This project included a team of PCVAD researchers from KSU, ISU and SDSU who utilized a single set of experimentally infected pigs to address several important questions related to pathobiology, immunology and immunodiagnostics of PCVAD. The four principal project objectives included:

Characterization of the virological and immunological response of vaccinated or naïve pigs following PCV2b and/or PCV2b-PRRS infection.Development of a quantitative ELISA for assessment of active and passive antibody levels. Development of a seroassay to differentiate infected from vaccinated animals (DIVA). Development of a monoclonal antibody panel for the differentiation of PCV genotypes and diagnostic tools. Results:
Objective 1:
· Experimental co-infection (dual challenge) with PCV2b and PRRS can result in clinical disease and death in conventional animals.
· PCV2b or PRRS infections alone do not result in significant clinical disease or death.
· PRRS infection potentiates PCV2b replication.
· PCV2b infection does not enhance PRRS replication.
· Temporal coincidence of PCV2b and PRRS co-infection contributes significantly to the mortality of animals.
· Vaccination of animals with a heterologous PCV2a subunit vaccine generates neutralizing antibodies and a protective immune response in vaccinated animals.
· Vaccination results in no apparent viremia following single or dual challenge, whereas non-vaccinated animals are viremic for at least 42 days post challenge.

Objective 2:
· A quantitative PCV2b ELISA was developed and then updated to use a fluorescent-microsphere immunoassay (MIA) using Luminex technology.
· PCV2 antibody quantitation results using the MIA compared favorably to the IFA gold standard.
· A quantitative MIA assay for PRRS was developed.
· MIA assays proved to be more rapid than IFA.
The MIA assay for PCV2 and PRRS (multiplex assay) is currently undergoing validation testing using the control and infection sera generated in the animal study from Objective 1.· Multiplex MIA are run as single sample against multiple pathogens in a single test system. Once fully developed diagnostic sero-testing should be more cost effective.
· Multiplex MIA as a herd profiling tool can be developed and used to monitor antibody levels to multiple pathogens to make informed management decisions.

Objective 3:
· A DIVA (differentiate infected from vaccinated animals) ELISA was developed to enable the discrimination between subunit vaccinated animals and naturally infected animals.
· A second DIVA ELISA based on PCV2b-ORF2 capsid reactivity is currently under development. This DIVA ELISA should be useable for animals vaccinated with baculovirus subunit vaccines as well as conventional whole virus vaccines.
Both DIVA tests are based on the control and infection sera generated in the animal study from Objective 1.
Objective 4:
· Monoclonal antibodies (MAb) to PCV2 were developed allowing for the differentiation of PCV1 and PCV2.
The MAbs developed for this project will be useful tools for future development of diagnostic assays The MAb are available to all swine disease researchers and diagnostic labs as important tools for current diagnostics and future diagnostic test development.
The tools and knowledge from this collaborative project are being integrated into disease management programs. The multi-university collaborative effort efficiently leveraged the intellectual and scientific resources of three leading institutions. The savings cost and time are a consequence of the incorporation of a single group of experimentally infected pigs combined with the collaboration of a multidisciplinary team of virologists, immunologists and diagnosticians.