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.
Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonella enterica 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.
If a glacier calves into the Arctic Ocean, does it make a sound? Some scientists say yes and have devised a clever way to use those sounds to calculate the size of the fallen ice chunks.
Bacterial mono-ADP-ribosyltransferases (ARTs) catalyze the singular transfer of an ADP-ribose moiety from an NAD+ molecule onto a target molecule. ARTs contain an ancient and highly conserved tertiary structure and have a wide variety of intracellular targets and effects. Some, but not all, bacterial ARTs have an AB5-type multimeric structure consisting of an enzymatically active subunit non-covalently situated atop of a non-toxic pentamer. The active, or A, subunit of AB5-type toxins has a catalytic action that contributes to bacterial pathogenicity, and it is sometimes, but not always, an ART. ArtAB is an ART with AB5-type structure from the virulent and highly antibiotic resistant Salmonella Typhimurium DT104. In the studies described here, we tested the hypothesis that the active subunit of ArtAB is structurally and enzymatically homologous to that of the well-characterized AB5-type ART pertussis toxin. ArtAB was purified from E. coli and was used to characterize ArtAB’s cellular effects, predicted structure, and biophysical properties. In addition, a set of single-residue mutants was constructed and purified to probe ArtAB’s active site. AB5-type toxins have long been studied for their immunogenic properties, and some of these bacterial munitions have been harnessed and repurposed as vaccines or vaccine adjuvants to prevent infectious disease. Their receptor-binding pentamer, abbreviated as B5, binds to, and facilitates entry into, host cells. In additional work presented here, we tested the hypothesis that the B5 subunit of cholera toxin (CTB) from Vibrio cholerae could be used to construct a safe and effective mucosal vaccine against Staphylococcus aureus-caused mastitis. We constructed a bovine vaccine by conjugating Staphylococcus aureus antigens to the CTB-based adjuvant platform, and the immunogenicity of the vaccine was characterized in a bovine clinical trial. Finally, clinical isolates of caprine S. aureus were screened for the presence of surface antigens that could be use in a caprine version of the vaccine against mastitis. The work on bacterial AB5-type ARTs presented here contributes to a growing global understanding of the bacterial ART family, lays a foundation for the potential incorporation of ArtAB in a vaccine against Salmonella, and advances the development of bovine and caprine vaccines against S. aureus-caused mastitis.
The SARS-CoV-2 virus has spread globally causing coronavirus disease 2019 (COVID-19). Rapidly and accurately identifying viral infections is an ongoing necessity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to produce a DNA allonamer with two distinct binding domains made allosteric through a linker section; one domain binds SARS-CoV-2 spike (S) protein, inducing a conformational change that allows the reporter domain to bind a fluorescent reporter molecule. We used bead-based fluorescence and immunofluorescence assays to confirm the allonamer's affinity and specificity for S-protein and confirmed that the allonamer can bind to S-proteins with mutations corresponding to those of the alpha, beta, gamma, and delta variants. We then developed the allonamer-based Quantum-Logic Aptamer Analyte Detection (Q-LAAD) test, a rapid, high-throughput antigen test for qualitative detection of SARS-CoV-2 in clinical settings. We validated Q-LAAD against retrospective and prospective clinical anterior nasal swab samples collected from symptomatic patients suspected of having COVID-19. Q-LAAD showed 97% sensitivity and 100% specificity compared to the RT-qPCR assay. Q-LAAD has a limit of detection (LOD) of 1.88 TCID50/mL, is cost-effective and convenient, and requires only a common fluorescence plate reader. Q-LAAD may be a useful clinical diagnostic tool in the fight against SARS-CoV-2.
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