Joan Pujols

Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome (PMWS).

The objective of the present study was to analyze, by flow cytometry, changes in PBMC subsets in pigs having postweaning multisystemic wasting syndrome (PMWS), a new condition associated to porcine circovirus type 2 (PCV2) infection. Thirteen acutely PMWS affected pigs were selected from a farm seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) and to Aujeszkys disease virus (ADV); 11 clinically healthy pigs were selected from a high health farm with no history of PMWS and free of the major swine pathogens, and used as a control group. All pigs were necropsied, and tissue samples were fixed in formalin; blood with EDTA anticoagulant was used to perform the flow cytometric analysis. PBMC were incubated with mAb against porcine CD3, CD4, CD8, CD25, CD45, IgM, SWC3, and SLA-Class II. Flow cytometric analysis showed substantial changes in leukocyte subsets in the peripheral blood of PMWS-affected pigs, which were characterized by an increase of monocytes, a reduction of T (mainly CD4(+)) and B-lymphocytes, and the presence of low-density immature granulocytes. Altogether, these changes would suggest an inability of acutely PMWS-affected pigs to mount an effective immune response.

Retrospective study on swine Torque teno virus genogroups 1 and 2 infection from 1985 to 2005 in Spain

A retrospective study to detect evidence of swine Torque teno virus (TTV) genogroups 1 and 2 infection in sera of pigs from the Spanish livestock between years 1985 and 2005 was carried out by means of PCR. Also, the molecular evolution of TTV genogroups 1 and 2 during the 20-year period studied using a 250-base sequence of the non-coding region of the viral genome was assessed. Both TTV genogroup genomes were found in pig sera from the first year of study. Taking into account the whole study period, 113 out of 162 animals (69.8%) were infected with one or the other genogroup, while 38 out of 162 pigs (23.5%) were co-infected with both genogroups. Moreover, TTV genogroup 2 (90 out of 162, 55.6%) was significantly more prevalent than genogroup 1 (54 out of 162, 33.3%). The non-coding region of swine TTV genome sequenced showed its adequacy as a molecular marker in swine TTV. This study represents the earliest evidence of TTV infection in pigs to date, 14 years before the initial description of this virus. Moreover, this is also the earliest evidence of TTV infection in any species.

Porcine epidemic abortion and respiratory syndrome (mystery swine disease). Isolation in Spain of the causative agent and experimental reproduction of the disease

In March of 1991, a disease that affected pregnant sows and caused a high mortality in unweaned piglets was detected in Spain. Based on the clinical signs observed, mystery swine disease, which had been described recently in Germany, Holland and Belgium, was suspected. From the samples obtained from the affected farm, a filtrable agent (0.22 micron) was isolated on cell culture. It produced cytopathic effects, its replication was intracytoplasmic, it was sensitive to chloroform, and cross-reacted with a Lelystad reference serum. When inoculated into pregnant sows, the agent produced inappetence for 2-4 days, without hyperthermia. One of the sows aborted at 100 days of gestation; the two others had delayed parturitions (days 115 and 116). There was a mixture of healthy piglets, mummified fetuses, stillbirths and weak piglets. Microscopic examination of the lungs of healthy piglets killed at 8 and 12 days of life revealed the presence of interstitial pneumonia. The sera from the three sows at 39 days after infection cross-reacted with the Lelystad virus (titres > or = 1/640), whereas pre-inoculation sera did not recognize it (titres < or = 1/10). This is the first report from Spain of the isolation of an agent (antigenically related to the Lelystad virus), capable of reproducing the disease previously designated as mystery swine disease.

Development of cell-mediated immunity to porcine circovirus type 2 (PCV2) in caesarean-derived, colostrum-deprived piglets.

Abstract The interaction between porcine circovirus type 2 (PCV2) and the pig immune system has been suggested to be a determinant event for the pathogenesis of postweaning multisystemic wasting syndrome (PMWS). To gain insight into the host immune mechanisms developed upon PCV2 infection, early innate and adaptive immune responses were examined in 1-week-old, caesarean-derived, colostrum-deprived piglets using a subclinical infection model of PCV2 in combination with lipopolysaccharide (LPS) as a potential immunostimulation factor. The use of LPS did not show any significant effect on the course of PCV2 infection, nor did in the evolution of the immunological parameters evaluated. Ex vivo responses were detected as early as 1 day post-infection (PI) and consisted of an elevation of the plasmatic levels of interleukin (IL)-8 in PCV2-inoculated pigs followed by an increase on plasmatic IFN-α at day 5 PI. Regarding IL-10, only one PCV2-inoculated pig was positive (day 7 PI); this pig was the only one in which viremia persisted until the end of the study. In vitro cytokine determination showed that, regardless of the treatment administrated to the pigs, an IL-10 release was observed when peripheral blood mononuclear cells (PBMC) cultures were stimulated with PCV2. Seroconvertion to PCV2 measured by an immunoperoxidase monolayer assay (IPMA) occurred between 7 and 14 days PI, whereas neutralizing antibodies (NA) did not appear until day 29 PI. PCV2 DNA was first detected in serum at day 7 PI, reaching the peak of viremia between days 14 and 21 PI, followed by a drop in viral load that was found coincident with the appearance of PCV2-specific IFN-γ-secreting cells (PCV2-IFN-γ-SC) and NA. Results from the present work suggest that viral clearance might be mediated by the development of PCV2-IFN-γ-SC in contribution to the PCV2-specific NA.

Cytokine profiles and phenotype regulation of antigen presenting cells by genotype-I porcine reproductive and respiratory syndrome virus isolates

The present study examined the immunological response of antigen presenting cells (APC) to genotype-I isolates of porcine reproductive and respiratory syndrome virus (PRRSV) infection by analysing the cytokine profile induced and evaluating the changes taking place upon infection on immunologically relevant cell markers (MHCI, MHCII, CD80/86, CD14, CD16, CD163, CD172a, SWC9). Several types of APC were infected with 39 PRRSV isolates. The results show that different isolates were able to induce different patterns of IL-10 and TNF-α. The four possible phenotypes based on the ability to induce IL-10 and/or TNF-α were observed, although different cell types seemed to have different capabilities. In addition, isolates inducing different cytokine-release profiles on APC could induce different expression of cell markers.

Molecular epidemiology and evolution of avian infectious bronchitis virus in Spain over a fourteen-year period

Abstract An in-depth molecular study of infectious bronchitis viruses (IBV) with particular interest in evolutionary aspects of IBV in Spain was carried out in the present study based on the S1 gene molecular characterization of twenty-six Spanish strains isolated over a fourteen-year period. Four genotypes were identified based on S1 gene sequence analyses and phylogenetic studies. A drastic virus population shift was demonstrated along time and the novel Italy 02 serotype was shown to have displaced the previous predominant serotype 4/91 in the field. Detailed analyses of synonymous to non-synonymous ratio of the S1 gene sequences of this new serotype Italy 02 suggested positive selection pressures might have contributed to the successful establishment of Italy 02 serotype in our country. In addition, differences on the fitness abilities of new emergent genotypes were indicated. Furthermore, intergenic sequences (IGs)-like motifs within S1 gene sequences of IBV isolates were suggested to enhance the recombination abilities of certain serotypes.

In silico prediction and ex vivo evaluation of potential T-cell epitopes in glycoproteins 4 and 5 and nucleocapsid protein of genotype-I (European) of porcine reproductive and respiratory syndrome virus.

T-cell epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) glycoproteins 4 (GP4), 5 (GP5) and nucleocapsid (N) were predicted using bioinformatics and later tested by IFN-gamma ELISPOT in pigs immunized with either a modified live vaccine (MLV) or DNA (open reading frames 4, 5 or 7). For MLV-vaccinated pigs, immunodominant epitopes were found in N but T-epitopes were also found in GP4 and GP5. For DNA-immunized pigs, some peptides were differently recognized. Using a large set of PRRSV sequences it was shown that N contains a conserved epitope and that for GP5, the genotype-I counterparts of previously reported epitopes of genotype-II strains were also immunogenic.

Genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype I strains

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been based on ORF5/GP5 and ORF7/N protein variations. Complete viral genome studies are limited and focused on a single or a few set of strains. Moreover, there is a general tendency to extrapolate results obtained from a single isolate to the overall PRRSV population. In the present study, six genotype-I isolates of PRRSV were sequenced from ORF1a to ORF7. Phylogenetic comparisons and the variability degree of known linear B-epitopes were done considering other available full-length genotype-I sequences. Cytokine induction of all strains was also evaluated in different cellular systems. Non structural protein 2 (nsp2) was the most variable part of the virus with 2 out of 6 strains harboring a 74 aa deletion. Deletions were also found in ORF3 and ORF4. Phylogenetic analyses showed that isolates could be grouped differently depending on the ORF examined and the highest similarity with the full genome cluster was found for the nsp9. Interestingly, most of predicted linear B-epitopes in the literature, particularly in nsp2 and GP4 regions, were found deleted or varied in some of our isolates. Moreover, 4 strains, those with deletions in nsp2, induced TNF-α and 3 induced IL-10. These results underline the high genetic diversity of PRRSV mainly in nsp1, nsp2 and ORFs 3 and 4. This variability also affects most of the known linear B-epitopes of the virus. Accordingly, different PRRSV strains might have substantially different immunobiological properties. These data can contribute to the understanding of PRRSV complexity.

Evolution of ORF5 of Spanish porcine reproductive and respiratory syndrome virus strains from 1991 to 2005

Abstract ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991–1995, 18 strains) and a second set (n =46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991–1995 to 89.5% in 2000–2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000–2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991–1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000–2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-β were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log2) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-β levels in serum for 7–14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.