For the reasons outlined under 7.1, the present project will develop tests for analysing piglet humoral immunity and the dsDNA replicative form of PCV2, which can be applied to identification of piglets at greater risk from PCV2 infections and the PCVD such as PMWS.
A. Characterization of anti-PCV2 antibodies (maternal-derived and piglet-derived).
The first aim is to apply the M-ELISA for analysing serum samples from piglets which became symptomatic or remained asymptomatic. These were in groups of vaccinated or not, and have already been collected from our collaborators in Canada (Dr. John Harding, University of Sasketchewan), the Netherlands (Dr. Norbert Stockhofe) and Zurich (Dr. Xaver Sydler). The largest number of samples has been supplied by Dr. John Harding, while Dr. Norebert Stockhofe has supplied in addition to the sera PBMC and/or lymph node cells and/or splenocytes from an experiment in which pigs were dually infected with PCV2 and PRRSV. Dr. Xaver Sydeler has supplied sera from pigs infected with PCV2, and will supply additional material from an experiment to be performed in collaboration with the IVI. Details of this material is given under 7.3.2.
The analysis by M-ELISA will look to distinguish maternal antibody and the piglet’s own developing immunity, by performing a kinetic study and monitoring both antibody titres and avidities. This M-ELISA will also be modified to allow distinction of anti-PCV2 IgG and IgM titres and avidities. The data will be analysed by developing a fast throughput Excel Worksheet allowing for the rapid definition of antibody titre and avidity.
B. Characterization of PCV2 dsDNA concentrations in vaccinated and non-vaccinated piglets.
In addition to the determination of anti-PCV2 antibody titres and avidities, current RT-PCR techniques will be adapted to measure the presence and concentration of PCV2 dsDNA. This will be in close collaboration with our partners in Zurich and Barcelona. It is foreseen that the student employed for this project will therefore spend some months in the laboratories of these two partners to execute the PCR analyses.
It is also important to determine if this serum dsDNA is itself immunomodulatory. Accordingly, non-immune and immune sera (from vaccinated pigs) will be spiked with different concentrations of recombinant dsDNA known to be immunomodulatory at particularly concentrations (Balmelli et al., 2009; Vincent et al., 2007). The immunomodulatory activity will be tested on porcine dendritic cell cultures as described (Balmelli et al., 2009; Vincent et al., 2007). Once the immunomodulatory levels of the dsDNA in sera have been defined, the serum samples will also be tested using the same assays on dendritic cell cultures. This will be related to the levels of dsDNA detected therein, to determine if this is potentially immunomodulatory or if there are other factors involved – as would be defined when sera were seen to be immunomodulatory in the absence of detectable dsDNA.
C. Relationship of anti-PCV2 antibody titres and affinities, and PCV2 dsDNA concentrations to piglet disease susceptibility.
The results obtained with the M-ELISA will be related to the outcome of the disease in the piglets. This information is already in the hands of our collaborators, and will be provided once the double blind testing of the sera is completed.
Related to the above, once the concentrations of the dsDNA have been determined, this information will be related to the disease status of the piglets from which the serum containing the dsDNA was obtained
