Dr Anna Eleonora Karagianni

A state-of-the-art multi-omics approach was applied to improve our understanding of the aetio-pathogenesis of a highly prevalent, performance-limiting disorder of racehorses: mild-to-moderate equine asthma (MMEA). This is a prerequisite to improving prophylactic, management, and therapeutic options for this condition. Although a number of risk factors have been identified, options for intervention are limited. This study applied a multi-omic approach to reveal key inflammatory pathways involved in inflammatory cell recruitment to the lower airways and highlight distinct MMEA inflammatory profiles. We compared bronchoalveolar lavage fluid (BALF) cell gene and protein expression data from horses with non-inflammatory BALF cytology with those isolated from horses with neutrophilic, mastocytic, mixed neutrophilic/mastocytic, and eosinophilic/mastocytic inflammation. The analyses on transcriptomic/proteomic data derived from BALF from horses with neutrophilic cytology showed enrichment in classical inflammatory pathways, and horses with mastocytic inflammation showed enrichment in pathways involved in hypersensitivity reactions related to nonclassical inflammation potentially mimicking a Th2-immune response. The mixed eosinophilic/mastocytic group also presented with a nonclassical inflammatory profile, whereas the mixed neutrophilic/mastocytic group revealed profiles consistent with both neutrophilic inflammation and hypersensitivity. Our adopted multi-omics approach provided a holistic assessment of the immunological status of the lower airways associated with the different cytological profiles of equine asthma.

Mild-to-moderate equine asthma is prevalent in young racehorses, particularly early in their training period. Although the precise etiopathogenesis remains undetermined, it is possible that the susceptibility of this population might partly reflect an exercise-associated immune derangement at the level of the airway. We performed a genome-wide basal gene expression scan on alveolar macrophages (AMs) isolated from Standardbred racehorses before and after commencement of competition race training with a view to identifying any exercise-associated gene expression modulation consistent with functional alterations, which might reflect training-associated immunological derangement. Microarray technology was used to analyze the basal gene expression profiles of bronchoalveolar fluid-derived AMs, harvested from six systemically healthy Standardbred racehorses before (T0) and after (T1) entry into training. In addition, AM lipopolysaccharide (LPS)-induced TNF-alpha and IL-10 release at T0 and T1 was assessed. Although the data revealed significant interhorse heterogeneity in relation to the magnitude of individual gene expression at each timepoint, within each horse, several inflammatory-related genes [e.g., chemokine ligands, interferons, and nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB)] declined in expression from T0 to T1. Entry into training did not significantly alter AM LPS-induced TNF-alpha or IL-10 release. The data support a direct effect of training on AM basal gene expression, particularly with respect to immune-related genes. The pattern of training-associated differential gene expression may indicate relative downregulation of inflammatory-related genes, consistent with an immunosuppressive effect of training and an increased susceptibility to opportunistic pathogens. (C) 2019 Elsevier Inc. All rights reserved.

Objective—To determine the prevalence of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) in dogs with confirmed or suspected immune-mediated hemolytic anemia (IMHA) or dogs infected with various vector-borne pathogens, including Rickettsia rickettsii, Bartonella henselae, Bartonella vinsonii subsp berkhoffii, Ehrlichia canis, Borrelia burgdorferi, and Leishmania infantum. Animals—55 dogs with confirmed or suspected IMHA, 140 dogs seroreactive for vector-borne pathogens, and 62 healthy dogs and dogs seronegative for vector-borne pathogens. Procedures—Samples were allocated to subgroups on the basis of the health status of the dogs and the degree of seroreactivity against various vector-borne pathogens. Serum samples were tested retrospectively via indirect immunofluorescence assay to determine pANCA status. Results—26 of 55 (47%) dogs with confirmed or suspected IMHA and 67 of 140 (48%) dogs seroreactive for vector-borne pathogens had positive results when tested for pANCA. Serum samples with the highest antibody concentrations against L infantum antigen had the highest proportion (28/43 [65%]) that were positive for pANCA. One of 20 (5%) dogs seronegative for tick-borne pathogens and 8 of 22 (36%) dogs seronegative for L infantum had positive results for pANCA. One of 20 (5%) healthy dogs had serum antibodies against pANCA. Conclusions and Clinical Relevance—pANCA were detected in a high percentage of dogs with IMHA and vector-borne infectious diseases. Therefore, pANCA may be a relatively nonspecific marker for dogs with inflammatory bowel disease, although they could represent a biomarker for immune-mediated diseases and infections.

Neutrophilic airway inflammation is highly prevalent in racehorses in training, with the term mild to moderate equine asthma (MMEA) being applied to the majority of such cases. Our proposed study is largely derived from the strong association between MMEA in racehorses and their entry into a race training program. The objectives of this study are to characterise the effect of training on the local pulmonary immune system by defining the gene and protein expression of tracheal wash (TW) derived samples from Thoroughbred racehorses prior to and following commencement of race training. Multiomics analysis detected 2138 differentially expressed genes and 260 proteins during the training period. Gene and protein sets were enriched for biological processes related to acute phase response, oxidative stress, haemopoietic processes, as well as to immune response and inflammation. This study demonstrated TW samples to represent a rich source of airway cells, protein and RNA to study airway immunity in the horse and highlighted the benefits of a multiomics methodological approach to studying the dynamics of equine airway immunity. Findings likely reflect the known associations between race-training and both airway inflammation and bleeding, offering further insight into the potential mechanisms which underpin training associated airway inflammation.

Abstract Background The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems. Results The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome. Conclusions This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals (n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses.

Airway inflammation is highly prevalent in horses, with the majority of non-infectious cases being defined as equine asthma. Currently, cytological analysis of airway derived samples is the principal method of assessing lower airway inflammation. Samples can be obtained by tracheal wash (TW) or by lavage of the lower respiratory tract (bronchoalveolar lavage (BAL) fluid; BALF). Although BALF cytology carries significant diagnostic advantages over TW cytology for the diagnosis of equine asthma, sample acquisition is invasive, making it prohibitive for routine and sequential screening of airway health. However, recent technological advances in sample collection and processing have made it possible to determine whether a wider range of analyses might be applied to TW samples. Considering that TW samples are relatively simple to collect, minimally invasive and readily available in the horse, it was considered appropriate to investigate whether, equine tracheal secretions represent a rich source of cells and both transcriptomic and proteomic data. Similar approaches have already been applied to a comparable sample set in humans; namely, induced sputum. Sputum represents a readily available source of airway biofluids enriched in proteins, changes in the expression of which may reveal novel mechanisms in the pathogenesis of respiratory diseases, such as asthma and chronic obstructive pulmonary disease. The aim of this study was to establish a robust protocol to isolate macrophages, protein and RNA for molecular characterization of TW samples and demonstrate the applicability of sample handling to rodent and human pediatric bronchoalveolar lavage fluid isolates. TW samples provided a good quality and yield of both RNA and protein for downstream transcriptomic/proteomic analyses. The sample handling methodologies were successfully applicable to BALF for rodent and human research. TW samples represent a rich source of airway cells, and molecular analysis to facilitate and study airway inflammation, based on both transcriptomic and proteomic analysis. This study provides a necessary methodological platform for future transcriptomic and/or proteomic studies on equine lower respiratory tract secretions and BALF samples from humans and mice.

Vector or reservoir species of five mollusc diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Mollusc species on or in which Mikrocytos mackini, Perkinsus marinus, Bonamia exitiosa, Bonamia ostreae and Marteilia refringens were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, this studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected molluscs was not found, these were defined as reservoir. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir mollusc species during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that M. mackini, P. marinus, B. exitiosa B. ostreae and M. refringens will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or at aquaculture establishments or through contaminated water supply can possibly transmit these pathogens. For transmission of M. refringens, the presence of an intermediate host, a copepod, is necessary.

Vector or reservoir species of five fish diseases listed in the Animal Health Law were identified, based on evidence generated through an extensive literature review (ELR), to support a possible updating of Regulation (EU) 2018/1882. Fish species on or in which highly polymorphic region-deleted infectious salmon anaemia virus (HPR increment ISAV), Koi herpes virus (KHV), epizootic haematopoietic necrosis virus (EHNV), infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV) were detected, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as a vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms or reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected fish was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors or reservoir fish species during transport was collected from scientific literature. For VHSV, IHNV or HPR increment ISAV, it was concluded that under transport conditions at temperatures below 25 & DEG;C, it is likely (66-90%) they will remain infective. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild, aquaculture establishments or through water supply can possibly transmit VHSV, IHNV or HPR increment ISAV into a non-affected area when transported at a temperature below 25 & DEG;C. The conclusion was the same for EHN and KHV; however, they are likely to remain infective under all transport temperatures.

Vector or reservoir species of three diseases of crustaceans listed in the Animal Health Law were identified based on evidence generated through an extensive literature review, to support a possible updating of Regulation (EU) 2018/1882. Crustacean species on or in which Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV) were identified, in the field or during experiments, were classified as reservoir species with different levels of certainty depending on the diagnostic tests used. Where experimental evidence indicated transmission of the pathogen from a studied species to another known susceptible species, the studied species was classified as vector species. Although the quantification of the risk of spread of the pathogens by the vectors or reservoir species was not part of the terms of reference, such risks do exist for the vector species, since transmission from infected vector species to susceptible species was proven. Where evidence for transmission from infected crustaceans was not found, these were defined as reservoirs. Nonetheless, the risk of the spread of the pathogens from infected reservoir species cannot be excluded. Evidence identifying conditions that may prevent transmission by vectors during transport was collected from scientific literature. It was concluded that it is very likely to almost certain (90-100%) that WSSV, TSV and YHV will remain infective at any possible transport condition. Therefore, vector or reservoir species that may have been exposed to these pathogens in an affected area in the wild or aquaculture establishments or by water supply can possibly transmit WSSV, TSV and YHV.

Spring Viraemia of Carp (SVC) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9 and Article 8 for listing animal species related to SVC. The assessment was performed following the ad hoc method for data collection and assessment previously developed by the AHAW panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment performed here, it is uncertain whether SVC can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (45–90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that SVC does not meet the criteria in Section 1 (Category A; 5–33% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 33–66%, 10–66%, 45–90% and 45–90% probability of meeting the criteria, respectively). The animal species to be listed for SVC according to Article 8 criteria are provided.

Infection with salmonid alphavirus (SAV) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to infection with SAV. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment, it was uncertain whether infection with salmonid alphavirus can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (50–80% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that infection with salmonid alphavirus does not meet the criteria in Section 1 (Category A; 5–10% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 50–90%, probability of meeting the criteria). The animal species to be listed for infection with SAV according to Article 8 criteria are provided.

Infectious pancreatic necrosis (IPN) was assessed according to the criteria of the Animal Health Law (AHL), in particular, the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9, and Article 8 for listing animal species related to IPN. The assessment was performed following a methodology previously published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment here performed, it is uncertain whether IPN can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (50–90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that IPN does not meet the criteria in Section 1 (Category A; 0–1% probability of meeting the criteria) and it is uncertain whether it meets the criteria in Sections 2, 3, 4 and 5 (Categories B, C, D and E; 33–66%, 33–66%, 50–90% and 50–99% probability of meeting the criteria, respectively). The animal species to be listed for IPN according to Article 8 criteria are provided.

Bacterial kidney disease (BKD) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to BKD. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to this assessment, BKD can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (66–90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that BKD does not meet the criteria in Sections 1, 2 and 3 (Categories A, B and C; 1–5%, 33–66% and 33–66% probability of meeting the criteria, respectively) but meets the criteria in Sections 4 and 5 (Categories D and E; 66–90% and 66–90% probability of meeting the criteria, respectively). The animal species to be listed for BKD according to Article 8 criteria are provided.

On request of the EU Commission, EFSA carried out an Extensive Literature Review (ELR) to provide a list of vector species or reservoirs species of pathogens of crustaceans, listed in Annex II to the AHL, aiming to update the Annex of Implementing Regulation (EU) 2018/1882. In this Technical Report, the detailed review protocol of the ELR and assessment of potential vector and reservoir species is described of the crustacean pathogens listed in Annex II to the AHL: Taura syndrome virus (TSV), Yellow head virus (YHV) or White spot syndrome virus (WSSV). In total 2,530 research publications were collected for abstract screening and from these, 110 were selected for further full text analysis. In the final data collection and assessment 34 relevant research publications were used for extracting information on vector and reservoir species of the above crustacean pathogens. The results for crustacean species for which scientific evidence indicates that a role as vector species or reservoir species is likely are presented as tables in the supplementary material of this report.

The systematic monitoring of health indicators has the potential to complement conventional (confirmation-based) surveillance systems, by extracting value from already existing data. A working group (WG) of experts on syndromic and early warning surveillance was formed by EFSA in May 2020 and tasked to develop and test prototype early-warning animal health surveillance systems covering the wider European region. The first successful pilot of the WG, described in this report, focused on the systematic monitoring of indicators of rabies occurrence. The working assumption of this pilot project is that the number of news articles in media referring to rabies would constitute a good indicator for monitoring sudden changes in occurrence and/or risk of rabies cases in neighbouring EU countries. EFSA, and the European Commission's Joint Research Centre (JRC) worked together on developing a user-friendly interface for the TIM (Tools for Innovation Monitoring) tool, and particularly the TIM NEWS application. An automatic search (across a predefined number of data sources) for rabies-related news items was already implemented in the tool. This was used to develop a “dashboard” with spatial and temporal visualisation of the results. This open dashboard will enable authorities involved in rabies risk assessment and management to monitor the daily distribution of media articles related to rabies published worldwide, which is assumed to contribute to disease and risk situation awareness.

The mononuclear phagocyte system (MPS) is a family of cells of related function that includes bone marrow progenitors, blood monocytes and resident tissue macrophages. Macrophages are effector cells in both innate and acquired immunity. They are a major resident cell population in every organ and their numbers increase in response to proinflammatory stimuli. Their function is highly regulated by a wide range of agonists, including lymphokines, cytokines and products of microorganisms. Macrophage biology has been studied most extensively in mice, yet direct comparisons of rodent and human macrophages have revealed many functional differences. In this review, we provide an overview of the equine MPS, describing the variation in the function and phenotype of macrophages depending on their location and the similarities and differences between the rodent, human and equine immune response. We discuss the use of the horse as a large animal model in which to study macrophage biology and pathological processes shared with humans. Finally, following the recent update to the horse genome, facilitating further comparative analysis of regulated gene expression between the species, we highlight the importance of future transcriptomic macrophage studies in the horse, the findings of which may also be applicable to human as well as veterinary research.

Reasons for performing study: Alveolar macrophages (AMs) are the first line of defence against pathogens in the lungs of all mammalian species and thus may constitute appropriate therapeutic target cells in the treatment and prevention of opportunistic airway infections. Therefore, acquiring a better understanding of equine macrophage biology is of paramount importance in addressing this issue in relation to the horse. Objectives: To compare the transcriptome of equine AMs with that of equine peritoneal macrophages (PMs) and to investigate the effect of lipopolysaccharide (LPS) on equine AM. Study design: Gene expression study of equine AMs. Methods: Cells from both bronchoalveolar and peritoneal lavage fluid were isolated from systemically healthy horses that had been submitted to euthanasia. Cells were cryopreserved. RNA was extracted and comparative microarray analyses were performed in AMs and PMs, and in AMs treated and untreated with LPS. Comparisons with published data derived from human AM studies were made, with particular focus on LPS-induced inflammatory status. Results: The comparison between AMs and PMs revealed the differential basal expression of 451 genes. Gene expression analysis revealed an alternative (M2) macrophage polarisation profile in AMs and a hybrid macrophage activation profile in PMs, a phenomenon potentially attributable to a degree of induced endotoxin tolerance. The gene expression profile of equine AMs following LPS stimulation revealed significant changes in the expression of 240 genes, including well-known upregulated inflammatory genes. This LPS-induced gene expression profile of equine AMs more closely resembles that of human rather than murine macrophages. Conclusions: This study improves current understanding of equine macrophage biology. These data suggest that the horse may represent a suitable animal model for the study of human macrophage-associated lung inflammation and data derived from human macrophage studies may have significant relevance to the horse.

The experimental vaccine for bovine malignant catarrhal fever consists of viable attenuated alcelaphine herpesvirus 1 (AlHV-1) derived by extensive culture passage, combined with an oil-in-water adjuvant, delivered intramuscularly. This immunisation strategy was over 80% effective in previous experimental and field trials and protection appeared to be associated with induction of virus-neutralising antibodies. Whether the vaccine virus is required to be viable at the point of immunisation and whether adjuvant is required to induce the appropriate immune responses remains unclear. To address these issues two studies were performed, firstly to analyse immune responses in the presence and absence of adjuvant and secondly, to investigate immune responses to vaccines containing adjuvant plus viable or inactivated AlHV-1. The first study showed that viable attenuated AlHV-1 in the absence of adjuvant induced virus-specific antibodies but the titres of virus-neutralising antibodies were significantly lower than those induced by vaccine containing viable virus and adjuvant, suggesting adjuvant was required for optimal responses. In contrast, the second study found that the vaccine containing inactivated (>99.9%) AlHV-1 induced similar levels of virus-neutralising antibody to the equivalent formulation containing viable AlHV-1. Together these studies suggest that the MCF vaccine acts as an antigen depot for induction of immune responses, requiring adjuvant and a suitable antigen source, which need not be viable virus. These observations may help in directing the development of alternative MCF vaccine formulations for distribution in the absence of an extensive cold chain.

Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer. ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially expressed in JSRV-infected lung compared to noninfected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and reverse transcription-quantitative PCR. A key finding was the activation of anterior gradient 2, yes-associated protein 1, and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity, including genes encoding cytokines, chemokines, and complement system proteins. In contrast, there was little evidence for the upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with the transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma. IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.

Infection with Gyrodactylus salaris was assessed according to the criteria of the Animal Health Law (AHL), in particular, the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid down in Article 9 and Article 8 for listing animal species related to infection with G. salaris. The assessment was performed following the ad hoc method for data collection and assessment previously developed by AHAW panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to the assessment here performed, it is uncertain whether infection with G. salaris can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (33–70% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that Infection with G. salaris does not meet the criteria in Section 1 and 3 (Category A and C; 1–5% and 10–33% probability of fulfilling the criteria, respectively) and it is uncertain whether it meets the criteria in Sections 2, 4 and 5 (Categories B, D and E; 33–80%, 33–66% and 33–80% probability of meeting the criteria, respectively). The animal species to be listed for infection with G. salaris according to Article 8 criteria are provided.

Introduction: Reliable biomarkers could aid antibiotic decisions in paediatric LRTI. The proteomic interrogation of airway secretions, which may better reflect the local inflammatory environ, could provide them Methods: Protein was extracted from BAL (Bronchoalveolar Lavage) supernatant (from non-bronchoscopic BAL) using a TrisHCL/SDS based method. A mBCA assay measured total protein concentration and extracts ran on total protein gel for quality control. Extracts were pooled due to low sample volume and underwent chemical labelled (iTRAQ & TMT) LC/MS Proteomics. Proteins were identified using UniProt database with analysis in R & IPA Biomarker selection was by (1) ranking eligible proteins by the size of fold change & significance, then (2) promoting proteins that showed similar characteristics in parallel experiments run with archived samples from murine models of LRTI and consistent good diagnostic performance (AUC ≥0.8) identified at systematic review Results: BAL from 10 children with LRTI (6 bacterial & 4 viral determined by expert review panel) and 6 controls was obtained. 1176 proteins identified. 975 proteins in the bacterial & 889 in the viral groups were differentially expressed compared to control. 500 proteins differentiated bacterial and viral LRTI. Bioinformatics identified top networks in both groups related to terms acute phase response, LXR/RXR activation, ER2 signalling & inflammation. The final biomarker panel consisted of: HP, AGT, PIGR & AX2 Conclusions: This novel minimally invasive BAL and proteomic methodology has identified HP, AGT, PIGR & AX2 as potential biomarkers to differentiate viral and bacterial LRTI within the paediatric BAL proteome.