African swine fever (ASF) is among the most devastating viral diseases of pigs and wild boar worldwide. In recent years, the disease has spread alarmingly. Despite intensive research activities, a commercialized vaccine is still not available, and efficacious live attenuated vaccine candidates raise safety concerns. From a safety perspective, inactivated preparations would be most favourable. However, both historical and more recent trials with chemical inactivation did not show an appreciable protective effect. Under the assumption that the integrity of viral particles could enhance presentation of antigens, we used gamma irradiation for inactivation. To this means, gamma irradiated ASFV “Estonia 2014” was adjuvanted with either Polygen™ or Montanide™ ISA 201 VG, respectively. Subsequently, five weaner pigs per preparation were immunized twice with a three-week interval. Six weeks after the first immunization, all animals were challenged with the highly virulent ASFV strain “Armenia 2008”. Although ASFV p72-specific IgG antibodies were detectable in all vaccinated animals prior challenge, no protection could be observed. All animals developed an acute lethal course of ASF and had to be euthanized at a moderate humane endpoint within six days. Indeed, the vaccinated pigs showed even higher clinical scores and a higher inner body temperature than the control group. However, significantly lower viral loads were detectable in spleen and liver of immunized animals at the time point of euthanasia. This phenomenon suggests an immune mediated disease enhancement that needs further investigation.

In the recent years, safety concerns regarding the administration of probiotics led to an increased interest in developing inactivated probiotics, also called “paraprobiotics”. Gamma irradiation represents a promising tool that can be used to produce safe paraprobiotics by inhibiting replication while preserving the structure, the metabolic activity, and the immunogenicity of bacteria. In this study, we evaluated the ability of four strains of lactic acid bacteria (LAB: Lacticaseibacillus casei, Lactobacillus acidophilus, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei) in preserving the metabolic activity and the immune modulation of swine porcine peripheral blood mononuclear cells, after gamma irradiation or heat inactivation. Our results show that all four strains retained the metabolic activity following gamma irradiation but not after heat inactivation. In terms of immune-modulatory capacity, irradiated L. acidophilus and Lc. paracasei were able to maintain an overall gene expression pattern similar to their live state, as heat inactivation did with Lc. casei. Moreover, we show that the two inactivation methods applied to the same strain can induce an opposed expression of key genes involved in pro-inflammatory response (e.g., IFNα and interleukin-6 for Lc. casei), whereas gamma irradiation of L. acidophilus and Lc. paracasei was able to induce a downregulation of the anti-inflammatory TGFβ. Taken together, our data show that immune modulation can be impacted not only by different inactivation methods but also by the strain of LAB selected. This study highlights that gamma irradiation harbors the potential to produce safe non-replicative metabolically active LAB and identifies immunomodulatory capacities that may be applied as vaccine adjuvants.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis with different clinical manifestations. The disease is associated with compromised animal welfare and results in substantial economic losses in poultry production worldwide. So far, immunological mechanisms of protection against colibacillosis are not comprehensively resolved. Therefore, the present study aimed to use an ex vivo model applying chicken mononuclear cells stimulated by live and inactivated APEC. For this purpose, an 8-color flow cytometry panel was set up to target viable chicken immune cells including CD45⁺, CD8α⁺, CD4⁺, TCR-γδ⁺, Bu-1⁺ cells and monocytes/macrophages along with the cytokines interferon gamma (IFN-γ) or interleukin 17A (IL-17A). The 8-color flow cytometry panel was applied to investigate the effect of live and two different types of inactivated APEC (formalin-killed APEC and irradiated APEC) on the cellular immune response. For that, mononuclear cells from spleen, lung and blood of 10-week-old specific pathogen-free layer birds were isolated and stimulated with live, irradiated or killed APEC. Intracellular cytokine staining and RT-qPCR assays were applied for the detection of IFN-γ and IL-17A protein level, as well as at mRNA level for spleenocytes. Ex vivo stimulation of isolated splenocytes, lung and peripheral blood mononuclear cells (PBMCs) from chickens with live, irradiated or killed APEC showed an increasing number of IFN-γ and IL-17A producing cells at protein and mRNA level. Phenotyping of the cells from blood and organs revealed that IFN-γ and IL-17A were mainly produced by CD8α⁺, TCR-γδ⁺ T cells as well as CD4⁺ T cells following stimulation with APEC. Expression level of cytokines were very similar following stimulation with live and irradiated APEC but lower when killed APEC were applied. Consequently, in the present study, an ex vivo model using mononuclear cells of chickens was applied to investigate the cellular immune response against APEC. The results suggest the relevance of IFN-γ and IL-17A production in different immune cells following APEC infection in chickens which needs to be further investigated in APEC primed birds.

Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

Gastrointestinal parasitic nematode (GIN) infections are the cause of severe losses to farmers in countries where small ruminants such as sheep and goat are the mainstay of livestock holdings. There is a need to develop effective and easy-to-administer anti-parasite vaccines in areas where anthelmintic resistance is rapidly rising due to the inefficient use of drugs currently available. In this review, we describe the most prevalent and economically significant group of GIN infections that infect small ruminants and the immune responses that occur in the host during infection with an emphasis on mucosal immunity. Furthermore, we outline the different prevention strategies that exist with a focus on whole and purified native parasite antigens as vaccine candidates and their possible oral–nasal administration as a part of an integrated parasite control toolbox in areas where drug resistance is on the rise.