AviPro® Megan® Vac 1 is derived from Salmonella enterica subsp. enterica serovar Typhimurium strain UK-1 and is attenuated by mutations affecting the cyaA and crp genes. These mutations were generated using Tn10-mediated mutagenesis and bacteriophage P22 HT int-mediated transduction. The combined disruption of cyaA and crp interferes with cAMP-dependent regulation and contributes to attenuation of S. Typhimurium while retaining sufficient viability and immunogenicity for use as a live vaccine.

The cyaA gene encodes adenylate cyclase (CyaA), the enzyme responsible for the synthesis of cyclic adenosine monophosphate (cAMP). cAMP participates, together with the cAMP receptor protein (CRP), in the global regulation of numerous metabolic and physiological processes in Salmonella. During development of the attenuated strain, a Tn10-associated mutation affecting cyaA was transferred by generalized transduction using bacteriophage P22 HT int.  The resulting modification disrupts normal cyaA function and is commonly described as a Δcya or ΔcyaA mutation. More recent whole-genome sequence analysis of the commercial AviPro® Megan® Vac 1 strain confirmed disruption of cyaA, corresponding to locus STM3939 in the S. Typhimurium LT2 reference genome, and identified residual IS10-like sequence at the disrupted locus.

The crp gene encodes the cyclic AMP receptor protein (CRP), a global transcriptional regulator that interacts with cAMP and controls expression of numerous genes involved in bacterial metabolism and other cellular functions. A Tn10-associated mutation affecting crp was similarly introduced using P22 HT int-mediated generalized transduction, followed by selection of tetracycline-sensitive, fusaric-acid-resistant derivatives. The resulting modification disrupts normal crp function and is commonly described as a Δcrp mutation. Whole-genome sequence analysis of the commercial AviPro® Megan® Vac 1 strain confirmed disruption of crp, corresponding to locus STM3466 in the S. Typhimurium LT2 reference genome, and identified residual IS10-like sequence at the disrupted locus.

Note:

Bacteriophage P22 HT int was used as a generalized transducing phage during construction of the attenuated strain. It served as a vehicle for transferring Tn10-associated cyaA and crp mutations into the S. Typhimurium recipient background and was not intended to remain as an intact genetic element in the final vaccine strain.
Tn10 was used as part of the mutagenesis and selection procedure. The transposon contains IS10 elements associated with genes conferring tetracycline resistance. Following transduction and selection, tetracycline-sensitive derivatives were obtained using fusaric-acid selection. Therefore, the tetracycline-resistance phenotype associated with Tn10 was not retained in the final attenuated strain.

Whole-genome sequencing of the commercial AviPro® Megan® Vac 1 strain subsequently identified IS10-like sequences at both the disrupted cyaA and crp loci. The residual transposon-derived sequences were determined to be at least 1,329 bp in length; however, their exact length and complete organization could not be resolved using the available short-read sequencing data. Genes conferring tetracycline resistance were not detected.