Date Published: May 8, 2007
Publisher: BioMed Central
Author(s): Valentina Rosu, Mark S Chadfield, Antonella Santona, Jens P Christensen, Line E Thomsen, Salvatore Rubino, John E Olsen.
Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested.
Mutants were constructed in S. Gallinarum by P22 transduction from Salmonella Typhimurium (S. Typhimurium) with deletion of the crp gene. The effect was characterized by measuring biochemical properties and by testing of invasion in a chicken loop model and by challenge of six-day-old chickens. Further, birds were immunized with the deleted strain and challenged with the wild type isolate.
The crp deletions caused complete attenuation of S. Gallinarum. This was shown by ileal loop experiments not to be due to significantly reduced invasion. Strains with such deletions may have vaccine potential, since oral inoculatoin with S. Gallinarum Δcrp completely protected against challenge with the same dose of wild type S. Gallinarum ten days post immunization. Interestingly, the mutations did not cause the same biochemical and growth changes to the two biotypes of S. Gallinarum. All biochemical effects but not virulence could be complemented by providing an intact crp-gene from S. Typhimurium on the plasmid pSD110.
Transduction of a Tn10 disrupted crp gene from S. Typhimurium caused attenuation in S. Gallinarum and mutated strains are possible candidates for live vaccines against fowl typhoid.
The avian host specific serotype Salmonella enterica serotype Gallinarum consists of two biovars, gallinarum and pullorum (S. Gallinarum and S. Pullorum) . They are considered the causative agents of two distinct diseases, fowl typhoid and pullorum disease, which occur especially in countries with less developed poultry industries . While many western countries have succeeded with elimination of the diseases by test and slaughter, developing countries are often left with only the strategy of prophylactic treatment with antibiotics. To avoid this use of antibiotics, development of safe and effective vaccines is a priority.
The importance of crp in pathogenicity of the avian host specific S. Gallinarum has never been investigated. Therefore the present paper aimed to study the effect of Δcrp in this serotype by transduction of DNA from S. Typhimurium deleted in this gene. To demonstrate the successful transduction, wild type, mutated and re-complemented strains of S. Gallinarum G9 and J91 and S. Pullorum 3 (Table 1) were analysed by a multiplex PCR in order to detect both the crp gene and the Tn10 insertions. A fragment of 273 base pairs was amplified inside the crp gene in the wild type strains, and inside the crp-allele on the plasmid pSD110 in the complemented strains. It further amplified a fragment of approx. 500 base pairs between the crp gene and one of the insertion sequences of Tn10 in all mutant and re-complemented strains. Results for S. Gallinarum G9 are shown in Figure 1. From the analysis we concluded that the wild type crp alleles had been exchanged with the inactivated genes in S. Gallinarum G9, J91 and S. Pullorum 3.
In conclusion transduction of a crp deletion from S. Typhimurium to S. Gallinarum by P22 transduction caused attenuation and the mutated strain may have vaccine potentials, since orally infected chickens survived challenge with wild type strains.
The following abbreviations were have been used for Salmonella serotypes and biovars:
The author(s) declare that they have no competing interests.
VR and AS performed mutations and characterized strains biochemically and by PCR. They performed virulence characterization in collaboration with MSC and JPC. LET performed RT-PCR. SR and JEO contributed significantly to the design of the study and JEO drafted the manuscript. All authors contributed to the wording of the final version of this manuscript.