Roland and his colleagues have instead produced a single-dose oral vaccine. The experimental vaccine strains in the current study make use of a technique known as delayed attenuation, developed in the laboratory of Roy Curtiss, who directs the Institute’s Center for Infectious Diseases and Vaccinology.

With delayed attenuation, the Salmonella strain enters the system with its native virulence intact, producing a strong, systemic immune response. Then, a key virulence-related gene switches off after a number of cell divisions, shutting down the bacterium’s disease-causing potential.

The trick in delayed attenuation is to reengineer the Salmonella virulence gene so that it requires the artificial sugar arabinose for effective functioning. Once the bacterial cell’s storehouse of arabinose is exhausted, the virulence gene essentially short-circuits and becomes inactive.

Three key virulence-related genes – crp, rfc and rfaH – were previously identified from studies with the related pathogen Salmonella typhimurium in mice. Vaccine strains were constructed and tested in chickens with each of these genes subject to delayed attenuation via arabinose depletion. These strains were also compared for effectiveness with strains in which the virulence genes had been deleted altogether, rather than attenuated and with the wild-type form of the pathogen.

The best results in terms of immunogenicity and safety were produced by the vaccine strain bearing a crp gene subject to delayed attenuation. The vaccine was avirulent and produced only minor internal lesions while offering superb protection from a lethal challenge of S. Gallinarum. Further, the presence of small quantities of arabinose in the birds’ drinking water was not sufficient to disable the crp attenuation mechanism or affect virulence or immunogenicity.

The crp delayed attenuation strain was also tested for effectiveness against a lethal challenge of the human pathogen Salmonella Enteritidis. After four days, no detectable trace of S. Enteritidis appeared in systemic organs including the liver and spleen, indicating strong protection by the vaccine.

The researchers noted that the vaccine strain in which the rfc gene was deleted still exhibited full virulence in chickens, indicating that contrary to the mouse model of Salmonella typhimurium, rfc is a non-essential component for S. Gallinarum virulence in birds. In contrast, the rfaH deletion mutant was attenuated and protective, while the strain with arabinose-regulated rfaH expression retained full virulence.

The study indicates that delayed-attenuation Salmonella vaccines of the kind explored here can have wide applicability for the effective protection from a range of infectious diseases. In future efforts, the group hopes to fine-tune a vaccine strain with more than one attenuating mutation, at least one of which remains unaffected by dietary components, thereby offering improved safety along with maximum immunogenicity.

“Our goal,” Roland says, “is to utilize 'high-tech' strategies to provide a 'low-tech,' easy-to-use, inexpensive vaccine, allowing everyone from backyard farmers to commercial hatcheries to vaccinate their flocks, resulting in better food security in the developing world.”

Richard.Harth@asu.edu