau élevé de séroconversion à partir de 3 jusqu’à 32-34 semaines post-vaccination par rapport à celui avec une seule immunisation (p < 0.02), mais il n’y avait plus de différence entre les groupes à 54 et 64 semaines d’âge. Il n’y a pas eu de corrélation entre les niveaux d’IgG et les taux d’isolement des salmonelles dans les organes et des jaunes d’œuf. La production des IgA n’a été observée que chez les oiseaux vaccinés avec 2 injections de MBL SE4C (p ≤ 0.04). Après l’infection expérimentale, la production des IgA a été significativement plus élevée aux jours 1 et 7 p.i dans l’oviducte des oiseaux vaccinés (sauf pour le groupe vacciné avec 2 injections de Layermune) par comparaison avec le groupe contrôle (p ≤ 0.03). Seule la bactérine MBL SE4C a eu un effet protecteur contre la contamination des jaunes d’œuf chez les oiseaux infectés. Ce vaccin réduit partiellement en utilisant deux immunisations, le taux d’excrétion fécale des salmonelles chez les oiseaux inoculés et les oiseaux horizontalement infectés (p ≤ 0.02). Cinq des protéines identifiées par la spectrométrie de masse sont considérées comme des protéines potentiellement candidates pour une étude plus approfondie de leur immonogénicité: Lipoamide dehydrogenase, Enolase (2-phosphoglycerate dehydratase) (2-phospho-D-glycerate hydro-lyase), Elongation factor Tu (EF-Tu), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et DNA protection during starvation protein. En général, les bactérines ont induit une immunité humorale (IgG et IgA) chez les poules pondeuses. Cette réponse immunitaire a protégé partiellement les oiseaux quant à l’élimination des salmonelles, la contamination des jaunes d’œuf, ainsi que la transmission horizontale. Dans cette étude, la bactérine MBL SE4C (avec deux immunisations) s’est montrée plus efficace pour protéger les oiseaux que la bactérine Layermune. Nos résultats apportent des informations objectives et complémentaires sur le potentiel de deux bactérines pour lutter contre SE chez les poules pondeuses. Étant donné la protection partielle obtenue en utilisant ces vaccins, l’identification des antigènes immunogènes a permis de sélectionner des protéines spécifiques pour l’élaboration éventuelle d’un vaccin plus efficace contre SE chez les volailles.

Production and immunogenicity of selected proteins of Salmonella Enteritidis

Au cours des dernières années, Salmonella Enteritidis est devenus les sérotypes les plus souvent isolés chez les patients canadiens, les cas étant liés à la consommation de viande de poulet et d’œufs crus. Les vaccins tués commercialement disponibles pour la volaille, stimulent mal l'immunité mucosale, tandis que l'utilisation de vaccins vivants reste controversée. Par conséquent, un vaccin sous-unitaire par voie orale peut être une solution. Cinq protéines bactériennes ont été choisies comme candidates potentielles et identifiées, soit Glyceraldehyde-3-phosphate dehydrogenase, Enolase, Lipoamide dehydrogenase, DNA protection during starvation protein et Elongation factor-Tu. Notre objectif a été de produire et de purifier ces protéines et de démontrer leur immunogénicité. Les gènes des protéines ont été amplifiés et clonés dans le vecteur pQE-30 pour expression dans Escherichia coli M15. La purification a été effectuée par FPLC. Des poules pondeuses SPF ont été séparées en 6 groupes et injectées par voie intramusculaire à different âges avec une des 5 protéines, ou le PBS chez le groupe témoin. Les œufs ont été ramassés pendant l'expérience et du sang a été prélevé à 36 semaines d'âge. Les anticorps IgY ont été extraits à partir du jaune d'oeuf et du sérum, et les IgA à partir du blanc d'oeuf. Des immunodots, westernblots et ELISA ont évalué l'immunogénicité des protéines et les niveaux d'anticorps induits . Nous avons constaté que ces cinq protéines pourraient stimuler la production d'anticorps spécifiques in vivo. GAPDH, Enolase et DPS ont induit des titres d'anticorps plus élevés que LpdA et EF-Tu.

Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retails markets of Coimbatore, South India

A study was conducted to determine the incidence of Salmonella enterica serovar Enteritidis and other Salmonella serovars on eggshell, egg contents and on egg-storing trays. A total of 492 eggs and 82 egg-storing trays were examined over a period of 1 year from different retail outlets of a residential area of Coimbatore city, South India. Salmonella contamination was recorded in 38 of 492 (7.7%) eggs out of which 29 was in eggshell (5.9%) and 9 in egg contents (1.8%). Around 7.5% of the egg-storing trays were also found to be contaminated with Salmonella. Serotyping of the Salmonella strains showed that 89.7% of the strains from eggshell, 100% of the strains from egg contents and 71.4% of the strains from egg-storing trays were Salmonella Enteritidis. Other serovarvars encountered were S. Cerro, S. Molade and S. Mbandaka from eggshell and S. Cerro from egg-storing trays. Seasonal variations in the prevalence pattern were identified with, a higher prevalence during monsoon months followed by post-monsoon and premonsoon. Further examination of the Salmonella strains was carried out by testing their antimicrobial sensitivity against 10 commonly used antimicrobials. Results revealed high prevalence of multiple antimicrobial resistance among these strains suggesting possible prior selection by use of antimicrobials in egg production

Salmonella cross contamination in retail chicken outlets and the efficacy of spice extracts on salmonella enteritidis growth inhibhition on various surfaces

The source of samonella cross contamination in 15 retail chicken outlets in aresidual area in coimbatore city ,sourthern India was studied. Chopping boards and the butchers hands were predominant followed by knives and the weighing balance tray. Serotyping of the salmonella strains revealed that all strains were salmonella enteritis, except one which was found to be salmonella cerro.The anti bacterial activity of commonly used spices were evaluated.

Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retails markets of Coimbatore, South India

A study was conducted to determine the incidence of Salmonella enterica serovar Enteritidis and other Salmonella serovars on eggshell, egg contents and on egg-storing trays. A total of 492 eggs and 82 egg-storing trays were examined over a period of 1 year from different retail outlets of a residential area of Coimbatore city, South India. Salmonella contamination was recorded in 38 of 492 (7.7%) eggs out of which 29 was in eggshell (5.9%) and 9 in egg contents (1.8%). Around 7.5% of the egg-storing trays were also found to be contaminated with Salmonella. Serotyping of the Salmonella strains showed that 89.7% of the strains from eggshell, 100% of the strains from egg contents and 71.4% of the strains from egg-storing trays were Salmonella Enteritidis. Other serovarvars encountered were S. Cerro, S. Molade and S. Mbandaka from eggshell and S. Cerro from egg-storing trays. Seasonal variations in the prevalence pattern were identified with, a higher prevalence during monsoon months followed by post-monsoon and premonsoon. Further examination of the Salmonella strains was carried out by testing their antimicrobial sensitivity against 10 commonly used antimicrobials. Results revealed high prevalence of multiple antimicrobial resistance among these strains suggesting possible prior selection by use of antimicrobials in egg production

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized L-histidine, L-glutamine, L-proline, L-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Studies into the role of the SEF14 fimbrial antigen in the pathogenesis of Salmonella enteritidis

To investigate the role of the SEF14 fimbrial antigen in pathogenesis, a single defined sefA (SEF14(-)) inactivated mutant of Salmonella enteritidis strain LA5 was constructed and tested in a number of biological assay systems. There was no significant difference between the wild-type strain and the isogenic SEF14(-) mutant in their abilities to adhere to and invade HEp-2 epithelial cells or their survival in mouse peritoneal macrophages, whereas the SEF14(-) mutant was ingested more rapidly by isolated human PMN. Both the strains colonized the intestine, invaded and spread systemically in 1 day-old chicks, laying hens and BALB/c mice equally well. A significantly greater number of chicks excreted the wildtype SEF14(+) strain during the first week following infection as compared to those infected with the SEF14(-) mutant. However, similar numbers of chicks excreted the two strains between 2 and 7 weeks after infection. These results indicate that possession of SEF14 fimbriae alone do not appear to play a significant role in the pathogenesis of S. enteritidis although its contribution to virulence may be dependent on the host species infected. (C) 1996 Academic Press Limited

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25 degrees C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17(-) mutans at 25 degrees C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17(-) mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

Expression of SEF17 fimbriae by Salmonella enteritidis

Specific immunological reagents were used to investigate the expression of SEF17 fimbriae by cultured strains of Salmonella enteriditis. Most strains of Salm. enteritidis tested expressed SEF17 when cultured at temperatures of 18-30 degrees C. However, two wild-type strains produced SEF17 when also grown at 37 degrees C and 42 degrees C. Colonization factor antigen agar was the optimum medium for SEF17 expression, whereas Drigalski and Sensitest agars poorly supported SEF17 production. Very fine fimbriae produced by a strain of Salm. typhimurium were specifically and strongly labelled by SEF17 monoclonal and polyclonal antibodies, indicating considerable antigenic conservation between the two. Curli fimbriae from Escherichin coli were similarly labelled. The production of these fimbriae corellated with the binding of fibronectin by the organism. Congo red binding by cultured bacteria was not a reliable criterion for the expression of SEF17 fimbriae.

Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis

Salmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.

Contribution of fimbriae and flagella of Salmonella enteritidis to colonization and invasion of chicks

Isogenic mutants of Salmonella enteritidis defective for the elaboration of fimbrial types SEF14, SEF17, SEF21 and flagella were used to study the contribution these organelles made to colonization, invasion and lateral transfer in young chicks. The caecum, liver and spleen were colonized within 24 h following oral inoculation of 1-day-old chicks with 10(5) wild-type S. enteritidis strain LA5. However, for some mutants, the numbers of organisms recovered from internal organs was reduced significantly, particularly at 24 h post-inoculum, which supported the hypothesis that the organelles contribute to invasion and dissemination to internal organs. Specifically, mutations affecting SEF17, SEF21 and flagella contributed to a delay in colonization of the spleen, and those affecting SEF21 and flagella delayed colonization of the liver. Lower numbers of bacteria were recovered from the caecum with mutants deficient in elaboration of SEF21. Sentinel birds were colonized by LA5 or EAV40 (14(-), 17(-), 21(-), fla(-)) directly from the environment within 2 days, although a consistent slight delay was observed with the multiple mutant. Overall, our data suggest a collective role for SEF17, SEF21 and flagella, but not SEF14, in the early stages of colonization and invasion of young chicks by S. enteritidis, but these surface appendages appear unnecessary for colonization of birds from their immediate environment.

Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degreesC for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degreesC, or 250 cells per egg when eggs were stored at 30 degreesC, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs. (C) 2001 Elsevier Science B.V. All rights reserved.

Caracterização por susceptibilidade a antimicrobianos, PCR-ribotipificação e RAPD de Salmonella Enteritidis envolvidas em surtos de doenças veiculadas por alimentos ocorridas no Rio Grande do Sul, nos anos de 2001 e 2002

A Salmonella é uma das principais causas de doenças transmitidas por alimentos em todo o mundo, sendo que no Rio Grande do Sul ela tem sido apontada como o principal agente de toxinfecções alimentares nos últimos anos. Nesse trabalho, foram caracterizadas linhagens de Salmonella isoladas de alimentos envolvidos em Salmoneloses ocorridas no Rio Grande do Sul, no período de 2001 a 2002. Entre os 85 isolados investigados, 79 (93%) foram sorotipificados como S. Enteritidis, enquanto os outros seis isolados foram classificados como S. Javiana (n=1), S. Infantis (n=1), S. Agona (n=1), S. Typhimurium (n=1) e S. enterica subsp. enterica (1,4,5) (n=2). A resistência das amostras de S. Enteritidis a dez agentes antimicrobianos foi investigada. De modo geral, altas porcentagens de sensibilidade foram verificadas. As maiores porcentagens de resistência foram apresentadas em relação ao ácido nalidíxico (21,5%), à gentamicina (12,7%) e à estreptomicina (11,4%). A resistência a um ou mais antimicrobianos foi verificada em 30 amostras (37,97%), o que permitiu que os isolados fossem agrupados em 32 perfis de susceptibilidade. Apenas duas amostras apresentaram resistência múltipla a quatro drogas. Quando os isolados de S. Enteritidis foram submetidos à PCR-Ribotipificação, somente dois perfis (R1 e R2) foram identificados, sendo que o perfil R1 agrupou 92,4% dos isolados. . Os mesmos isolados também foram analisados por RAPD, sendo possível identificar quatro perfis de bandas (A a D). O perfil A agrupou 81% das amostras, enquanto os perfis B, C e D agruparam 9%, 5% e 5% dos isolados, respectivamente. Os resultados das análises de PCR-Ribotipificação e de RAPD sugerem que uma mesma linhagem de S. Enteritidis foi isolada a partir de alimentos envolvidos em Salmoneloses ocorridas em diferentes cidades do Estado durante o período de 2001 a 2002.