Enric Mateu

The challenge of PRRS immunology.

Abstract Porcine reproductive and respiratory syndrome (PRRS) is one of the most challenging subjects of research in veterinary viral immunology, and the immune response against PRRS virus (PRRSV) still is poorly understood. Infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an antibody-dependent enhancement of disease. In contrast, development of neutralising antibodies (NAs) is delayed and generation of cell-mediated immune responses, such as PRRSV-specific interferon (IFN)-γ secreting cells, is initially erratic. In spite of this, induction of strong and rapid NAs and IFN-γ responses seem to be required for effective vaccination. PRRSV strongly modulates the host’s immune responses. The virus inhibits key cytokines, such as IFN-α, and may induce regulatory cytokines, such as interleukin (IL)-10. Development of NAs seems to be impaired by the existence of a decoy epitope close to the main neutralisation epitope in glycoprotein 5. This ability to modulate the host immune response probably varies among strains or isolates. The genetic diversity of the virus is very high and it has been shown that this diversity can have serious implications for the development of vaccines, since the immunity induced by one strain may be only partial against a different strain, even within the same genotype. With this panorama, the development of newer and universally efficacious PRRSV vaccines is challenging, but the present state of knowledge allows optimism if collaborative efforts are undertaken in the scientific community.

Porcine circovirus type 2 (PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins.

The efficacy of recently developed porcine circovirus type 2 (PCV2) vaccines has not been tested yet against PCV2 isolates of the two proposed genotypes. In the present work, the efficacy of a subunit vaccine containing PCV2 capsid protein was evaluated by using a challenge model with four different PCV2 isolates of different genotype and geographic origin. The vaccine prevented the development of viremia in all cases as well as significantly decreased nasal and faecal shedding of the virus. Also, the vaccine elicited PCV2-specific neutralizing antibodies to PCV2 even in the presence of maternally derived immunity.

Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: An immune riddle.

Summary.Postweaning multisystemic wasting syndrome (PMWS) is a disease of pigs first recognised in North America in 1997 and subsequently reported worldwide that is caused by Porcine circovirus 2 (PCV2), a member of the family Circoviridae. The most consistent feature of PMWS is a generalized depletion of lymphocytes. Secondary infections with opportunistic organisms are common. There is evidence that the destruction of thymic lymphocytes has a central role in the pathogenesis of PMWS. Pigs with PMWS have altered cytokine responses to mitogens and recall antigens. It remains unknown what cells are primarily infected and are permissive for the replication of PCV2. Macrophages and dendritic cells commonly contain virus in their cytoplasm but may not be the primary source of the large amounts of virus found in tissues of diseased pigs. There is evidence that PCV2, like mammalian parvoviruses, requires cells in the S phase of the cell cycle for replication. It has been difficult to reproduce PMWS experimentally although some protocols have been developed which involve antigenic stimulation with other agents that presumably increase the number of permissive cells entering S phase of the cell cycle. In addition to reviewing the literature attempts are made to identify key unresolved areas that should be the focus of future research.

Certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most costly pathogens for the swine industry. Since its emergence some 20 years ago, much has been learned about the immunobiology of PRRSV. Although vaccines are available, they do not provide full and universal protection against PRRSV infection. In the present review, current knowledge on the viruss immunobiology will be discussed including: role of viral receptors, innate immune response to the virus, regulation of the immune response by PRRSV, and the characteristics and role of adaptive immunity. In addition, some hypotheses for future research in this area are presented.

Changes in CD4+, CD8+, CD4+ CD8+, and Immunoglobulin M-Positive Peripheral Blood Mononuclear Cells of Postweaning Multisystemic Wasting Syndrome-Affected Pigs and Age-Matched Uninfected Wasted and Healthy Pigs Correlate with Lesions and Porcine Circovirus Type 2 Load in Lymphoid Tissues

ABSTRACT Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4+, CD8+, CD4+ CD8+ (double-positive [DP]), and immunoglobulin M-positive (IgM+) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyers patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8+ and DP cell subsets compared to the other groups (P < 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (P < 0.05) as well as to the relative decrease in IgM+ and CD8+ cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response.

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.

Several Salmonella enterica subsp. enterica Serotype 4,5,12:i:− Phage Types Isolated from Swine Samples Originate from Serotype Typhimurium DT U302

ABSTRACT Pulsed-field gel electrophoresis, plasmid profiling, and phage typing were used to characterize and determine possible genetic relationships between 48 Salmonella enterica subsp. enterica isolates of pig origin collected in Catalonia, Spain, from 1998 to 2000. The strains were grouped into 23 multidrug-resistant fljB-lacking S. enterica serovar 4,5,12:i:− isolates, 24 S. enterica serovar Typhimurium isolates, and 1 S. enterica serovar 4,5,12:−:− isolate. After combining the XbaI and BlnI macrorestriction profiles (XB profile), we observed 29 distinct subtypes which were grouped into seven main patterns. All 23 of the 4,5,12:i:− serovar strains and 10 serovar Typhimurium isolates were found to have pattern AR, and similarities of >78% were detected among the subtypes. Three of the serovar Typhimurium DT U302 strains (strains T3, T4, and T8) were included in the same 4,5,12:i:− serovar cluster and shared a plasmid profile (profile I) and a pattern of multidrug resistance (resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, gentamicin, and trimethoprim-sulfamethoxazole) commonly found in monophasic isolates. This led us to the conclusion that strains of the S. enterica 4,5,12:i:− serovar might have originated from an S. enterica serovar Typhimurium DT U302 strain.

Effect of Acidified Feed on the Prevalence of Salmonella in Market-age Pigs

Two trials were carried out to determine the effect of feed acidification upon Salmonella carriage in market‐age pigs. In the first trial, the administration for the last 14 weeks of the fattening period of a commercial pelleted feed added with 0.6% lactic acid plus 0.6% formic acid (Lac‐Formic‐1.2) was compared to an unacidified standard diet (STD). A second experiment was carried out in two herds of growing pigs (Herd I, 3000 pigs; Herd II, 900 pigs) in which three different diets were assayed during the last 8–9 weeks of the fattening period: a diet containing 0.8% formic acid (Formic‐0.8), a diet containing 0.4% lactic acid plus 0.4% formic acid (Lac‐Formic‐0.8) and a STD. In the first experiment, serological evolution of the infection was examined by ELISA and microbiological cultures (rectal samples and mesenteric lymph nodes) were also done. Feed intake by pen and the individual weight of the animals were also measured. In the second trial, blood, rectal samples and mesenteric lymph nodes were collected at slaughter in both herds (30 pigs per experimental group). In the first experiment, the acidified diet (Lac‐Formic‐1.2) reduced Salmonella carriers in mesenteric lymph nodes (Fisher’s exact P < 0.01). In the second trial, Lac‐Formic‐0.8 diet significantly reduced Salmonella seroprevalence compared to the STD (P = 0.001) in both herds. Also Lac‐Formic‐0.8 and Formic‐0.8 diets in Herd II showed a lower faecal excretion and Salmonella carriage in mesenteric lymph nodes than the STD (P < 0.05). Our results suggest that the administration of a combination of lactic and formic acids at the levels used in this study could be used to reduce Salmonella prevalence in finishing pigs.

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.