Evaluation of PRRSv specific, maternally derived and induced immune response in Ingelvac PRRSFLEX EU vaccinated piglets in the presence of maternally transferred immunity.

In this study, we analyzed PRRS virus (PRRSv) specific lymphocyte function in piglets vaccinated with Ingelvac PRRSFLEX EU® at two and three weeks of age in the presence of homologous maternal immunity. Complete analysis of maternal immunity to PRRSv was evaluated postpartum, as well as passive transfer of antibodies and T cells to the piglet through colostrum intake and before and after challenge with a heterologous PRRSv at ten weeks of age. Maternal-derived antibodies were detected in piglets but declined quickly after weaning. However, vaccinated animals restored PRRSv-specific antibody levels by anamnestic response to vaccination. Cell analysis in colostrum and milk revealed presence of PRRSv-specific immune cells at suckling with higher concentrations found in colostrum than in milk. In addition, colostrum and milk contained PRRSv-specific IgA and IgG that may contribute to protection of newborn piglets. Despite the presence of PRRSv-specific Peripheral Blood Mononuclear cells (PBMCs) in colostrum and milk, no PRRSv-specific cells could be detected from blood of the piglets at one or two weeks of life. Nevertheless, cellular immunity was detectable in pre-challenged piglets up to 7 weeks after vaccination while the non-vaccinated control group showed no interferon (IFN) γ response to PRRSv stimulation. After challenge, all piglets developed a PRRSv-specific IFNγ-response, which was more robust at significantly higher levels in vaccinated animals compared to the primary response to PRRSv in non-vaccinated animals. Cytokine analysis in the lung lumen showed a reduction of pro-inflammatory responses to PRRSv challenge in vaccinated animals, especially reduced interferon (IFN) α levels. In conclusion, vaccination of maternally positive piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX EU induced a humoral and cellular immune response to PRRSv and provided protection against virulent, heterologous PRRSv challenge.

Splenocyte Infusion and Whole-Body Irradiation for Induction of Peripheral Tolerance in Porcine Lung Transplantation: Modifications of the Preconditioning Regime for Improved Clinical Feasibility.

BACKGROUND: Preoperative low-dose whole-body irradiation (IRR) with 1.5 and 7 Gy thymic IRR of the recipient, combined with a perioperative donor splenocyte infusion lead to reliable donor specific peripheral tolerance in our allogeneic porcine lung transplantation model. To reduce the toxicity of this preconditioning regime, modifications of the IRR protocol and their impact on allograft survival were assessed. METHODS: Left-sided single lung transplantation from major histocompatibility complex and sex mismatched donors was performed in 14 adult female minipigs. Recipient animals were exposed to 3 different protocols of nonmyeloablative IRR within 12 hours before transplantation. All animals were administered a donor splenocyte infusion on the day of lung transplantation. Intravenous pharmacologic immunosuppression was withdrawn after 28 postoperative days. Allograft survival was monitored by chest radiographs and bronchoscopy. RESULTS: IRR prolonged transplant survival in a dose- and field-dependent manner. Shielding of the bone marrow from IRR (total lymphoid IRR at 1.5 and 7 Gy thymic IRR) significantly reduced protocol toxicity defined as thrombocytopenia and consecutive increased bleeding propensity, but had a less effective impact on graft survival. Whole-body IRR at 0.5 and 7 Gy thymic IRR proved to be ineffective for reliable tolerance induction. Eventually, high levels of circulating CD4+CD25high regulatory T cells were present in long-term survivors. CONCLUSIONS: These data show that the infusion of donor-specific alloantigen in combination with IRR is efficient once a threshold dose is exceeded.

Mortality Due to Porcine Reproductive and Respiratory Syndrome Virus in Immunocompromised Göttingen Minipigs (Sus scrofa domestica).

Porcine reproductive and respiratory syndrome virus (PRRSV) infection was diagnosed in 6 Göttingen minipigs (Sus scrofa domestica) with severe interstitial pneumonia. The virus was defined as a North American (NA) subtype virus, which is common in the commercial pig population and might be derived from a widely used attenuated live-virus vaccine in Europe. The ORF5 sequence of the isolated PRRSV was 98% identical to the vaccine virus. The affected pigs were part of a lung transplantation model and received tacrolimus and steroids as well as irradiation or CD8 antibody for immunosuppression. The likely source of the infection was pigs that were shedding the identified PRRSV, which were housed in a separate room of the same building. This case report provides evidence that a virus closely related to an attenuated live vaccine might cause severe pneumonia and death in PRRSVseronegative pigs receiving immunosuppressive treatment. We recommend strict barrier housing for immunocompromised pigs.

Vaccination of piglets at 2 and 3 weeks of age with Ingelvac PRRSFLEX® EU provides protection against heterologous field challenge in the face of homologous maternally derived antibodies.

BACKGROUND: Due to difficulties in eradicating porcine reproductive and respiratory syndrome (PRRS) linked to biosecurity challenges, transmission of the virus and the lack of efficient DIVA vaccines, successful control of PRRS requires a combination of strict management measures and vaccination of both sows and piglets. The present study aimed to assess the efficacy of a recently developed MLV vaccine (Ingelvac PRRSFLEX® EU) in piglets at 2 and 3-weeks of age in the presence of homologous maternally derived antibodies as the dams were vaccinated with the same vaccine strain (ReproCyc® PRRS EU). METHODS: The study was carried out on a Hungarian farrow to finish farm naturally infected with PRRSv. The study was designed as a blind, placebo controlled side by side trial. ORF5 sequence similarity of the vaccine strain and the resident field strain was 87.8 %. PRRS specific real-time quantitative PCR was performed from serum samples to measure both the viral load and the frequency of virus positive animals. RESULTS: At the time of the natural infection observed in the control group at 10-12 weeks of age, the number of viraemic animals did not increase significantly in the vaccinated group. To understand the infection dynamics, positive PCR samples with low Ct values were sequenced (ORF5) and the data analysis indicated the circulation of wild type virus in both groups, however wild type virus was only found in non-vaccinated animals. CONCLUSIONS: Our data indicate that piglets vaccinated at as early as 2 weeks of age with Ingelvac PRRSFLEX® EU were protected both in terms of proportion of viraemic animals and viraemia levels. It has to be highlighted that these results were achieved in piglets with high levels of homologous maternally derived antibodies (MDA) at the time of vaccination.

Allogeneic CD4+CD25high T cells regulate obliterative bronchiolitis of heterotopic bronchus allografts in both porcinized and humanized mouse models.

BACKGROUND: Bronchiolitis obliterans syndrome is caused by a fibroproliferative process in lung allografts resulting in irreversible damage. In this study, we induced obliterative bronchiolitis and studied the contribution of regulatory T cells to its development in immune-deficient mice receiving heterotopic porcine bronchus transplants, and major histocompatibility complex-mismatched porcine peripheral blood mononuclear cell. Furthermore, we aimed to corroborate our findings in a humanized mouse model. METHODS: Heterotopic bronchus transplantation was performed in 33 NOD.rag(−/−)γc(−/−) mice, using miniature pigs as tissue donors.The recipient mice then either received saline (negative control), unsorted MHC-mismatched PBMC (positive control), PBMC enriched with CD4(+)CD25(high) cells or PBMC depleted of CD4(+)CD25(high) cells for reconstitution. The results were validated in 28 NOD.rag(−/−)γc(−/−) mice undergoing heterotopic human bronchus transplantation and reconstitution with allogeneic human PBMC. RESULTS: Histological lesions similar to those typical for obliterative bronchiolitis developed in vivo after reconstitution with allogeneic PBMC and were more severe in animals engrafted with PBMC depleted of CD4(+)CD25(high) cells. In contrast, the group reconstituted with PBMC enriched with CD4(+)CD25(high) cells showed well-preserved histology. The results of the humanized model confirmed those obtained in the porcinized model. CONCLUSIONS: In conclusion, both porcinized and humanized mouse models of heterotopic subcutaneous bronchus transplantation imitate the in vivo development of bronchiolitis obliterans syndrome-like lesions and reveal its sensitivity to T-cell regulation.

Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence.

The ability of the bacterial pathogen Actinobacillus pleuropneumoniae to grow anaerobically allows the bacterium to persist in the lung. The ArcAB two-component system is crucial for metabolic adaptation in response to anaerobic conditions, and we recently showed that an A. pleuropneumoniae arcA mutant had reduced virulence compared to the wild type (F. F. Buettner, A. Maas, and G.-F. Gerlach, Vet. Microbiol. 127:106-115, 2008). In order to understand the attenuated phenotype, we investigated the ArcA regulon of A. pleuropneumoniae by using a combination of transcriptome (microarray) and proteome (two-dimensional difference gel electrophoresis and subsequent mass spectrometry) analyses. We show that ArcA negatively regulates the expression of many genes, including those encoding enzymes which consume intermediates during fumarate synthesis. Simultaneously, the expression of glycerol-3-phosphate dehydrogenase, a component of the respiratory chain serving as a direct reduction equivalent for fumarate reductase, was upregulated. This result, together with the in silico analysis finding that A. pleuropneumoniae has no oxidative branch of the citric acid cycle, led to the hypothesis that fumarate reductase might be crucial for virulence by providing (i) energy via fumarate respiration and (ii) succinate and other essential metabolic intermediates via the reductive branch of the citric acid cycle. To test this hypothesis, an isogenic A. pleuropneumoniae fumarate reductase deletion mutant was constructed and studied by using a pig aerosol infection model. The mutant was shown to be significantly attenuated, thereby strongly supporting a crucial role for fumarate reductase in the pathogenesis of A. pleuropneumoniae infection.