RESUMEN
Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Capripoxvirus , Dermatosis Nodular Contagiosa , Virus de la Dermatosis Nodular Contagiosa , Pruebas de Neutralización , Infecciones por Poxviridae , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Virus de la Dermatosis Nodular Contagiosa/inmunología , Virus de la Dermatosis Nodular Contagiosa/genética , Capripoxvirus/inmunología , Capripoxvirus/genética , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Ovinos , Dermatosis Nodular Contagiosa/prevención & control , Dermatosis Nodular Contagiosa/inmunología , Dermatosis Nodular Contagiosa/virología , Infecciones por Poxviridae/veterinaria , Infecciones por Poxviridae/inmunología , Infecciones por Poxviridae/prevención & control , Infecciones por Poxviridae/virología , Enfermedades de las Ovejas/virología , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/prevención & control , CabrasRESUMEN
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.
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Antihelmínticos , Enfermedades Transmisibles , Enfermedades Gastrointestinales , Nematodos , Infecciones por Nematodos , Enfermedades de las Ovejas , Animales , Ovinos , Inmunidad Mucosa , Rumiantes , Infecciones por Nematodos/prevención & control , Infecciones por Nematodos/veterinaria , Enfermedades Gastrointestinales/tratamiento farmacológico , Cabras , Enfermedades Transmisibles/tratamiento farmacológico , Antihelmínticos/farmacología , Enfermedades de las Ovejas/prevención & controlRESUMEN
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity. Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes. The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-γ and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-γ secretion assessed using ELISA showed a significantly higher titer (**p < 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.
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Monocitos , Vacunas Antirrábicas , Bovinos , Animales , Leucocitos Mononucleares , Células Dendríticas , Antígeno Ki-67/metabolismo , Inmunogenicidad Vacunal , Antígenos/metabolismo , Diferenciación Celular , Células CultivadasRESUMEN
The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1ß and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1ß was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.
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Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Animales , Pollos , Femenino , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/veterinaria , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & control , AerosolesRESUMEN
H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection.
RESUMEN
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.
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Fiebre Porcina Africana , Vacunas Virales , Fiebre Porcina Africana/prevención & control , Virus de la Fiebre Porcina Africana , Animales , Rayos gamma , Inmunogenicidad Vacunal , Porcinos , Vacunación , Vacunas Atenuadas/inmunología , Proteínas Virales , Vacunas Virales/inmunologíaRESUMEN
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.
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Infecciones por Escherichia coli , Enfermedades de las Aves de Corral , Animales , Pollos , Citocinas/metabolismo , Escherichia coli , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Leucocitos Mononucleares/metabolismo , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/metabolismoRESUMEN
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-κß p65 (NF-κß), 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-κß, between IL-18 and IL-15, and between NF-κß and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 ß, 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.