A porcine reproductive and respiratory syndrome virus candidate vaccine based on the synthetic attenuated virus engineering approach is attenuated and effective in protecting against homologous virus challenge.

Current porcine reproductive and respiratory syndrome virus (PRRSV) vaccines sometimes fail to provide adequate immunity to protect pigs from PRRSV-induced disease. This may be due to antigenic differences among PRRSV strains. Rapid production of attenuated farm-specific homologous vaccines is a feasible alternative to commercial vaccines. In this study, attenuation and efficacy of a codon-pair de-optimized candidate vaccine generated by synthetic attenuated virus engineering approach (SAVE5) were tested in a conventional growing pig model. Forty pigs were vaccinated intranasally or intramuscularly with SAVE5 at day 0 (D0). The remaining 28 pigs were sham-vaccinated with saline. At D42, 30 vaccinated and 19 sham-vaccinated pigs were challenged with the homologous PRRSV strain VR2385. The experiment was terminated at D54. The SAVE5 virus was effectively attenuated as evidenced by a low magnitude of SAVE5 viremia for 1-5 consecutive weeks in 35.9% (14/39) of the vaccinated pigs, lack of detectable nasal SAVE5 shedding and failure to transmit the vaccine virus from pig to pig. By D42, all vaccinated pigs with detectable SAVE5 viremia also had detectable anti-PRRSV IgG. Anti-IgG positive vaccinated pigs were protected from subsequent VR2385 challenge as evidenced by lack of VR2385 viremia and nasal shedding, significantly reduced macroscopic and microscopic lung lesions and significantly reduced amount of PRRSV antigen in lungs compared to the non-vaccinated VR2385-challenged positive control pigs. The nasal vaccination route appeared to be more effective in inducing protective immunity in a larger number of pigs compared to the intramuscular route. Vaccinated pigs without detectable SAVE5 viremia did not seroconvert and were fully susceptible to VR2385 challenge. Under the study conditions, the SAVE approach was successful in attenuating PRRSV strain VR2385 and protected against homologous virus challenge. Virus dosage likely needs to be adjusted to induce replication and protection in a higher percentage of vaccinated pigs.

Development of a novel fluorescent microbead-based immunoassay and comparison with three enzyme-linked immunoassays for detection of anti-Erysipelothrix spp. IgG antibodies in pigs with known and unknown exposure.

A novel fluorescent microbead immunoassay (FMIA) using the recombinant polypeptide SpaA415 was developed for detection of anti-Erysipelothrix spp. IgG in pig sera. The diagnostic performance of the FMIA was evaluated on samples from pigs with known and unknown Erysipelothrix spp. exposure and compared to an in-house enzyme-linked immunosorbent assay (ELISA-1) based on the same capture antigen, and two commercially available ELISAs (ELISA-2 and ELISA-3). Sera from pigs experimentally infected with Erysipelothrix rhusiopathiae serotype 1a (n=60) or 19 (n=12), sera from pigs vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12) or a commercial bacterin based on serotype 2 (n=12), and 90 field samples were utilized. The sensitivity on 22 true positive samples collected in the later stages of infection/post-vaccination was 100% for the FMIA and ELISA-1, 63.6% for ELISA-2 and 81.8% for ELISA-3. The earliest antibody response was detected 7days post inoculation with the FMIA (77.8%) and ELISA-1 (11.1%), and at 14days post-vaccination (dpv) with FMIA (50%) and ELISA-1 (50%). On field samples, a higher seroprevalence was found in pigs older than 21days with all four assays. Kappa analysis indicated that the FMIA and ELISA-1 had almost complete agreement whereas the agreement was slight with ELISA-2 and fair with ELISA-3. The sensitivity of both immunoassays based on the rSpaA415 antigen was higher compared to that of the two commercial ELISAs. The rSpaA415 FMIA has great potential as an inexpensive ELISA alternative for detection of antibodies against E. rhusiopathiae in the future.

Development and evaluation of an enzyme-linked immunosorbent assay based on a recombinant SpaA protein (rSpaA415) for detection of anti-Erysipelothrix spp. IgG antibodies in pigs.

The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for detection of anti-Erysipelothrix spp. IgG in pig sera by utilizing recombinant polypeptide SpaA415 (rSpaA415) based on surface protective antigen (Spa) A (SpaA) of Erysipelothrix spp. The sensitivity of the rSpaA415 ELISA was evaluated on sera from pigs experimentally infected with E. rhusiopathiae serotype 1a (n=72), serotype 19 (n=12), or experimentally vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12), a commercial bacterin based on serotype 2 (n=12), or an experimental bacterin based on serotype 2 (n=300). Specificity was tested using 221 negative control samples. The earliest antibody response was detected at 7 days post-inoculation (dpi) and 14 days post-vaccination (dpv). At the cutoff of 0.9 sample optical density, the sensitivity was 96.5% and the specificity was 100%. In experimentally infected pigs, the sensitivity of the rSpaA415 ELISA ranged from 5.5 to 100% which improved as dpi increased. Antimicrobial treatment, administered prior to appearance of clinical signs, decreased assay sensitivity. In vaccinated pigs, the rSpaA415 ELISA had a sensitivity of 48.3-100%. Serum samples from rabbits each hyperimmunized with one of the 28 Erysipelothrix spp. serotypes were used to determine cross-reactivity with strains expressing SpaB, SpaC or no currently recognized Spa protein and antibodies against E. tonsillarum were not detected. These data suggest that the novel rSpaA415 ELISA test is a useful tool to detect anti-IgG antibodies against different serotypes of E. rhusiopathiae in infected or vaccinated pigs without cross-reacting with the economically less important E. tonsillarum strains.

Relationship between glucose transport and metabolism in isolated bovine mammary epithelial cells.

Glucose transport by isolated bovine mammary epithelial cells involves translocation across the cell membrane into a compartment that exchanges slowly with the bulk cytosol. The significance to glucose metabolism of this compartmentalization was examined by generation, modeling, and analysis of transport and metabolism data. Net uptake of 5 mM 3-O-methyl-d-glucose by isolated bovine mammary epithelial cells was measured at 37 degrees C. Time-course curves were better fitted by a double exponential equation than a single exponential equation and were subjected to compartmental analysis to obtain glucose transport model parameters. Lactose synthesis and glucose oxidation rates and cellular concentrations of intermediary metabolites, glucose-6-phosphate and glucose-1-phosphate, were measured at varied media glucose concentrations. A model that integrates both glucose transport and metabolism under-predicted the rates of lactose synthesis and glucose oxidation by a factor of 3. To account for the observed glucose use rates, glucose must be available for phosphorylation once translocated across the cell membrane (intermediate compartmentalization of translocated glucose does not exclude access to hexokinase). Metabolic control analysis indicated that, at physiological glucose concentrations, phosphorylation by hexokinase exerts 80% of the control of glucose metabolism to lactose and CO(2), and transport exerts the remaining 20%.