Efficient priming of CD4 T cells by Langerin-expressing dendritic cells targeted with porcine epidemic diarrhea virus spike protein domains in pigs.

Porcine epidemic diarrhea virus (PEDV) first emerged in the United States in 2013 causing high mortality and morbidity in neonatal piglets with immense economic losses to the swine industry. PEDV is an alpha-coronavirus replicating primarily in porcine intestinal cells. PEDV vaccines are available in Asia and Europe, and conditionally-licensed vaccines recently became available in the United States but the efficacies of these vaccines in eliminating PEDV from swine populations are questionable. In this study, the immunogenicity of a subunit vaccine based on the spike protein of PEDV, which was directly targeted to porcine dendritic cells (DCs) expressing Langerin, was assessed. The PEDV S antigen was delivered to the dendritic cells through a single-chain antibody specific to Langerin and the targeted cells were stimulated with cholera toxin adjuvant. This approach, known as "dendritic cell targeting," greatly improved PEDV S antigen-specific T cell interferon-γ responses in the CD4posCD8pos T cell compartment in pigs as early as 7days upon transdermal administration. When the vaccine protein was targeted to Langerinpos DCs systemically through intramuscular vaccination, it induced higher serum IgG and IgA responses in pigs, though these responses require a booster dose, and the magnitude of T cell responses were lower as compared to transdermal vaccination. We conclude that PEDV spike protein domains targeting Langerin-expressing dendritic cells significantly increased CD4 T cell immune responses in pigs. The results indicate that the immunogenicity of protein subunit vaccines can be greatly enhanced by direct targeting of the vaccine antigens to desirable dendritic cell subsets in pigs.

Prevalence of virulence factors in enterotoxigenic Escherichia coli isolated from pigs with post-weaning diarrhoea in Europe.

BACKGROUND: Post-weaning diarrhoea (PWD), due to Escherichia coli, is an important cause of economic losses to the pig industry primarily as a result of mortality and worsened productive performance. In spite of its relevance, recent data about the prevalence of virulence genes and pathotypes among E. coli isolates recovered from cases of PWD in Europe are scarce. RESULTS: This study investigates the prevalence of fimbrial and toxin genes of E. coli by PCR among 280 farms with PWD across Europe. A total of 873 samples collected within the first 48 h after the onset of PWD (occurring 7-21 days post weaning) were submitted to the laboratory for diagnostic purposes. Isolation and identification of E. coli were performed following standard bacteriological methods and PCR assays for the detection of genes encoding for fimbriae (F4, F5, F6, F18 and F41) and toxins (LT, STa, STb and Stx2e). The prevalence of fimbriae and toxins among E. coli isolates from cases of PWD was: F4 (45.1 %), F18 (33.9 %), F5 (0.6 %), F6 (0.6 %), F41 (0.3 %), STb (59.1 %), STa (38.1 %), LT (31.9 %) and Stx2e (9.7 %). E. coli isolates carrying both fimbrial and toxin genes were detected in 52.5 % of the cases (178 out of 339 isolates), with 94.9 % of them being classified as enterotoxigenic E. coli (ETEC). The most common virotype detected was F4, STb, LT. CONCLUSIONS: This study confirms that ETEC is frequently isolated in pig farms with PWD across Europe, with F4- and F18-ETEC variants involved in 36.1 % and 18.2 % of the outbreaks, respectively.

Performance of a commercial Swine influenza virus H1N1 and H3N2 antibody enzyme-linked immunosorbent assay in pigs experimentally infected with European influenza viruses.

The IDEXX Swine influenza virus H1N1 and H3N2 enzyme-linked immunosorbent assays (ELISAs) are used worldwide, but their capacity to detect antibodies to European Swine influenza viruses (SIVs) has not been documented. A total of 313 well-defined sera from SIV seronegative pigs and pigs experimentally infected with European SIVs were used to compare the performance of both ELISAs and the hemagglutination inhibition (HI) test. The ELISAs largely failed to detect pigs that had been infected with H1N1 (0/42 positive in H1N1 ELISA) or H3N2 only (9/18 positive in H3N2 ELISA; group 1). Higher ELISA detection rates were found after consecutive infection of pigs with either H1N1 or H3N2 and 1 other subtype (7/40 and 11/22 positive in H1N1 and H3N2 ELISA, respectively; group 2). Of 39 pigs that had been vaccinated twice with 1 of 4 commercial SIV vaccines (group 3), 25 tested positive in the H1N1 and 4 in the H3N2 ELISA. Pigs that had received a single vaccination after a prior infection with H1N1 and/or H3N2 (group 4) were more frequently positive than group 1 or 3 pigs (23/24 and 15/24 positive in H1N1 and H3N2 ELISA, respectively). Both the H1N1 and H3N2 ELISA showed a low sensitivity (39% and 35%, respectively) relative to the HI test. Because pigs in the field are frequently infected and/or vaccinated with multiple SIV subtypes and variants, they are more likely to test positive in the ELISAs. However, the interpretation of ELISA results will be difficult, and HI remains the method of choice.

Seroprevalence of H1N1, H3N2 and H1N2 influenza viruses in pigs in seven European countries in 2002-2003.

OBJECTIVES: Avian-like H1N1 and human-like H3N2 swine influenza viruses (SIV) have been considered widespread among pigs in Western Europe since the 1980s, and a novel H1N2 reassortant with a human-like H1 emerged in the mid 1990s. This study, which was part of the EC-funded 'European Surveillance Network for Influenza in Pigs 1', aimed to determine the seroprevalence of the H1N2 virus in different European regions and to compare the relative prevalences of each SIV between regions. DESIGN: Laboratories from Belgium, the Czech Republic, Germany, Italy, Ireland, Poland and Spain participated in an international serosurvey. A total of 4190 sow sera from 651 farms were collected in 2002-2003 and examined in haemagglutination inhibition tests against H1N1, H3N2 and H1N2. RESULTS: In Belgium, Germany, Italy and Spain seroprevalence rates to each of the three SIV subtypes were high (> or =30% of the sows seropositive) to very high (> or =50%), except for a lower H1N2 seroprevalence rate in Italy (13.8%). Most sows in these countries with high pig populations had antibodies to two or three subtypes. In Ireland, the Czech Republic and Poland, where swine farming is less intensive, H1N1 was the dominant subtype (8.0-11.7% seropositives) and H1N2 and H3N2 antibodies were rare (0-4.2% seropositives). CONCLUSIONS: Thus, SIV of H1N1, H3N2 and H1N2 subtype are enzootic in swine producing regions of Western Europe. In Central Europe, SIV activity is low and the circulation of H3N2 and H1N2 remains to be confirmed. The evolution and epidemiology of SIV throughout Europe is being further monitored through a second 'European Surveillance Network for Influenza in Pigs'.

Serological profiles after consecutive experimental infections of pigs with European H1N1, H3N2, and H1N2 swine influenza viruses.

Swine influenza viruses (SIVs) of H1N1, H3N2, and H1N2 subtypes, with antigenically different hemagglutinins, are currently cocirculating in pigs in Europe. This study aimed to determine whether the hemagglutination inhibition (HI) test, which is the primary serological test for SIV, is sufficiently specific to discriminate between infections with the three subtypes. In experiment 1, pigs were consecutively inoculated with European H1N1, H3N2, and H1N2 SIVs by the intranasal route, or with the respective subtypes only. In a second experiment, a commercial, inactivated H1N1- and H3N2- based SIV vaccine was administered once to pigs previously infected with one to three SIV subtypes or to influenza-naive pigs. Sequential serum samples were examined in HI and virus-neutralizing (VN) tests to the three strains used for pig inoculations. Of the 160 sera collected after infection with one or two SIV subtypes, only 8 showed cross-reactive antibodies to the remaining subtype(s) in the HI test, and 11 in the VN test. Consecutive inoculations with H1N1 and H1N2 or vice versa were followed by a significant rise in preexisting antibody titers to the first subtype after the second inoculation. When dually infection-immune pigs were inoculated with the third, remaining SIV subtype, nasal virus excretion was undetectable or reduced and the serological response was absent to moderate. A single vaccination of infection-immune pigs resulted in a dramatic rise in HI and VN antibody titers to any of the previously encountered subtypes, whereas SIV-naive pigs barely seroconverted. Most important, pigs previously infected with H1N1 but not with H1N2 developed crossreactive antibodies to H1N2 after the vaccination. In conclusion, the HI test remains adequate for the differential diagnosis of infections with H1N1, H3N2, and H1N2 in European swine populations if it is properly used and if the SIV vaccination status is taken into account.

The combination of PRRS virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs.

This paper reviews in vivo studies on the interaction between porcine reproductive and respiratory syndrome virus (PRRSV) and LPS performed in the authors' laboratory. The main aim was to develop a reproducible model to study the pathogenesis of PRRSV-induced multifactorial respiratory disease. The central hypothesis was that respiratory disease results from an overproduction of proinflammatory cytokines in the lungs. In a first series of studies, PRRSV was shown to be a poor inducer of TNF-alpha and IFN-alpha in the lungs, whereas IL-1 and the anti-inflammatory cytokine IL-10 were produced consistently during infection. We then set up a dual inoculation model in which pigs were inoculated intratracheally with PRRSV and 3-14 days later with LPS. PRRSV-infected pigs developed acute respiratory signs for 12-24h upon intratracheal LPS inoculation, in contrast to pigs inoculated with PRRSV or LPS only. Moreover, peak TNF-alpha, IL-1 and IL-6 titers were 10-100 times higher in PRRSV-LPS inoculated pigs than in the singly inoculated pigs and the cytokine overproduction was associated with disease. To further prove the role of proinflammatory cytokines, we studied the effect of pentoxifylline, a known inhibitor of TNF-alpha and IL-1, on PRRSV-LPS induced cytokine production and disease. The clinical effects of two non-steroidal anti-inflammatory drugs (NSAIDs), meloxicam and flunixin meglumine, were also examined. Pentoxifylline, but not the NSAIDs, significantly reduced fever and respiratory signs from 2 to 6h after LPS. The levels of TNF-alpha and IL-1 in the lungs of pentoxifylline-treated pigs were moderately reduced, but were still 26 and 3.5-fold higher than in pigs inoculated with PRRSV or LPS only. This indicates that pathways other than inhibition of cytokine production contributed to the clinical improvement. Finally, we studied a mechanism by which PRRSV may sensitize the lungs for LPS. We hypothesized that PRRSV would increase the amount of LPS receptor complex in the lungs leading to LPS sensitisation. Both CD14 and LPS-binding protein, two components of this complex, increased significantly during infection and the amount of CD14 in particular was correlated with LPS sensitisation. The increase of CD14 was mainly due to infiltration of strongly CD14-positive monocytes in the lungs. The PRRSV-LPS combination proved to be a simple and reproducible experimental model for multifactorial respiratory disease in pigs. To what extent the interaction between PRRSV and LPS contributes to the development of complex respiratory disease is still a matter of debate.

Apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines.

Apoptosis was studied in the lungs of pigs during an infection with a European strain of porcine reproductive and respiratory syndrome virus (PRRSV) and it was examined if cytokines were involved in the induction of apoptosis. Twenty-two 4- to 5-week-old gnotobiotic pigs were inoculated intranasally with 10(6.0) TCID50 of the Lelystad virus and euthanised between 1 and 52 days post inoculation (PI). The lungs and broncho-alveolar lavage (BAL) cells were assessed both for virus replication and apoptosis; BAL fluids were examined for interleukin (IL)-1, tumour necrosis factor-alpha and IL-10. Double-labellings were conducted to determine the relation between virus replication and apoptosis and to identify the apoptotic cells. Apoptosis occurred in both infected and non-infected cells. The percentages of infected cells, which were apoptotic, ranged between 9 and 39% in the lungs and between 13 and 30% in the BAL cells. The majority of apoptotic cells were non-infected. Non-infected apoptotic cells in the lungs were predominantly monocytes/macrophages, whereas those in the broncho-alveolar spaces were predominantly lymphocytes. The peak of apoptosis in the lungs at 14 days PI was preceded by a peak of IL-1 and IL-10 production at 9 days PI, suggesting a possible role of these cytokines in the induction of apoptosis in non-infected interstitial monocytes/macrophages. However, the latter hypothesis was not confirmed in vitro, since blood monocytes or alveolar macrophages did not undergo apoptosis after treatment with recombinant porcine IL-1 or IL-10.