Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection.

The impact of the Fusarium mycotoxin deoxynivalenol (DON) on the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) vaccination and infection was investigated. Forty-two weaned piglets were separated into seven groups and received three different diets: Low DON (1.09 ppm), High DON (2.81 ppm) or No DON. These three treatments were split further into either vaccinated (Ingelvac PRRSFLEX EU) and challenged with PRRSV 28 days post-vaccination, or only infected at day 28. A seventh group received no DON, no vaccination, and no infection. Two weeks after challenge infection, when pigs were euthanized, the number of IFN-γ producing lymphocytes in the blood of vaccinated animals was lower in pigs on High DON compared to animals on Low DON or No DON. Intracellular cytokine staining showed that vaccinated animals fed with the Low DON diet had higher frequencies of TNF-α/IFN-γ co-producing CD4+ T cells than the other two vaccinated groups, particularly in lung tissue. Vaccinated animals on High DON had similar viral loads in the lung as the non-vaccinated groups, but several animals of the Low DON or No DON group receiving vaccination had reduced titers. In these two groups, there was a negative correlation between lung virus titers and vaccine-specific TNF-α/IFN-γ co-producing CD4+ T cells located either in lung tissue or blood. These results indicate that after PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects.

Typhlitis induced by Histomonas meleagridis affects relative but not the absolute Escherichia coli counts and invasion in the gut in turkeys.

Unlike in chickens, dynamics of the gut microbiome in turkeys is limitedly understood and no data were yet published in context of pathological changes following experimental infection. Thus, the impact of Histomonas meleagridis-associated inflammatory changes in the caecal microbiome, especially the Escherichia coli population and their caecal wall invasion in turkeys was investigated. Birds experimentally inoculated with attenuated and/or virulent H. meleagridis and non-inoculated negative controls were divided based on the severity of macroscopic caecal lesions. The high throughput amplicon sequencing of 16SrRNA showed that the species richness and diversity of microbial community significantly decreased in severely affected caeca. The relative abundances of operational taxonomic units belonging to Anaerotignum lactatifermentans, E. coli, and Faecalibacterium prausnitzii were higher and paralleled with a decreased abundances of those belonging to Alistipes putredinis, Streptococcus alactolyticus, Lactobacillus salivarius and Lactobacillus reuteri in birds with the highest lesion scores. Although the relative abundance of E. coli was higher, the absolute count was not affected by the severity of pathological lesions. Immunohistochemistry showed that E. coli was only present in the luminal content of caecum and did not penetrate even severely inflamed and necrotized caecal wall. Overall, it was demonstrated that the fundamental shift in caecal microbiota of turkeys infected with H. meleagridis was attributed to the pathology induced by the parasite, which only led to relative but not absolute changes in E. coli population. Furthermore, E. coli cells did not show tendency to penetrate the caecal tissue even when the intestinal mucosal barriers were severely compromised.

Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken.

The protozoan parasite Histomonas meleagridis is the causative agent of the re-emerging disease histomonosis of chickens and turkeys. Due to the parasite's extracellular occurrence, a type-2 differentiation of H. meleagridis-specific T cells has been hypothesized. In contrast, a recent study suggested that IFN-γ mRNA+ cells are involved in protection against histomonosis. However, the phenotype and cytokine production profile of H. meleagridis-specific T cells still awaits elucidation. In this work, clonal cultures of a virulent monoxenic strain of H. meleagridis were used for infecting chickens to detect IFN-γ protein and IL-13 mRNA by intracellular cytokine staining and PrimeFlow™ RNA Assays, respectively, in CD4+ and CD8ß+ T cells. Infection was confirmed by characteristic pathological changes in the cecum corresponding with H. meleagridis detection by immunohistochemistry and H. meleagridis-specific antibodies in serum. In splenocytes stimulated either with H. meleagridis antigen or PMA/ionomycin, IFN-γ-producing CD4+ T cells from infected chickens increased in comparison to cells from non-infected birds 2 weeks and 5 weeks post-infection. Additionally, an increase of IFN-γ-producing CD4-CD8ß- cells upon H. meleagridis antigen and PMA/ionomycin stimulation was detected. Contrariwise, frequencies of IL-13 mRNA-expressing cells were low even after PMA/ionomycin stimulation and mainly had a CD4-CD8ß- phenotype. No clear increase of IL-13+ cells related to H. meleagridis infection could be found. In summary, these data suggest that H. meleagridis infection induces a type-1 differentiation of CD4+ T cells but also of non-CD4+ cells. This phenotype could include γδ T cells, which will be addressed in future studies.

Comparative investigation of IFN-γ-producing T cells in chickens and turkeys following vaccination and infection with the extracellular parasite Histomonas meleagridis.

The re-emerging disease histomonosis is caused by the protozoan parasite Histomonas meleagridis that affects chickens and turkeys. Previously, protection by vaccination with in vitro attenuated H. meleagridis has been demonstrated and an involvement of T cells, potentially by IFN-γ production, was hypothesized. However, comparative studies between chickens and turkeys on H. meleagridis-specific T cells were not conducted yet. This work investigated IFN-γ production within CD4+, CD8α+ and TCRγδ+ (chicken) or CD3ε+CD4-CD8α- (turkey) T cells of spleen and liver from vaccinated and/or infected birds using clonal cultures of a monoxenic H. meleagridis strain. In infected chickens, re-stimulated splenocytes showed a significant increase of IFN-γ+CD4+ T cells. Contrariwise, significant increments of IFN-γ-producing cells within all major T-cell subsets of the spleen and liver were found for vaccinated/infected turkeys. This indicates that the vaccine in turkeys causes more intense systemic immune responses whereas in chickens protection might be mainly driven by local immunity.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.