Selection of method is crucial for the diagnosis of porcine circovirus type 2 associated reproductive failures.

During a 2-month period a newly repopulated Danish pig herd experienced an increase in numbers of stillborn and mummies, caused by porcine circovirus type 2 (PCV2) associated reproductive failure. Based on recordings of data over time, the progression of the clinical outbreak was studied and the diagnostic value of different techniques was evaluated. Foetal hearts (38 cases and 13 controls) were examined by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) for the detection of PCV2; and total immunoglobulin G (IgG) was measured in pleura cavity fluid. PCV2 IHC was positive in 14/38 of the case foetuses, which were delivered during a 9 days period early in the outbreak. On the basis of the results obtained by IHC and PCR, the foetuses were divided into 3 categories: PCV2 negative; moderately positive (10(4) to 10(7) copies per 500 ng DNA); and massively positive for PCV2 (>10(7) copies per 500 ng DNA). All control- and IHC positive foetuses were included in the negative and massively positive groups, respectively. Ten case foetuses had elevated IgG levels, which did not correlate with the IHC or PCR results. Based on the clustering of the IHC positive foetuses, it is suggested that IHC only is suited for diagnosing acute stages of reproductive failure, whereas quantitative PCR can be used as a sensitive diagnostic method within a wider time span. It seems that IgG measurements are unpredictable as indication of intrauterine infection with PCV2.

Examination for a viral co-factor in postweaning multisystemic wasting syndrome (PMWS).

In order to test the hypothesis that a putative co-factor for the development of postweaning multisystemic wasting syndrome (PMWS) in pigs could be of viral origin, we performed extensive virological examinations on organ material from pigs diagnosed with PMWS originating from within a Danish PMWS-transmission study. Virus isolation attempts were carried out on a large panel of different cell types including primary pig kidney cells and lung macrophages, primary rabbit kidney cells and seven established cell lines (MARC-145, ST117, PK15, BHK21, HeLa, Vero, and MDCK). Although these represent cells with susceptibility to a wide range of known viruses, the results did not provide evidence for a specific virus other than PCV2 contributing to the development of PMWS. Furthermore, in order to test whether specific genotypes of PCV2 may trigger the switch from PCV2 infection to clinical disease, we compared complete DNA genome sequences of PCV2 derived from PMWS-positive as well as PMWS-negative pigs. On the basis of the DNA sequences, the PCV2 isolates were divided into two groups. Group 1 consisting of one isolate originating from a herd unaffected by PMWS, with group 2 consisting of nine isolates originating from four PMWS-affected herds, four PMWS-positive pigs plus one unaffected herd. The PCV2 genomes from the two groups showed 95.5% identity. Alignment analyses of the sequences encoding the replicase and capsid protein from group 1 and group 2 PCV2 isolates showed two amino acid differences encoded in the replicase protein, while 19 amino acid differences were predicted among the capsid protein sequences. The PCV2 DNA sequence analysis supports recent observations from studies in USA as well as Europe, which suggest that strain variations may influence the clinical outcome of PCV2 infection.

The Flo11p-deficient Saccharomyces cerevisiae strain background S288c can adhere to plastic surfaces.

The effects of four types of plastic surfaces and four pre-incubation media, containing high/low glucose and +/- amino acids, on adhesion of Saccharomyces cerevisiae BY4742 wild type and Deltaflo11 mutant (strain background S288c) were investigated. No difference in adhesive ability between the two yeast strains was observed in any of our experiments, thus confirming that FLO11 is not operational in the S. cerevisiae S288c strain background. The adhesive abilities of both yeast strains depended on the plastic type and pre-incubation conditions. The poorest adhesion was observed on hydrophilic polystyrene, whereas hydrophobic polystyrene resulted in moderate adhesion. The best adhesion of both yeast strains was observed on polystyrene surfaces with combined hydrophilic/hydrophobic domains. When amino acids were present in the pre-incubation media, lack of glucose increased the cell surface hydrophobicity and enhanced the adhesion to all four types of polystyrene. Lack of amino acids in the pre-incubation media increased the cell surface hydrophobicity and enhanced the adhesion especially to polystyrene surfaces with combined hydrophilic/hydrophobic domains. Our results suggest that glucose and amino acid starvation induces other genes than FLO11 in S. cerevisiae S288c coding for hydrophobic cell surface constituents with adhesive properties to especially moderately hydrophobic plastic surfaces.

Identification of the receptor-binding protein in 936-species lactococcal bacteriophages.

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.

Identification of Lactococcus lactis genes required for bacteriophage adsorption.

The aim of this work was to identify genes in Lactococcus lactis subsp. lactis IL1403 and Lactococcus lactis subsp. cremoris Wg2 important for adsorption of the 936-species phages bIL170 and phi 645, respectively. Random insertional mutagenesis of the two L. lactis strains was carried out with the vector pGh9:ISS1, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In L. lactis IL1403 integration was obtained in the ycaG and rgpE genes, whereas in L. lactis Wg2 integration was obtained in two genes homologous to ycbC and ycbB of L. lactis IL1403. rgpE and ycbB encode putative glycosyltransferases, whereas ycaG and ycbC encode putative membrane-spanning proteins with unknown functions. Interestingly, ycaG, rgpE, ycbC, and ycbB are all part of the same operon in L. lactis IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that phi645 binds to and infects L. lactis Wg2, L. lactis IL1403, and L. lactis IL1403 strains with pGh9:ISS1 integration in ycaG and rgpE, whereas bIL170 binds to and infects only L. lactis IL1403 and cannot infect Wg2. These results indicate that phi 645 binds to a WPS structure present in both L. lactis IL1403 and L. lactis Wg2, whereas bIL170 binds to another WPS structure not present in L. lactis Wg2. Binding of bIL170 and phi 645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and phi 645 that showed no homology in the C-terminal part.