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》

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》

Control of Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing attenuated Salmonella Gallinarum mutant strain.

The ideal live vaccine to control Salmonella in commercial chicken flocks should engender protection against various strains. The purpose of the present study was to confirm the attenuation of a Salmonella Gallinarum (SG) mutant strain with deletion on genes cobS and cbiA, that are involved in the biosynthesis of cobalamin. Furthermore, evaluate its use as a live vaccine against Salmonella. For the evaluation of the vaccine efficacy, two experiments were conducted separately. Birds from a commercial brown line of chickens were used to perform challenge with SG wild type strain and birds from a commercial white line of chickens were used to perform challenge with Salmonella Enteritidis (SE) wild type strain. In both experiments, the birds were separated in three groups (A, B and C). Birds were orally vaccinated with the SG mutant as the following programme: group A, one dose at 5 days of age; group B, one dose at 5 days of age and a second dose at 25 days of age; and group C, birds were kept unvaccinated as controls. At 45 days of age, birds from all groups, including the control, were challenged orally by SG wild type (brown line) or SE wild type (white line). Lastly, another experiment was performed to evaluate the use of the SG mutant strain to prevent caecal colonization by SE wild type on 1-day-old broiler chicks. Mortality and systemic infection by SG wild type strain were assessed in brown chickens; faecal shedding and systemic infection by SE wild type were assessed in white chickens and caecal colonization was assessed in broiler chicks. Either vaccination with one or two doses of SG mutant, were capable to protect brown chickens against SG wild type. In the experiment with white chickens, only vaccination with two doses of SG mutant protected the birds against challenge with SE wild type. Although, SG mutant could not prevent caecal colonization in 1-day-old broiler chicks by the challenge strain SE wild type. Overall, the results indicated that SG mutant is a promising Salmonella live vaccine candidate that demonstrated good efficacy to control the infection by two serotypes of major importance to the poultry industry.

Penha Filho RA ，de Paiva JB ，da Silva MD ，de Almeida AM ，Berchieri A Jr ... -  《-》

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》

A Salmonella Enteritidis hilAssrAfliG deletion mutant is a safe live vaccine strain that confers protection against colonization by Salmonella Enteritidis in broilers.

Consumption of contaminated poultry meat is an important cause of Salmonella infections in humans. Therefore, there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonization-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the safety and efficacy of a Salmonella Enteritidis ΔhilAssrAfliG strain as a colonization-inhibition strain for protection of broilers against Salmonella Enteritidis was evaluated. After administration of the Salmonella Enteritidis ΔhilAssrAfliG strain to day-old chickens, this strain could not be isolated from the gut, internal organs or faeces after 21 days of age. In addition, administration of this strain to one-day-old broiler chickens decreased faecal shedding and caecal and internal organ colonization of a Salmonella Enteritidis challenge strain administered one day later using a seeder bird model. To our knowledge, this is the first report of an attenuated Salmonella strain for which both the safety and efficacy has been shown in long-term experiments (until slaughter age) in broiler strain can potentially be used as a live colonization-inhibition strain for controlling Salmonella Enteritidis infections in broilers.

De Cort W ，Geeraerts S ，Balan V ，Elroy M ，Haesebrouck F ，Ducatelle R ，Van Immerseel F ... -  《-》