SUMMARYPigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands.

CLINICAL mastitis has the largest economic impact on the dairy cattle industry. Despite intensive bacteriological research, 20 to 35 per cent of clinical cases of bovine mastitis have an unknown aetiology (Miltenburg and others 1996, Barkema and others 1998). Although viral infections have occasionally been associated with bovine mastitis (Siegler and others 1984, Yoshikawa and others 1997), it is generally considered that viruses do not play a role in the aetiology of bovine mastitis (Watts 1988, Radostits and others 1994 (BHV-1) per millilitre of milk, served as positive controls. A milk sample without viruses and a plain cell culture control, served as negative controls. After the second passage, a haemadsorption reaction, for the detection of, for example, Orthomyxoviridae and Paramyxoviridae, was performed on EBTr cells with 0-2 per cent guinea pig erythrocytes, and incubated at 370C for one hour. An EBTr cell culture inoculated with parainfluenza virus 3 was used as a positive control. Electron microscopy (EM) was performed after the second passages for all four cell types. A 400 mesh carbon-coated nickel grid with a collodion film was floated on a drop of the inoculated cell cultures for five minutes, drained onto filter paper and stained with 2 per cent phosphotungstic acid (pH 6-8). The grids were examined by transmission EM after drying.Serum samples collected from mastitis cows were examined for antibodies against BHV-1 by ELISA (Kramps and others 1994), against bovine herpesvirus 2 (BHV-2) by a 24-hour virus neutralisation test (Bushnell and Edwards 1988), against bovine herpesvirus 4 (BHV-4) by ELISA (Wellenberg and others 1999), against bovine respiratory syncytial virus (BRSV) by ELISA (Westenbrink and others 1985), against bovine viral diarrhoea virus (BVDV) by ELISA (Westenbrink and others 1986), against bovine leukaemia virus (BLV) by ELISA (Pourquier), and against adenovirus type 3 by ELISA (BIO-X). In case blocking percentages, ELISA coefficients or optical density values indicated a significant increase in antibody titre, the serum samples were titrated by serial two-fold dilution steps. The control cows were only examined for antibodies against BHV-4, because only a few cows with clinical mastitis seroconverted (where seroconversion is defined as a seronegative acute serum and a seropositive convalescent serum) against viruses other than BHV-4. Serum samples containing antibodies against BHV-4, were titrated by serial two-fold dilution steps. A four-fold (two dilution steps) higher antibody titre in convalescent serum compared with acute serum is defined as a significant increase.Bacteriological culture of the milk samples was performed according to standards of the National Mastitis Council (Harmon and others 1990). Milk samples (0-01 ml) were inoculated on 6 per cent blood agar plates (both aerobically and anaerobically), on TCT medium (Thallium sulphate, Crystal Violet, Staphylococcus f-toxin; Merck) and on MacConkey number 3 agar (Oxoid). The plates were incubated at 370C and bacteri...

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy naïve individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10(6) PBMC. HIV could not be isolated using up to 30x10(6) patient PBMC. One individual was heterozygous for CCR5 Delta32, but CCR5 expression on CD4+ T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4+ T helper cells were demonstrated by proliferation of CD4+ T cells and intracellular staining for IL-2 and IFNgamma after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection.