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》

The Salmonella pathogenicity island 1 and Salmonella pathogenicity island 2 type III secretion systems play a major role in pathogenesis of systemic disease and gastrointestinal tract colonization of Salmonella enterica serovar Typhimurium in the chicken.

Salmonella enterica serovar Typhimurium infection of chickens is a major public and animal health problem. In young chicks, S. Typhimurium infection results in severe systemic infection; in older chicks, infection results in prolonged gastrointestinal tract colonization. Here we determined the role of the Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) type III secretion systems in systemic infection and gastrointestinal tract colonization of the chicken though experimental infection of chicks with a S. Typhimurium strain with mutations in the genes encoding the secretion system machinery of SPI-1 (spaS) and SPI-2 (ssaU) that prevent secretion of effector proteins. In 1-day-old chicks, mutation of SPI-2 lead to a decrease in both systemic bacterial numbers and pathology, although no difference in gastrointestinal numbers was observed. Mutation of SPI-1 had little effect in 1-day old chicks. In 1-week-old animals the SPI-2 mutants could not be detected systemically and colonized the gastrointestinal tract only in low numbers in comparison with the parent strain, and was cleared in 1 week. The SPI-1 mutant showed greatly reduced levels of systemic infection, and colonized the gastrointestinal tract at a lower level than the parent strain. The findings show that the SPI-2 type III secretion system is required for systemic S. Typhimurium infection in both infection models, and that it plays a significant role in gastrointestinal colonization. The SPI-1 system is involved in both systemic infection and gastrointestinal colonization, but does not appear absolutely essential for either infection process.

Jones MA ，Hulme SD ，Barrow PA ，Wigley P ... -  《AVIAN PATHOLOGY》

Long-term colonisation-inhibition studies to protect broilers against colonisation with Salmonella Enteritidis, using Salmonella Pathogenicity Island 1 and 2 mutants.

Mutants in the Salmonella Pathogenicity Island 1 (hilA and sipA) and 2 (ssrA) were tested for their potential to induce protection against infection by homologous virulent Salmonella Enteritidis challenge strain, administered 24h later, in chickens. Although they colonised the internal organs to a significantly lower degree compared to the wild type strain, both a sipA and a ssrA mutant persistently colonised the gut when inoculated to newly hatched chicks. After inoculation of 1-day-old chicks with a sipA or a ssrA mutant and subsequent challenge with a wild type Salmonella Enteritidis 24h later, a significant degree of resistance against caecal and internal organ colonisation by the challenge strain was found. The protection lasted for the full 6 weeks of study, but due to their persistence, the sipA and ssrA mutants are not useful to induce broiler protection. After inoculation of newly hatched chicks with a hilA mutant no positive cloacal swabs could be detected anymore at 4 weeks post-inoculation and the hilA mutant was almost completely cleared from the gut. When newly hatched chicks were inoculated with a hilA mutant and challenged 24h later, the excretion of the virulent challenge strain was significantly reduced and the intestinal colonisation of the challenge strain was inhibited to a high level until the age of 9 days. Moreover, the hilA mutant exerted a significant profound inhibition of internal organ colonisation by the virulent challenge strain throughout the study period. The approach of vaccination with a hilA mutant strain can be a valuable basis for development of vaccine strains for broilers protection.

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

The Salmonella Pathogenicity Island 2 regulator ssrA promotes reproductive tract but not intestinal colonization in chickens.

Using a deletion mutant in the regulator of SPI-2, ssrA, we investigated the role of SPI-2 in invasion, intestinal colonization and reproductive tract infection of chickens by Salmonella Enteritidis. The ssrA mutant was fully invasive in phagocytic and non-phagocytic cells but failed to persist within chicken macrophages. The ability of Salmonella Enteritidis to cause disease in orally infected 1-day-old chicks was not altered when ssrA was deleted. Furthermore, caecal colonization was not affected, while spleen and liver showed reduced colonization. Following intra-peritoneal and intravenous infection of 1-day-old chicks, internal organ colonization was strongly reduced. After intravenous inoculation in adult laying hens bacterial numbers of the ssrA mutant were significantly lower in oviducts and ovaries as compared to the wild type strain. The chickens showed less reproductive tract lesions and the recovery of egg production were faster compared to the wild type strain infected chickens. These findings indicate that the SPI-2 regulator ssrA promotes reproductive tract colonization, but is not essential for intestinal colonization of chickens with the host non-specific serotype Enteritidis.

Bohez L ，Gantois I ，Ducatelle R ，Pasmans F ，Dewulf J ，Haesebrouck F ，Van Immerseel F ... -  《VETERINARY MICROBIOLOGY》