Effects of crp deletion in Salmonella enterica serotype Gallinarum

Abstract

Background: 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.

Methods: 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.

Results: 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 Deltacrp 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.

Conclusion: 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.

Figure 1

Multiplex PCR with primers crp-1, crp-2 and IS10as2. A fragment of 273 base pairs was produced inside the crp gene from the wild type Salmonella enterica serotype Gallinarum biovar gallinarum G9 (lanes 1 and 4) and crp⁺ from pSD110 in the re-complemented strain (lane 3 and 6). A fragment of 500 base pairs was amplified between crp-1 and one of the IS sequences in Tn10 in the mutant (lane 2 and lane 5) and the re-complemented strain (lanes 3 and 6).

Figure 2

Intestinal invasion of the wild type Salmonella enterica serotype Gallinarum biovar gallinarum (G9) and Δcrp and Δcrp re-complemented with plasmid pSD110 in small intestine of hens. Experiments were replicated to allow rotation of the individual strains in different positions. Counts are expressed as log₁₀ colony forming units (CFU) per biopsy of 84-mm² according to Aabo et al. [12]. The dose used was approximately log₁₀ 7.8 per loop. The invasion of the complemented strain was significantly different from the two other strains by comparison of mean CFU, as indicated by an asterix (p < 0.05). Similar results were obtained with the wild type J91 and its mutated variants.