Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonellaenterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and d-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella.

Staphylococcus aureus (S. aureus) is a primary agent of bovine mastitis and a source of significant economic loss for the dairy industry. We previously reported antigen-specific immune induction in the milk and serum of dairy cows following vaccination with a cholera toxin A2 and B subunit (CTA2/B) based vaccine containing the iron-regulated surface determinant A (IsdA) and clumping factor A (ClfA) antigens of S. aureus (IsdA + ClfA-CTA2/B). The goal of the current study was to assess the efficacy of this vaccine to protect against S. aureus infection after intramammary challenge. Six mid-lactation heifers were randomized to vaccinated and control groups. On days 1 and 14 animals were inoculated intranasally with vaccine or vehicle control, and on day 20 animals were challenged with S. aureus. Clinical outcome, milk quality, bacterial shedding, and somatic cell count (SCC) were followed for ten days post-challenge. Vaccinated animals did not show signs of clinical S. aureus mastitis and had lower SCCs compared to control animals during the challenge period. Reductions in bacterial shedding were observed but were not significant between groups. Antibody analysis of milk and serum indicated that, upon challenge, vaccinated animals produced enhanced IsdA- and ClfA-CTA2/B specific immunoglobulin G (IgG) responses, while responses to CTA2/B alone were not different between groups. Responses after challenge were largely IgG1 against the IsdA antigen and mixed IgG1/IgG2 against the ClfA antigen. In addition, there was a significant increase in interferon gamma (IFN-γ) expression from blood cells in vaccinated animals on day 20. While preliminary, these findings support evidence of the induction of active immunity by IsdA + ClfA-CTA2/B, and further assessment of this vaccine is warranted.