Vaccination with a Streptomycin-Resistant Strain of Salmonella enteric serovar Enteritidis lacking pefA and spvC Genes Reduces Cecal Colonization and Organ Invasion in SPF Chicks

Abstract

Salmonellosis is one of the most important food-borne diseases and remains an important pathogen of poultry. In this study was evaluated the protection of a vaccine containing a streptomycin-resistant strain of Salmonella entericas erovar Enteritidis lacking pefA and spvC genes with respect to cecal colonization, organ invasion and excretion in SPF chicks and the potential use as a vaccine candidate was tested. Streptomycin-mutant strain was obtained by exposure to high concentration of streptomycin. Parent and resistant strain were evaluated phenotypically by measuring biochemical properties, growth rate and antibiotic resistance, and genetically for expression of twenty-three genes. Mutant strain was tested in SPF chickens by testing excretion, and by challenge using a wild-type isolate three weeks after immunization evaluating cecal colonization and organ invasion. Streptomycin-resistant strain showed lack of expression of pefA and spvC when compared to the parent strain. DNA sequencing of a PCR-amplified gyrA fragment detected one point of mutation Ser15→Phe. Biochemical properties did not change. Growth rate differences were observed between parent and mutant strains, showing a generation time was increased five-fold in the mutant strain.  Excretion of the vaccine strain was reduced 50% at the second week compared to the positive control group and no excretion of the vaccine strain was detected at the third week. Cecal colonization and organ invasion were significantly reduced in the vaccinated group, 80% and 70%, respectively. The vaccine strain was not detected in cecal and organ samples at the end of the trial. Attenuated strains produced by selecting for resistance to streptomycin have been described in mice. This study showed that streptomycin-resistant strain may be an important factor in the attenuation, suggesting that after exposure to streptomycin the parent isolate lost the expression of pefA and spvC genes and it could be a vaccine candidate to protect chicks against a Salmonella Enteritidis challenge.

Key words: Salmonella Enteritidis, streptomycin-resistant strain, potential vaccine strain