Development and preliminary validation of an antibody filtration-assisted single-dilution chemiluminometric immunoassay for potency testing of Piscirickettsia salmonis vaccines.

Challenge with live pathogens could be substituted by serology for many veterinary diseases, however little progress has been made in the development of alternative batch vaccine potency tests for fish. This study reports the development and preliminary validation of a single-dilution filtration-assisted chemiluminometric immunoassay (SD FAL-ELISA) applied to measure anti Piscirickettsia salmonis IgM in individual or pooled serum and mucus samples. The assay was set up to test a single-dilution of the sample. Serum SD FAL-ELISA yielded a sensitivity of 90% and a specificity of 96%. SD FAL-ELISA was applied to evaluate pooled and individual samples from P. salmonis challenge assessments. Relative-light units values (RLU) obtained by SD FAL-ELISA were proportional to antibody levels in serum. RLU values obtained from pooled and individual serum samples increased with the observed relative percent survival (RPS) values, indicating a correlation between protection and specific IgM levels. Results obtained for specific IgM in mucus samples was not related to the RPS, but discriminated the vaccine that yielded high RPS (86.4%) from the others (40.9 and 54.5%). This is the first report on the development of an indirect high-throughput serological assessment for P. salmonis vaccine potency testing using both pooled or individual serum and cutaneous mucus samples.

Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle.

Serological assessment of the heterologous response among Foot-and-Mouth Disease Virus (FMDV) strains is mainly performed by virus neutralization test (VNT), liquid phase blocking ELISA and complement fixation assay. In this study two high-throughput ELISA techniques, avidity and IgG subtype ELISA, were developed and used to further characterize heterologous antibody responses in cattle during vaccination and challenge. Both assays were applied to a set of previously characterized sera from animals immunized with an inactivated A24 Cruzeiro/Brazil/55 (A24 Cruzeiro) strain monovalent FMDV vaccine and challenged with the heterologous A/Argentina/2001 (A/Arg/01) strain. Single dilution avidity ELISA assessment showed that animals that were protected against A/Arg/01 challenge had higher avidity antibodies to this heterologous strain than non-protected cattle. Animals with low or even undetectable anti-A/Arg/01 serum-neutralizing titers that passed the heterologous challenge presented higher IgG1/IgG2 ratio than non-protected animals. In this study, the three assessments (VNT and both ELISAs) discriminated between protected and not protected animals against a heterologous challenge. The combination of these techniques may be applied to complement current indirect serological vaccine-matching assessments. The measurement of these qualitative parameters may provide additional information to understand the mechanisms underlying FMD heterologous responses and the induction of cross-protection in cattle.

The immune enhancement of a novel soy lecithin/ß-glucans based adjuvant on native Neospora caninum tachyzoite extract vaccine in mice.

Efficient, cost-effective and safe Th1-immunity-inducing vaccine formulations are paramount for achieving protection against Neospora caninum. In this study, a new adjuvant (Providean-AVEC) was used in the development of a N. caninum vaccine and evaluated in a mouse model. Soluble N. caninum tachyzoite native protein extract (sNcAg) was selected as vaccine antigen based on its capacity to activate production of pro-inflammatory cytokines on dendritic cells. Vaccines containing 4 and 0.4 µg of sNcAg, and Providean-AVEC, ISCOM-Matrix or aluminum hydroxide (Alum) were tested in BALB/c mice. While mice vaccinated with 4µg of sNcAg + Providean-AVEC developed specific antibodies shortly after the first dose, the rest of the high antigen payload formulations only induced seroconversion after the booster. Mice immunized with the high payload ISCOM vaccine (4 µg sNcAg) or with either low or high payload Providean-AVEC formulations (0.4 µg and 4 µg sNcAg, respectively) elicited higher IgG2a than IgG1 serum levels, and IFN-γ anamnestic responses with a Th1-cytokine biased profile. These animals had no histological signs of cerebral lesions and parasite burden assessed by quantitative real-time PCR was not detected. Vaccine preparations including Providean-AVEC as adjuvant limited N. canimum multiplication even with only a tenth of antigen payload compared to vaccines containing other adjuvants. Using adjuvants to specifically activate dendritic cells, combined with a careful antigen selection can enhance cellular responses to inert N. caninum vaccines.