Genetically modified attenuated salmonid alphavirus: A potential strategy for immunization of Atlantic salmon.

Pancreas disease (PD) is a serious challenge in European salmonid aquaculture caused by salmonid alphavirus (SAV). In this study, we report the effect of immunization of Atlantic salmon with three attenuated infectious SAV3 strains with targeted mutations in a glycosylation site of the envelope E2 protein and/or in a nuclear localization signal in the capsid protein. In a pilot experiment, it was shown that the mutated viral strains replicated in fish, transmitted to naïve cohabitants and that the transmission had not altered the sequences. In the main experiment, the fish were immunized with the strains and challenged with SAV3 eight weeks after immunization. Immunization resulted in infection both in injected fish and 2 weeks later in the cohabitant fish, followed by a persistent but declining load of the mutated virus variants in the hearts. The immunized fish developed clinical signs and pathology consistent with PD prior to challenge. However, fish injected with the virus mutated in both E2 and capsid showed little clinical signs and had higher average weight gain than the groups immunized with the single mutated variants. The SAV strain used for challenge was not detected in the immunized fish indicating that these fish were protected against superinfection with SAV during the 12 weeks of the experiment.

The early intestinal immune response in experimental neonatal ovine cryptosporidiosis is characterized by an increased frequency of perforin expressing NCR1(+) NK cells and by NCR1(-) CD8(+) cell recruitment.

Cryptosporidium parvum, a zoonotic protozoan parasite, causes important losses in neonatal ruminants. Innate immunity plays a key role in controlling the acute phase of this infection. The participation of NCR1+ Natural Killer (NK) cells in the early intestinal innate immune response to the parasite was investigated in neonatal lambs inoculated at birth. The observed increase in the lymphocyte infiltration was further studied by immunohistology and flow cytometry with focus on distribution, density, cellular phenotype related to cytotoxic function and activation status. The frequency of NCR1+ cells did not change with infection, while their absolute number slightly increased in the jejunum and the CD8+/NCR1- T cell density increased markedly. The frequency of perforin+ cells increased significantly with infection in the NCR1+ population (in both NCR1+/CD16+ and NCR1+/CD16- populations) but not in the NCR1-/CD8+ population. The proportion of NCR1+ cells co-expressing CD16+ also increased. The fraction of cells expressing IL2 receptor (CD25), higher in the NCR1+/CD8+ population than among the CD8+/NCR1- cells in jejunal Peyer's patches, remained unchanged during infection. However, contrary to CD8+/NCR1- lymphocytes, the intensity of CD25 expressed by NCR1+ lymphocytes increased in infected lambs. Altogether, the data demonstrating that NK cells are highly activated and possess a high cytotoxic potential very early during infection, concomitant with an up-regulation of the interferon gamma gene in the gut segments, support the hypothesis that they are involved in the innate immune response against C. parvum. The early significant recruitment of CD8+/NCR1- T cells in the small intestine suggests that they could rapidly drive the establishment of the acquired immune response.

Characterization of NCR1+ cells residing in lymphoid tissues in the gut of lambs indicates that the majority are NK cells.

Natural killer (NK) cells are important for immune protection of the gut mucosa. Previous studies have shown that under pathologic conditions NK cells, T cells and dendritic cells are found co-localised in secondary lymphoid organs where their interaction coordinates immune responses. However, in the gut-associated lymphoid tissues (GALTs), there are few detailed reports on the distribution of NK cells. Sheep harbour several types of organised lymphoid tissues in the gut that have different functions. The ileal Peyer's patch (IPP) functions as a primary lymphoid tissue for B cell generation, while the jejunal Peyer's patches (JPPs) and colon patches (CPs) are considered secondary lymphoid tissues. In the present study, we analysed tissues from healthy lambs by flow cytometry and in situ multicolour immunofluorescence, using recently described NCR1 antibodies to identify ovine NK cells. Most NCR1+ cells isolated from all tissues were negative for the pan T cell marker CD3, and thus comply with the general definition of NK cells. The majority of NCR1+ cells in blood as well as secondary lymphoid organs expressed CD16, but in the GALT around half of the NCR1+ cells were negative for CD16. A semi-quantitative morphometric study on tissue sections was used to compare the density of NK cells in four compartments of the IPPs, JPP and CPs. NCR1+ cells were found in all gut segments. Statistical analysis revealed significant differences between compartments of the primary lymphoid organ IPP and the secondary lymphoid organs of the JPPs and CP. NK cells co-localised and made close contact with T cells, dendritic cells and other NK cells, but did not show signs of proliferation. We conclude that NK cells are present in all investigated segments of the sheep gut, but that presence of other innate lymphoid cells expressing NCR1 cannot be excluded.

Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORγt+IL-17+ Tregs and improve metabolism.

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration. Intake of the Methylococcu-based diet markedly boosts Parabacteroides abundances in a manner depending on adaptive immunity, and upregulates triple positive (Foxp3+RORγt+IL-17+) regulatory T cells in the small and large intestine. Collectively, these data point to the potential for leveraging the use of McB lysates to improve immunometabolic homeostasis.

Phenotypic characterisation of intestinal dendritic cells in sheep.

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.

Gut microbiota profiling in Norwegian weaner pigs reveals potentially beneficial effects of a high-fiber rapeseed diet.

Rapeseed meal (RSM) is an alternative feed ingredient to soybean meal (SBM) in pig diets. However, knowledge on the effect of RSM on gut health, especially in relation to changes in gut microbiota is still limited. In our study, Norwegian Landrace weaner pigs were fed with either a control diet (CON) based on wheat, barley and SBM, or a high-fiber experimental diet where SBM was replaced by RSM (RSF). We found no large differences in the gut microbiota of pigs fed the two diets, suggesting that RSF does not disturb the gut microbiota and the normal gut function. The relative abundance of SCFA-producing phylotypes and colon-health related phylotypes increased in the large intestine of RSF-fed pigs. Among them, Lachnospira and Coprococcus were negatively associated with the presence of neutrophils in the colon wall. The higher abundance of these bacteria in colon of RSF pigs may suggest an anti-inflammatory stimulus effect of the RSF diet. The gut microbiota of RSF-fed pigs was relatively unaltered following episodes of diarrhea suggesting that the RSF diet may promote robustness in weaner pigs and reduce the risk of dysbiosis.

Transient Migration of Large Numbers of CD14(++) CD16(+) Monocytes to the Draining Lymph Node after Onset of Inflammation.

The dynamics of skin-draining cells following infection or vaccination provide important insight into the initiation of immune responses. In this study, the local recruitment and activation of immune cells in draining lymph nodes (LNs) was studied in calves in an adjuvant-induced inflammation. A transient but remarkably strong recruitment of monocytes was demonstrated after onset of inflammation, constituting up to 41% of live cells in the draining LNs after 24 h. Numerous CD14(+) cells were visualized in subcutaneous tissues and draining LNs, and the majority of these cells did not express dendritic cell-associated markers CD205 and CD11c. In the LNs, recruited cells were predominately of a CD14(++) and CD16(+) phenotype, consistent with an intermediate monocyte subset characterized to possess a high inflammatory potential. Moreover, monocytes from the draining LN showed a high expression of genes coding for pro-inflammatory cytokines, including IL-1ß, IL-6, TNFa, and TGFß. Shortly after their appearance in the LN cortical areas, the monocytes had moved into the medulla followed by an increase in peripheral blood. In conclusion, this study provides novel information on in vivo monocyte recruitment and migration after onset of inflammation.

NCR1+ cells appear early in GALT development of the ovine foetus and acquire a c-kit+ phenotype towards the end of gestation.

The amount, distribution and phenotype of ovine NCR1+ cells were investigated during developing GALT from day 70 of gestation. Antibodies against CD3 and CD79 were used to identify the compartments of GALT, and the localization of NCR1+ cells were correlated within these structures. Markers CD34 and c-kit, in addition to Ki67, were used to investigate possible origin and the stage of development of the NCR1+ cells. NCR1+ cells were present as single cells in the subepithelial tissue as early as 70 days of gestation, and were predominantly present in the T cell rich IFAs and domes as these intestinal wall compartments developed. While NCR1+ cells proliferated more intensively at mid-gestation (70-104 days), the number of NCR1+ cells also expressing c-kit, increased at the end of gestation. In conclusion, NCR1+ cells appeared early in T cell areas of the gut and displayed a phenotype consistent with intermediate stages of cNK cells and/or a subpopulation of ILC22.

Exosome-producing follicle associated epithelium is not involved in uptake of PrPd from the gut of sheep (Ovis aries): an ultrastructural study.

In natural or experimental oral scrapie infection of sheep, disease associated prion protein (PrP(d)) often first accumulates in Peyer's patch (PP) follicles. The route by which infectivity reaches the follicles is unknown, however, intestinal epithelial cells may participate in intestinal antigenic presentation by delivering exosomes as vehicles of luminal antigens. In a previous study using an intestinal loop model, following inoculation of scrapie brain homogenate, inoculum associated PrP(d) was detected by light microscopy shortly (15 minutes to 3.5 hours) after inoculation in the villous lacteals and sub-mucosal lymphatics. No PrP(d) was located within the follicle-associated epithelium (FAE), sub-FAE domes or the PP follicles. To evaluate this gut loop model and the transportation routes in more detail, we used electron microscopy (EM) to study intestinal tissues exposed to scrapie or control homogenates for 15 minutes to 10 days. In addition, immuno-EM was used to investigate whether exosomes produced in the FAE may possess small amounts of PrP(d) that were not detectable by light microscopy. This study showed that the integrity of the intestinal epithelium was sustained in the intestinal loop model. Despite prominent transcytotic activity and exosome release from the FAE of the ileal PP in sheep, these structures were not associated with transportation of PrP(d) across the mucosa. The study did not determine how infectivity reaches the follicles of PPs. The possibility that the infectious agent is transported across the FAE remains a possibility if it occurs in a form that is undetectable by the methods used in this study. Infectivity may also be transported via lymph to the blood and further to all other lymphoid tissues including the PP follicles, but the early presence of PrP(d) in the PP follicles during scrapie infection argues against such a mechanism.