Influence of Salmonella enterica serovar enteritidis infection on the development of the cecum microbiota in newly hatched chicks.

Terminal restriction fragment length polymorphism and quantitative PCR showed that the cecal microbiota of chicks up to the age of 21 days was dominated by representatives of the orders Enterobacteriales, Clostridiales, and Lactobacillales. Salmonella enterica serovar Enteritidis infection caused the greatest changes in the gut microbiota when 1-day-old chicks were infected, compared with the infection of 4- and 16-day-old chicks.

Vaccination of chickens with Salmonella Pathogenicity Island (SPI) 1 and SPI2 defective mutants of Salmonella enterica serovar Enteritidis.

In this study we were interested in the vaccine potential of two attenuated mutants of Salmonella enterica serovar Enteritidis for poultry. The first mutant was attenuated by the removal of the whole Salmonella Pathogenicity Island 1 (SPI1) and the second mutant was devoid of the whole SPI2. These 2 mutants were used for oral vaccination of 2 chicken lines; Lohmann Brown and ISA Brown. Chickens were vaccinated orally on day 1 of life, revaccinated on day 21 and challenged on day 42. The challenge was performed either orally or intravenously. Despite a slightly different response between the two chicken lines, both the mutants gave protection to poultry against S. Enteritidis challenge as documented by findings such as the bacterial counts in tissues, spleen weight, antibody production and cytokine response (namely IL-17 and IL-22). When the 2 mutants were compared, vaccination with the SPI1 mutant proved to be more effective in the protection of poultry against S. Enteritidis challenge than the vaccination with the SPI2 mutant. On the other hand, vaccination with the SPI2 mutant stimulated a slightly higher antibody production and such a mutant might therefore be a better choice if Salmonella is used as a vector for the delivery of heterologous antigens with a desired stimulation of the humoral part of the immune system.

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils.

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1ß and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.

Comparative analysis of Salmonella enterica serovar Enteritidis mutants with a vaccine potential.

If any new live Salmonella vaccine is introduced in the future, it is quite probable that detailed characterisation of its attenuation will be required. In this study we therefore compared 34 isogenic mutants of S. Enteritidis in aroA, aroD, galE, ssrA, sseA, phoP, rpoS, ompR, htrA, clpP, lon, rfaL, rfaG, rfaC, hfq, sodCI, hilA, sipA, avrA, sopB, sopA, sopE, sifA, shdA, fliC, fur, relA, spoT, rel-spoT, misL, rmbA, STM4258, STM4259 and spvBC genes for their resistance to stresses likely to be expected in the host and for their virulence and immunogenicity in Balb/C mice. We found that the cold and bile resistances essentially did not correlate with the resistances to other stress factors. Resistance to acid pH, heat, polymyxin and serum correlated with each other and also with the attenuation. When the residual virulence and immunogenicity were both considered, mutants in htrA, ompR, aroA, aroD and lon performed the best in mice. Furthermore, when a detailed comparison of polymyxin and serum sensitive mutants was performed, the serum sensitive mutants were more immunogenic.

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection.

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.

Evolution of antibiotic resistance in Salmonella enterica serovar typhimurium strains isolated in the Czech Republic between 1984 and 2002.

In a collection of 66 Salmonella enterica serovar Typhimurium strains isolated between 1984 and 2002 in the Czech Republic, genes coding for antibiotic resistance were determined by using specific PCRs. We found that the pentadrug-resistant ACSSuT clone first appeared in the Czech Republic in 1990. A new variant of the aadA gene designated aadA21 is described, the 5' end of which was identical to aadA2 and the 3' end of which was identical to aadA1.

Rapid detection of Salmonella in field samples by nested polymerase chain reaction.

A simple and universal protocol for the rapid detection of Salmonella spp. in various samples using nested polymerase chain reaction (PCR) was developed. The protocol takes advantage of the rapid purification and concentration of Salmonella by centrifugation onto a layer of 60% sucrose solution. DNA was released by treatment with proteinase K and Triton X-100. Even without pre-enrichment, the detection limit for the nested PCR was 10(5) CFU g-1 of faeces. After pre-enrichment, it was possible to detect Salmonella in faeces where their original concentration was approximately 10(2) CFU g-1 of faeces and in meat samples, it was possible to detect Salmonella in samples where their original concentration was less than 10 cells g-1 of minced meat.

[Properties of Salmonella isolates in the Czech Republic]. / Vlastnosti salmonelových izolátu v Ceské republice.

Based on a grant project "Use and importance of epidemiological markers in Salmonella enteritidis and Salmonella typhimurium in the spread of salmonelloses in children under two years of age" implemented in 1995 to 1997, the authors investigated epidemiological markers in 1,186 salmonella isolates; the strains were isolated from faeces of 838 sick children, from 266 faeces of their contacts, from 49 specimens of incriminated foods and from 33 smears from the children's environment. Of 1,186 Salmonella isolates 999 were strains of S. enteritidis, 39 strains of S. typhimurium and 148 strains were not identified. The markers of Salmonella isolates were investigated from the aspect of biotyping--98% S. enteritidis were formed by the biovar Jena. 2% by biovar Essen; sensitivity to antibiotics--94.5% Salmonella strains were sensitive to 12 selected antibiotics, 2.9% were resistant and in 2.6% the resistance was in the intermediate zone; phagotyping--in 808 strains of S. enteritidis PT 8--88% predominated, in S. typhimurium DT 104 and DT 141; assessment of plasmid profiles--in strains of S. enteritidis plasmid 55 kb predominated, in three strains of S. typhimurium a plasmid size 95 kb; virulence--was compared in 43 strains isolated from hospitalized children with a severe clinical course with 39 strains from children treated at home. In vitro tests revealed that hospitalization of affected children was associated with virulence of the strains (SE phagotype 8) and not with age. The presented results are discussed with regard to the epidemiological situation in the Czech Republic and in the world.

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enteritidis.

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enterica serovar Enteritidis (S. enteritidis) is described. The procedure consists of plasmid DNA purification, its digestion with restriction endonuclease TaqI, electrophoresis, charge-coupled device camera scanning of the gels, and an analysis of the restriction patterns with the software Gel Manager. The system allowed us to analyse, in detail, results of plasmid profiling in more than 600 field strains of S. enteritidis. In addition to plasmid-free and virulence plasmid only containing strains, 15 additional plasmid types were detected. All the images and detailed protocols are available at the Web site http://www.clark.cz/vri/salmon.htm.

World Health Organisation--supervised interlaboratory comparison of ELISAs for the serological detection of Salmonella enterica serotype Enteritidis in chickens.

A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELISAs used for the serological detection of Salmonella enteritica serotype Enteritidis in chickens. The aim was to ascertain how far agreement could be reached on the interpretation of optical density readings for high titre, intermediate titre and low titre sera. Two sets of sera were sent to 14 participants. The first set compared high, medium and low titre sera raised in specified-pathogen-free and commercial broiler breeder chickens. The second set comprised 20 sera of different antibody titres raised in commercial birds reared under laboratory conditions and sent blind. Both indirect and double-antibody sandwich blocking ELISAs were used with a number of different detecting antigens. With a few exceptions good agreement was reached on the interpretation of results obtained from high and low titre sera from the optical density obtained with a single serum dilution. Differences were observed in the interpretation of medium titre sera. The results suggested that most ELISAs produce reasonably comparable results and that practical problems may arise from interpretation of the results mainly as a result of the choice of the criteria used for differentiating sera obtained from infected and uninfected chickens. These problems are discussed.

Differentiation of Salmonella enteritidis and S. typhimurium by plasmid profile analysis and restriction endonuclease analysis of chromosomal DNA.

Infections with Salmonella enteritidis and S. typhimurium are frequent causes of food-borne diseases in man and are responsible for considerable economic losses in the poultry industry (Fantasia et al., 1991; Pohl et al., 1991). Methods for the more careful differentiation and typing of these two serotypes are necessary for the investigation of the spread and transmission of the infection. Conventional methods of Salmonella differentiation (bioassays, phage-typing, resistance to antibiotics) often lack the necessary resolution potential (Wray et al., 1987). Plasmid profile analysis and restriction analysis of chromosomal DNA, based on restriction fragment length polymorphism (RFLP) have been used for the differentiation of Salmonellae (Wray et al., 1987; Nastasi et al., 1988; Franklin et al., 1990; Helmuth von et al., 1990; Martinetti and Altwegg, 1990). Rapid and unsophisticated analysis of the content of plasmid DNA tends to be preferred. However, some strains lack plasmids or may lose them during laboratory passages (Nastasi et al., 1988; Hartstein et al., 1991). On the other hand, restriction analysis of chromosomal DNA yields more constant and reliable information on bacterial strains (Tveten et al., 1991). The aim of this study was to compare 18 field strains of S. enteritis and 12 strains of S. typhimurium on the basis of plasmid profile analysis and restriction endonuclease analysis of chromosomal DNA. Plasmid DNA content was determined and chromosomal DNA, isolated from bacterial cells immobilized in low-melting agarose, was digested with restriction endonuclease Pst I in 18 and 12 field strains of S. enteritidis and S. typhimurium, respectively. The resulting fragments were separated by pulse-field electrophoresis in agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)