Characteristics of invasion-reduced hilA gene mutant of Salmonella Enteritidis in vitro and in vivo.

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is a facultative intracellular pathogen that causes huge losses in poultry industry and also food poisoning in humans due to its being a food-borne pathogen. Functions of Invasion-related genes need to be explored, as invasion is a key step for Salmonella infection. In this study, a transposon mutant library of Salmonella Enteritidis isolate SM6 was constructed and screened for the invasion-related genes via incubation with Caco-2 cells. Three stably attenuated mutants were identified for significantly reduced invasion with insertions all in hilA (hyperinvasive locus A) gene. We constructed and evaluated the hilA deletion mutant in vivo and in vitro. SM6△hilA showed significantly reduced ability to invade Caco-2 cells and decreased pathogenicity in chicks. However, the bacterial load and pathological damage in the cecum were significantly higher than those in the SM6 in vivo. Present results provide new evidences for pathogenicity research on Salmonella Enteritidis.

Lv S ，Si W ，Yu S ，Li Z ，Wang X ，Chen L ，Zhang W ，Liu S ... -  《-》

Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein.

Invasion of Salmonella into intestinal epithelial cells is believed to be essential for the pathogenesis of Salmonella infections. Invasion is mediated by genes located on the Salmonella pathogenicity Island I (SPI-1), which are needed for assembling a type three secretion system, that mediates injection of bacterial proteins into the cytosol of epithelial cells, resulting in cytoskeletal rearrangements and as a consequence invasion. HilA is the key regulator of the Salmonella Pathogenicity Island I. To assess the role of hilA in colonization of gut and internal organs in poultry, animals were infected with 10(8) CFU of a delta hilA mutant of S. Enteritidis and its parent strain at day of hatch. Very low numbers of delta hilA mutant strain were able to colonize the internal organs shortly after infection, but they were not eliminated from internal organs at 4 weeks post-infection. At that time, the colonization level of the wild type bacteria in internal organs was decreased to the same low level compared with delta hilA mutant strain bacteria. Shedding of the delta hilA mutant strain and colonization of the caeca was seriously decreased relative to the parent strain starting from Day 5 post-infection. At 4 weeks post-infection, the delta hilA mutant strain was more or less eliminated from the chicken gut, while the parent strain was still shed to a high level and colonized the caeca to a high extent (more than 10(7) CFU/g). It is concluded that hilA is involved in long-term shedding and colonization of S. Enteritidis in the chicken caeca.

Bohez L ，Ducatelle R ，Pasmans F ，Botteldoorn N ，Haesebrouck F ，Van Immerseel F ... -  《VETERINARY MICROBIOLOGY》

The effect of oral administration of a homologous hilA mutant strain on the long-term colonization and transmission of Salmonella Enteritidis in broiler chickens.

The effect of pre-treatment with a homologous live Salmonella hilA mutant strain on the long-term colonization and transmission of Salmonella Enteritidis in broilers was evaluated. For this purpose, three treatment groups of newly hatched broilers were created. Each group consisted of 4 pens with 25 birds per pen. The first and second groups were orally inoculated with a Salmonella Enteritidis hilA mutant strain (Nal r) whereas the third group was not. In the second and third group, 20% of the birds were challenged 1 day later with a Salmonella Enteritidis wild type strain (Strep r). The Salmonella Enteritidis hilA mutant strain showed no residual virulence in the chicken host and was largely cleared from the chickens at 6 weeks of age. A significant long-term inhibition of faecal shedding and caecal and internal organ colonization of the wild type Salmonella Enteritidis strain was observed in the birds pre-treated with the hilA mutant strain. Although pre-treatment with a hilA mutant strain could not fully prevent the spread of Salmonella Enteritidis amongst the broilers, a significant reduction of transmission was observed in comparison to the non-pre-treated groups. The observed colonization-inhibition (CI) indicates that administration of live attenuated hilA mutant Salmonella strains to newly hatched chicks might, in combination with other protective control measures, contribute to the control of Salmonella infections in broilers.

Bohez L ，Dewulf J ，Ducatelle R ，Pasmans F ，Haesebrouck F ，Van Immerseel F ... -  《VACCINE》

Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enterica serovar Enteritidis.

Van Immerseel F ，De Buck J ，Boyen F ，Bohez L ，Pasmans F ，Volf J ，Sevcik M ，Rychlik I ，Haesebrouck F ，Ducatelle R ... -  《-》

Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles. I. Salmonella enteritidis colonization and virulence gene hilA response.

The objectives of this study were to enumerate Salmonella enterica serovar Enteritidis colonization in fecal, cecal, and internal organs, and to compare the level of virulence gene expression (hilA) of experimentally challenged laying hens fed different dietary molt-induction regimens. Twelve Salmonella-free Single Comb Leghorn hens (>50 wk old) hens were randomly assigned to each of 6 treatment groups designated based on diet in 2 trials: 1) feed withdrawal Salmonella Enteritidis-positive (FW+), 2) fully fed Salmonella Enteritidis-positive (FF+), 3) 100% alfalfa crumble Salmonella Enteritidis-positive (ALC+), 4) feed withdrawal Salmonella Enteritidis-negative, 5) fully fed Salmonella Enteritidis-negative, and 6) 100% alfalfa crumble Salmonella Enteritidis-negative. A forced molt was induced by a 12-d alfalfa diet and a feed-withdrawal regimen. On d 4 of the molt, all hens in groups 1, 2, and 3 were challenged by crop gavage with 1 mL of inocula containing approximately 10(6) cfu of nalidixic acid- and novobiocin-resistant Salmonella Enteritidis (phage type 13A). At the conclusion of both trials, all hens were euthanized and Salmonella Enteritidis colonization was enumerated in the cecal contents, liver, spleen, and ovaries. In addition, fecal (d 4 and 8) and cecal samples (necropsy at d 12) were collected postchallenge from treatment groups 1, 2, and 3 (Salmonella Enteritidis-positive) to quantify hilA expression by PCR. In both trials, all nonchallenged birds were Salmonella Enteritidis-negative; therefore, no further analysis was done. In trial 1, a 2-fold reduction in Salmonella Enteritidis colonization was observed in the ALC+ hens (log10 Salmonella Enteritidis of 1.99) compared with the FW+ hens (log(10) Salmonella Enteritidis of 3.89). In trial 2, a 4-fold reduction in Salmonella Enteritidis colonization was observed in the ALC+ hens (log(10) Salmonella Enteritidis of 1.27) compared with the FW+ hens (log(10) Salmonella Enteritidis of 5.12). In trial 2, Salmonella Enteritidis colonization in spleens was higher (P <or= 0.05) in FW+ hens compared with ALC+ and FF+ hens. Relative expression of hilA was higher (P <or= 0.05) in FW+ compared with FF+ hens, whereas the FF+ and ALC+ groups were not different (P > 0.05). In trial 2, hilA expression in FW+ hens was higher (P <or= 0.05) for d 6, 11, and 12, respectively, when compared with ALC+ and FF+ hens. The results of these studies support the concept that changes in the gastrointestinal tract microenvironment, such as those created during feed deprivation, encourage Salmonella Enteritidis virulence and susceptibility in molted hens.

Dunkley KD ，McReynolds JL ，Hume ME ，Dunkley CS ，Callaway TR ，Kubena LF ，Nisbet DJ ，Ricke SC ... -  《-》