Influence of age on the effectiveness of PCV2 vaccination in piglets with high levels of maternally derived antibodies.

Two field studies were conducted to investigate the influence of age on the efficacy of vaccination against Porcine Circovirus Diseases (PCVD) in animals with high levels of maternally derived antibodies (MDA). A total of 416 piglets (Study 1) and 600 piglets (Study 2) were randomly allocated to one of three groups. Two groups in each study received a single dose of a PCV2 subunit vaccine, one group at 1 week old and the other at 3 weeks of age. The third group was left untreated. Animals vaccinated at 3 weeks of age showed a significantly higher average daily weight gain and significantly reduced viraemia following PCV2 infection than the respective control groups. This difference was not observed in pigs vaccinated at 1 week of age. Furthermore, only animals vaccinated at 3 weeks of age showed an increased serological response and a higher frequency of IgM-positive animals compared with controls. The data indicated that PCV2 vaccination in the presence of high MDA levels is efficacious when used in 3-week old but not in 1-week old pigs. As the range of MDA titres of pigs vaccinated at both 1 and 3 weeks of age were comparable, the data suggest that PCV2 vaccine efficacy was independent of the level of MDA. It appears that other age-related factors affecting the active and passive transfer of immunity may perhaps have interfered with the efficacy of the vaccine in 1-week old piglets. These findings have implications for future PCV2 vaccine testing and administration strategies.

Development of a multiplexed microcapillary liquid chromatography system for high-throughput proteome analysis.

Comprehensive proteome analysis requires the identification (and quantification) of the proteins in samples consisting of thousands of proteins spanning a range of abundance of several orders of magnitude. The currency of proteome analysis by mass spectrometry is the peptides generated by protein proteolysis. The high sample complexity of such samples requires a large separation capacity, which is commonly achieved by fractionation of the mixture followed by further serial separations of each fraction. The sample throughput of proteome analysis is therefore limited by the need to sequentially process large numbers of samples. We have developed a novel four-plexed microcapillary liquid chromatography system for automated, high-throughput separation of complex peptide samples. The system supports the concurrent separation of four different samples by directing identically split solvent-gradient flows into four microcapillary C18 columns. The simple design of the system achieves multiplexed separation without the need for extra solvent pumps. Peak resolution, reproducibility, and parallel separating capacity of the system were investigated using standard peptides. The applicability of the system to high-throughput protein expression profiling was demonstrated in qualitative and quantitative analyses of protein expression in S. cerevisiae grown on two different carbon sources using the isotope-coded affinity tag (ICAT) reagent and matrix-assisted desorption/ionization quadrupole time-of-flight mass spectrometry.

Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae.

Using an integrated genomic and proteomic approach, we have investigated the effects of carbon source perturbation on steady-state gene expression in the yeast Saccharomyces cerevisiae growing on either galactose or ethanol. For many genes, significant differences between the abundance ratio of the messenger RNA transcript and the corresponding protein product were observed. Insights into the perturbative effects on genes involved in respiration, energy generation, and protein synthesis were obtained that would not have been apparent from measurements made at either the messenger RNA or protein level alone, illustrating the power of integrating different types of data obtained from the same sample for the comprehensive characterization of biological systems and processes.

Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.

The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.