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This update on the African swine fever (ASF) outbreaks in the EU demonstrated that out of all tested wild boar found dead, the proportion of positive samples peaked in winter and summer. For domestic pigs only, a summer peak was evident. Despite the existence of several plausible factors that could result in the observed seasonality, there is no evidence to prove causality. Wild boar density was the most influential risk factor for the occurrence of ASF in wild boar. In the vast majority of introductions in domestic pig holdings, direct contact with infected domestic pigs or wild boar was excluded as the route of introduction. The implementation of emergency measures in the wild boar management zones following a focal ASF introduction was evaluated. As a sole control strategy, intensive hunting around the buffer area might not always be sufficient to eradicate ASF. However, the probability of eradication success is increased after adding quick and safe carcass removal. A wider buffer area leads to a higher success probability; however it implies a larger intensive hunting area and the need for more animals to be hunted. If carcass removal and intensive hunting are effectively implemented, fencing is more useful for delineating zones, rather than adding substantially to control efficacy. However, segments of fencing will be particularly useful in those areas where carcass removal or intensive hunting is difficult to implement. It was not possible to demonstrate an effect of natural barriers on ASF spread. Human‐mediated translocation may override any effect of natural barriers. Recommendations for ASF control in four different epidemiological scenarios are presented., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2018.EN-1521/full

A rapid qualitative assessment has been done by performing a theoretical analysis on the transmission of low pathogenic avian influenza (LPAI) via fresh meat from poultry reared or kept in captivity for the production of meat (raw poultry meat) or raw table eggs. A predetermined transmission pathway followed a number of steps from a commercial or non‐commercial poultry establishment within the EU exposed to LPAI virus (LPAIV) to the onward virus transmission to animals and humans. The combined probability of exposure and subsequent LPAIV infection via raw poultry meat containing LPAIV is negligible for commercial poultry and humans exposed via consumption whereas it is very unlikely for non‐commercial poultry, wild birds and humans exposed via handling and manipulation. The probability of LPAIV transmission from an individual infected via raw poultry meat containing LPAIV is negligible for commercial poultry and humans, whereas it is very unlikely for non‐commercial poultry and wild birds. The combined probability of exposure and subsequent LPAIV infection via raw table eggs containing LPAIV is negligible for commercial poultry and humans and extremely unlikely to negligible for non‐commercial poultry and wild birds. The probability of LPAIV transmission from an individual infected via raw table eggs containing LPAIV is negligible for commercial poultry and humans and very unlikely to negligible for non‐commercial poultry and wild birds. Although the presence of LPAIV in raw poultry meat and table eggs is very unlikely to negligible, there is in general a high level of uncertainty on the estimation of the subsequent probabilities of key steps of the transmission pathways for poultry and wild birds, mainly due to the limited number of studies available, for instance on the viral load required to infect a bird via raw poultry meat or raw table eggs containing LPAIV.

African swine fever virus (ASFV) has been notified in the Baltic countries and the eastern part of Poland from the beginning of 2014 up to now. In collaboration with the ASF‐affected Member States (MS), EFSA is updating the epidemiological analysis of ASF in the European Union which was carried out in 2015. For this purpose, the latest epidemiological and laboratory data were analysed in order to identify the spatial–temporal pattern of the epidemic and a risk factors facilitating its spread. Currently, the ASF outbreaks in wild boar in the Baltic countries and Poland can be defined as a small‐scale epidemic with a slow average spatial spread in wild boar subpopulations (approximately from 1 in Lithuania and Poland to 2 km/month in Estonia and Latvia). The number of positive samples in hunted wild boar peaks in winter which can be explained by human activity patterns (significant hunting activity over winter). The number of positive samples in wild boar found dead peaks in summer. This could be related to the epidemiology of the disease and/or the biology of wild boar; however, this needs further investigation. Virus prevalence in hunted wild boar is very low (0.04–3%), without any apparent trend over time. Apparent virus prevalence at country level in wild boar found dead in affected countries ranges from 60% to 86%, with the exception of Poland, where values between 0.5% and 1.42%, were observed. Since the beginning of the epidemic, the apparent antibody prevalence in hunted wild boar has always been lower than the apparent virus prevalence, indicating an unchanged epidemiological/immunological situation. The risk factor analysis shows an association between the number of settlements, human and domestic pigs population size or wild boar population density and the presence of ASF in wild boar for Estonia, Latvia and Lithuania.

Between 16 February and 15 May 2018, three highly pathogenic avian influenza (HPAI) A(H5N6) and 11 HPAI A(H5N8) outbreaks in poultry holdings, one HPAI A(H5N6) and one HPAI A(H5N8) outbreak in captive birds, and 55 HPAI A(H5N6) wild bird events were reported in Europe. There is no evidence to date that HPAI A(H5N6) viruses circulating in Europe are associated with clades infecting humans. Fewer HPAI wild bird cases have been detected than during the same period of previous year. Most of mortality events among wild birds involved single birds and species listed in the revised list of target species for passive surveillance. Raptor species constitute 74% of the HPAI‐infected wild birds found dead. Those raptor species probably became infected by hunting or scavenging HPAI virus‐positive birds, and so raptor cases may predominate later in the course of an HPAI epidemic. Despite the important HPAI virus incursion via wild birds there have been few associated HPAI A(H5N6) outbreaks in poultry. Fifteen low pathogenic avian influenza (LPAI) outbreaks were reported in three Member States. The risk of zoonotic transmission to the general public in Europe is considered to be very low. The situation in Africa and the Middle East should be closely monitored with regards to HPAI A(H5N1) and A(H5N8). Uncontrolled spread of the virus and subsequent further genetic evolution in regions geographically connected to Europe may increase uncertainty and the risk for further dissemination of virus. Long‐distance migrating wild birds from southern Africa, e.g. the common tern (Sterna hirundo), may be included in targeted active surveillance schemes at a few priority locations in Europe in order to detect HPAI A(H5)‐infected migrating birds early. However, the risk of HPAI introduction from non‐EU countries via migratory wild birds to Europe is still considered to be much lower for wild birds crossing the southern borders than for those crossing the north‐eastern borders.

EFSA assisted four countries in the analysis of epidemiological data on African swine fever (ASF), collected until September 2017. The temporal analysis demonstrated that the average proportions of PCR and antibody‐ELISA positive samples from the hunted wild boar remained below 3.9 and 6.6, respectively. A peak in the ASF incidence was observed 6 months after the first observed case, followed by a significant reduction of the number of cases and low levels of African swine fever virus (ASFV) circulation at the end of 38 months follow‐up period at different spatial resolutions. The spatial analysis concluded that human‐mediated spread of ASFV continues to play a critical role in the ASF epidemiology, despite all measures currently taken. ‘Wild boar density’, ‘total road length’ (as proxy for human activity) and ‘average suitable wild boar habitat availability’ were identified as predictors for the occurrence of ASF in Estonia by a Bayesian hierarchical model, whereas ‘wild boar density’ and ‘density of pig farms’ were predictors according to a generalised additive model. To evaluate the preventive strategies proposed in EFSA's Scientific Opinion (2015) to stop the spread of ASFV in the wild boar population, a simulation model, building on expert knowledge and literature was used. It was concluded that reduction of wild boar population and carcass removal to stop the spread of ASFV in the wild boar population are more effective when applied preventively in the infected area. Drastic depopulation, targeted hunting of female wild boar and carcass removal solely implemented as measures to control ASF in the wild boar population need to be implemented in a highly effective manner (at or beyond the limit of reported effectivity in wild boar management) to sustainably halt the spread of ASF., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1312/full

No human infections due to highly pathogenic avian influenza (HPAI) A(H5N8) or A(H5N6) viruses - detected in wild birds and poultry outbreaks in Europe - have been reported so far and the risk of zoonotic transmission to the general public in Europe is considered very low. Between 16 November 2018 and 15 February 2019, two HPAI A(H5N8) outbreaks in poultry establishments in Bulgaria, two HPAI A(H5N6) outbreaks in wild birds in Denmark and one low pathogenic avian influenza (LPAI) A(H5N3) in captive birds in the Netherlands were reported in the European Union (EU). Genetic characterisation of the HPAI A(H5N6) viruses reveals that they cluster with the A(H5N6) viruses that have been circulating in Europe since December 2017. The wild bird species involved were birds of prey and were likely infected due to hunting or scavenging infected wild waterfowl. However, HPAI virus was not detected in other wild birds during this period. Outside the EU, two HPAI outbreaks were reported in poultry during the reporting period from western Russia. Sequence information on an HPAI A(H5N6) virus found in a common gull in western Russia in October 2018 suggests that the virus clusters within clade 2.3.4.4c and is closely related to viruses that transmitted zoonotically in China. An increasing number of outbreaks in poultry and wild birds in Asia, Africa and the Middle East was observed during the time period for this report. Currently there is no evidence of a new HPAI virus incursion from Asia into Europe. However, passive surveillance systems may not be sensitive enough if the prevalence or case fatality in wild birds is very low. Nevertheless, it is important to encourage and maintain a certain level of passive surveillance in Europe testing single sick or dead wild birds and birds of prey as they may be sensitive sentinel species for the presence of HPAI virus in the environment. A well-targeted active surveillance might complement passive surveillance to collect information on HPAI infectious status of apparently healthy wild bird populations.

The A(H5N8) highly pathogenic avian influenza (HPAI) epidemic occurred in 29 European countries in 2016/2017 and has been the largest ever recorded in the EU in terms of number of poultry outbreaks, geographical extent and number of dead wild birds. Multiple primary incursions temporally related with all major poultry sectors affected but secondary spread was most commonly associated with domestic waterfowl species. A massive effort of all the affected EU Member States (MSs) allowed a descriptive epidemiological overview of the cases in poultry, captive birds and wild birds, providing also information on measures applied at the individual MS level. Data on poultry population structure are required to facilitate data and risk factor analysis, hence to strengthen science‐based advice to risk managers. It is suggested to promote common understanding and application of definitions related to control activities and their reporting across MSs. Despite a large number of human exposures to infected poultry occurred during the ongoing outbreaks, no transmission to humans has been identified. Monitoring the avian influenza (AI) situation in other continents indicated a potential risk of long‐distance spread of HPAI virus (HPAIV) A(H5N6) from Asia to wintering grounds towards Western Europe, similarly to what happened with HPAIV A(H5N8) and HPAIV A(H5N1) in previous years. Furthermore, the HPAI situation in Africa with A(H5N8) and A(H5N1) is rapidly evolving. Strengthening collaborations at National, EU and Global levels would allow close monitoring of the AI situation, ultimately helping to increase preparedness. No human case was reported in the EU due to AIVs subtypes A(H5N1), A(H5N6), A(H7N9) and A(H9N2). Direct transmission of these viruses to humans has only been reported in areas, mainly in Asia and Egypt, with a substantial involvement of wild bird and/or poultry populations. It is suggested to improve the collection and reporting of exposure events of people to AI., This publication is linked to the following EFSA Journal article: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2017.4991/full This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1282/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1283/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1284/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1285/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1286/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1287/full

Low pathogenic avian influenza (LPAI) has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of LPAI to be listed, Article 9 for the categorisation of LPAI according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to LPAI. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective levels. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, LPAI can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL. The disease would comply with the criteria as in Sections 3 and 5 of Annex IV of the AHL, for the application of the disease prevention and control rules referred to in points (c) and (e) of Article 9(1). The animal species to be listed for LPAI according to Article 8(3) criteria are all species of domestic poultry and wild species of mainly Anseriformes and Charadriiformes, as indicated in the present opinion.

The infection with Brucella abortus, Brucella melitensis and Brucella suis has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of the infection with B. abortus, B. melitensis and B. suis to be listed, Article 9 for the categorisation of the infection with B. abortus, B. melitensis and B. suis according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to the infection with B. abortus, B. melitensis and B. suis. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, the infection with B. abortus, B. melitensis and B. suis can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL. The disease complies with the criteria as in Sections 2, 3, 4 and 5 of Annex IV of the AHL, for the application of the disease prevention and control rules referred to in points (b), (c), (d) and (e) of Article 9(1). The animal species to be listed for the infection with B. abortus, B. melitensis and B. suis according to Article 8(3) criteria are several mammal species, as indicated in the present opinion.

A new fungus, Batrachochytrium salamandrivorans (Bsal), was identified in wild populations of salamanders in the Netherlands and Belgium, and in kept salamander populations in Germany and the United Kingdom. EFSA assessed the potential of Bsal to affect the health of wild and kept salamanders in the EU, the effectiveness and feasibility of a movement ban of traded salamanders, the validity, reliability and robustness of available diagnostic methods for Bsal detection, and possible alternative methods and feasible risk mitigation measures to ensure safe international and EU trade of salamanders and their products. Bsal was isolated and characterised in 2013 from a declining fire salamander (Salamandra salamandra) population in the Netherlands. Based on the available evidence, it is likely that Bsal is a sufficient cause for the death of S. salamandra both in the laboratory and in the wild. Despite small sample sizes, the available experimental evidence indicates that Bsal is associated with disease and death in individuals of 12 European and 3 Asian salamander species, and with high mortality rate outbreaks in kept salamanders. Bsal experimental infection was detected in individuals of at least one species pertaining to the families Salamandridae, Plethodontidae, Hynobiidae and Sirenidae. Movement bans constitute key risk mitigation measures to prevent pathogen spread into naïve areas and populations. The effectiveness of a movement ban is mainly dependent on the import volumes, possibility of Bsal to remain viable outside susceptible/tolerant species, and the capacity to limit illegal movements. Duplex real‐time PCR can be used to detect Bsal DNA, but has not been fully validated. Quarantining salamanders, enacting legislation that requires testing of animals to demonstrate freedom from Bsal, before movement can take place, restricting salamander movements, tracking all traded species, hygienic procedures/biosecurity measures before and during movements, and increasing public awareness are relevant measures for ensuring safe intra‐EU and international trade of salamanders.

Highly pathogenic avian influenza (HPAI) H5N8 is currently causing an epizootic in Europe, infecting many poultry holdings as well as captive and wild bird species in more than 10 countries. Given the clear clinical manifestation, passive surveillance is considered the most effective means of detecting infected wild and domestic birds. Testing samples from new species and non‐previously reported areas is key to determine the geographic spread of HPAIV H5N8 2016 in wild birds. Testing limited numbers of dead wild birds in previously reported areas is useful when it is relevant to know whether the virus is still present in the area or not, e.g. before restrictive measures in poultry are to be lifted. To prevent introduction of HPAIV from wild birds into poultry, strict biosecurity implemented and maintained by the poultry farmers is the most important measure. Providing holding‐specific biosecurity guidance is strongly recommended as it is expected to have a high impact on the achieved biosecurity level of the holding. This is preferably done during peace time to increase preparedness for future outbreaks. The location and size of control and in particular monitoring areas for poultry associated with positive wild bird findings are best based on knowledge of the wider habitat and flight distance of the affected wild bird species. It is recommended to increase awareness among poultry farmers in these established areas in order to enhance passive surveillance and to implement enhanced biosecurity measures including poultry confinement. There is no scientific evidence suggesting a different effectiveness of the protection measures on the introduction into poultry holdings and subsequent spread of HPAIV when applied to H5N8, H5N1 or other notifiable HPAI viruses., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2016.EN-1142/full

Between 1 September and 15 November 2017, 48 A(H5N8) highly pathogenic avian influenza (HPAI) outbreaks in poultry holdings and 9 H5 HPAI wild bird events were reported within Europe. A second epidemic HPAI A(H5N8) wave started in Italy on the third week of July and is still ongoing on 15 November 2017. The Italian epidemiological investigations indicated that sharing of vehicles, sharing of personnel and close proximity to infected holdings are the more likely sources of secondary spread in a densely populated poultry area. Despite the ongoing human exposures to infected poultry during the outbreaks, no transmission to humans has been identified in the EU. The report includes an update of the list of wild bird target species for passive surveillance activities that is based on reported AI‐infected wild birds since 2006. The purpose of this list is to provide information on which bird species to focus in order to achieve the most effective testing of dead birds for detection of H5 HPAI viruses. Monitoring the avian influenza situation in other continents revealed the same risks as in the previous report (October 2016‐August 2017): the recent human case of HPAI A(H5N6) in China underlines the continuing threat of this avian influenza virus to human health and possible introduction via migratory wild birds into Europe. Close monitoring is required of the situation in Africa with regards to HPAI of the subtypes A(H5N1) and A(H5N8), given the rapidity of the evolution and the uncertainty on the geographical distribution of these viruses. Interactions between EFSA and member states have taken place to initiate discussions on improving the quality of data collections and to find a step‐wise approach to exchange relevant (denominator) data without causing an additional resource burden.

Escherichia coli (E. coli) O157:H7 can cause haemorrhagic colitis and the haemolytic uremic syndrome in humans. Ruminants are the main reservoir for this bacterium: they can harbour the bacteria in the gastrointestinal tract without showing clinical symptoms. The reason for this persistence is still unclear, although it has been suggested that E. coli O157:H7 can suppress the immune system. To investigate the effects on the immune system of ruminants, an infection model is needed that mimics a long-term infection as it can occur in both sheep and cattle. As the terminal rectum has recently been identified as a primary colonisation site in cattle, we developed a rectal inoculation model for sheep and used this model to study immune responses against selected virulence factors of E. coli O157:H7 (intimin, EspA and EspB). Sheep were infected and re-infected when E. coli O157:H7 excretion was no longer detectable. The animals did not develop serum or local antibody responses but showed a cellular response against EspA and intimin respectively 9 and 16 days after infection. This response was also present 5 days after re-infection, albeit lower, and did not prevent animals from being re-infected. These results demonstrate that E. coli O157:H7 can be persistently present in the large intestine of sheep without inducing a clear protective immune response.

Infections with F4 + enterotoxigenic Escherichia coli (ETEC) causes severe diarrhoea in piglets, resulting in morbidity and mortality. F4 fimbriae are the key virulence factors mediating the attachment of F4 + ETEC to the intestinal epithelium. Intestinal epithelial cells (IEC) are recently being recognized as important regulators of the intestinal immune system through the secretion of cytokines, however, data on how F4 + ETEC affect this cytokine secretion are scarce. By using ETEC strains expressing either polymeric, monomeric or F4 fimbriae with a reduced polymeric stability, we demonstrated that polymeric fimbriae are essential for adhesion to porcine IEC and the secretion of IL-6 and IL-8 by IEC. Remarkably, this cytokine secretion was not abrogated following stimulation with an F4-negative strain. Since this strain expresses flagellin, TLR5 mediated signalling could be involved. Indeed, porcine IEC express TLR5 and purified flagellin induced IL-6 and IL-8 secretion, indicating that, as for other pathogens, flagellin is the dominant virulence factor involved in the induction of proinflammatory responses in IEC. These results indicate a potential mucosal adjuvant capacity of ETEC-derived flagellin and may improve rational vaccine design against F4 + ETEC infections.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 strains are associated with haemor raghic colitis and haemolytic uremic syndrome (HUS) in humans. Cattle are a reservoir of E. coli O157:H7. We studied the ability of bovine and human lactoferrin, two natural antimicrobial proteins present in milk, to inhibit E. coli O157:H7 growth and attachment to a human epithelial colorectal adenocarcinoma cell line (Caco-2). The direct antibacterial effect of bLF on E. coli O157:H7 was stronger than that of hLF. Nevertheless, both lactofer - rins had bacteriostatic effects even at high concentrations (10 mg/ml), suggesting blocking of LF activity by a yet undefined bacterial defence mechanism. Additionally, both lactoferrins significantly inhibited E. coli O157:H7 attachment to Caco-2 cells. However, hLF was more effective than bLF, probably due to more efficient binding of bLF to intelectin present on human enterocytes leading to uptake and thus removal of bLF from the extracellular environment. Inhibition of bacterial attachment to Caco-2 cells was at least partly due to the catalytic effect of lactoferrins on the type III secreted proteins EspA and EspB

Avian influenza viruses infect domestic poultry and wild birds as well as humans. In poultry, depending on whether these viruses are of high pathogenicity (HPAI) or low pathogenicity (LPAI), the infection can cause different clinical signs, with HPAI causing high mortality in poultry flocks. In order to ensure early detection of avian influenza viruses, surveillance in poultry and wild birds is considered essential. In 2010, the European Commission provided some guidelines to Member States (MSs) on how this surveillance should be carried out, both in poultry and wild birds. EFSA received a mandate from the Commission to collate, validate, analyse, and summarise in an annual report the data resulting from the ongoing avian influenza surveillance programmes established in the different MSs. To deliver on this mandate, EFSA, in collaboration with the Standing Working Group on AI, initiated its activities with the drafting of a scientific report where the future vision of this collection framework was presented. Initial and later drafts of this report were shared with MS representatives in order to get feedback on the practicalities concerning the collection and submission of avian influenza surveillance data to EFSA. In the present report, the data that MSs are legally requested to submit to EFSA (‘mandatory’) and also the data that would be important to collect in order to optimise the outputs (‘desirable’) are described. A number of actions that would lead to the optimal data collection are also presented and the added value to MSs is discussed. A step‐by‐step implementation of the outlined actions is anticipated, with a description of the initial collection framework for 2019 being included in this report.

Very few antigens have been described that induce an intestinal immunity when given orally. Our laboratory demonstrated that oral administration of isolated F4 (K88) fimbriae of Escherichia coli to F4-receptor positive (F4R + ) pigs induces protective mucosal immunity against challenge infection. However, presence of F4-receptors (F4R) on villous enterocytes is a prerequisite for inducing the immune response, as no F4-specific antibody-secreting cells (ASC) can be induced in F4R − pigs. In this study, the in vivo binding of isolated F4 fimbriae (F4) to the gut epithelium was examined in F4R + and F4R − pigs. It was further investigated whether binding of F4 to the F4R results in endocytosis in and translocation across the gut epithelium using microscopy. F4 did not adhere to the intestinal epithelium of F4R − pigs, whereas it strongly adhered to the villous epithelium and the follicle-associated epithelium (FAE) of the jejunum and ileum of F4R + pigs. Following binding to F4R, F4 was endocytosed by villous enterocytes, follicle-associated enterocytes and M cells. Transcytosis of F4 across the epithelium resulted in the appearance of F4 in the lamina propria and dome region of the jejunal and ileal PP. This is the first study showing transcytosis of fimbriae across the gut epithelium. This receptor-dependent transcytosis can explain the success of F4 fimbriae as oral immunogen for inducing protective immunity in F4R + pigs strengthening the importance of receptor-dependent endocytosis and translocation in oral vaccine strategies. Further identification of the receptor responsible for this transport is in progress.

Currently, no vaccines are available for edema disease and post-weaning diarrhoea (PWD) in pigs. In the present study, a subunit vaccine containing the F18 fimbrial adhesin FedF was studied. Hereto, recombinant FedF was produced as a fusion protein with maltose-binding protein. Even though the produced MBPFedF was shown to attach in vitro to enterocytes, almost no FedF-specific immune response could be detected after oral administration to piglets. The delivery of FedF to the intestinal mucosa was improved by conjugating the MBPFedF to F4 fimbriae. Indeed, this conjugation induced a systemic and local FedF-specific immune response and led to a reduction in excretion after infection with F18+ E. coli. Although complete protection was not observed, the conjugation between FedF and F4 fimbriae can be considered as a first step towards the development of a combined vaccine against F4+ and F18+ E. coli infections.

F18(+)Escherichia coli have the ability to colonize the gut and cause oedema disease or post-weaning diarrhoea by adhering to specific F18 receptors (F18R) on the porcine epithelium. Although it is well established that a DNA polymorphism on base pair 307 of the FUT1 gene, encoding an alpha(1,2)fucosyltransferase, accounts for the F18R phenotype, the F18R nature is not elucidated yet. The aim of the present study was to investigate the correlation between the presence of H-2 histo-blood group antigens (HBGAs) or its derivative A-2 HBGAs on the porcine gut epithelium and F18(+)E. coli adherence. A significant positive correlation was found between expression of both the H-2 (r=0.586, P

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/ faeG 19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/ faeG 19. Subsequently, pcDNA1/ faeG 19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4 + E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

F18 + Escherichia coli can cause post-weaning diarrhoea and oedema disease in pigs. These diseases are responsible for substantial economic losses, but a vaccine is not available. A good knowledge of the characteristic of the fimbriae is useful for the development of a vaccine composed of the fimbrial virulence factor. F18 fimbriae are composed of the major subunit FedA and the minor subunits FedE and the adhesin FedF. In the present study monoclonal antibodies (mAbs) against FedA and FedF were produced. In addition to their diagnostic value, these mAbs revealed a weaker interaction between FedA and FedF compared to the subunit–subunit interactions in other fimbriae, like type 1 and P pili. Further experiments are needed to investigate if this weak interaction could be one of the reasons for the slow colonisation of the small intestinal mucosa by F18 + E. coli .

A recently developed oral immunisation model in pigs in which F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a protective intestinal immunity, was used to determine the optimal inductive sites of the F4-specific intestinal immune response. Hereto, pigs were immunised with F4 orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small intestine, the highest number of ASC was found following jejunal immunisation, followed by ileal, oral and colonic immunisation. To determine the signifance of Peyer's patches in the induced immune response, F4 was injected into the jejunal Peyer's patches (JPP), lamina propria (LP) and ileal Peyer's patches (IPP). Immunisation in the JPP induced the highest number ASC in the small intestine, whereas immunisation in the LP and IPP resulted in lower intestinal antibody responses. In conclusion, we have shown that the JPP are the major inductive sites of the F4-specific intestinal antibody response. This knowledge could be important when using the pig as an animal model for vaccination studies.

F18+Escherichia coli cause postweaning diarrhoea and oedema disease in newly weaned piglets. Protection against these diseases can be established by preventing the fimbrial adhesion of these bacteria to the enterocytes of the porcine intestine. To test a vaccine against F18+E. coli consisting of the adhesin of F18 fimbriae, FedF, the conservation of the FedF subunit had to be examined. Therefore, the fedF sequence of 37 F18+E. coli isolates from different countries was determined and compared to the fedF gene of the F18ab reference strain F107/86. The amino acid sequence of the mature FedF from the individual F18+E. coli isolates was 96–100% identical to that from E. coli F107/86, but the overall homology was 90.4%. Hyper variable regions were not found in the FedF sequence. The FedF sequence was conserved over the different countries and between the two antigenic variants, F18ab and F18ac, suggesting that F18ab and F18ac strains have the same receptor. Furthermore, the conserved C-terminal region in the FedF adhesin suggests that the F18 fimbriae, in analogy with type 1 and P pili, are assembled by a donor strand mechanism. In conclusion, the reported conservation of FedF supports the usefulness of the fimbrial adhesin as a subunit vaccine against F18+E. coli infection.

Piglets are susceptible to F4 (K88)+ enterotoxigenic Escherichia coli (ETEC)-induced neonatal and post-weaning diarrhoea. The F4 fimbriae are composed of some minor subunits and the major subunit FaeG that also constitutes the adhesin. Parenteral vaccination of sows with an F4-containing vaccine protects the suckling piglets against neonatal F4+ ETEC-induced diarrhoea, but no commercial (mucosal) vaccine exists against F4+ ETEC-induced weaning diarrhoea. To develop a vaccine, a bioactive F4-receptor (F4R) binding FaeG molecule is required that binds to the F4R following oral immunization and induces a FaeG-specific immune response. The present study reports the altered binding of the FaeG-specific monoclonal antibody IMM01 with bioactive versus non-bioactive F4 fimbrial adhesin FaeG. The correlation of altered IMM01 binding with altered FaeG bioactivity permits the use of an IMM01-based ELISA as a fast, specific and sensitive in vitro selection for potent F4 or (recombinant) FaeG antigen formulations, useful in an F4+ ETEC vaccine.

Oral immunization of newly weaned piglets with recombinant F4 (K88) fimbrial adhesin FaeG induces a F4-specific immune response, significantly reducing F4 + Escherichia coli excretion following challenge. In order to use FaeG subunits in an oral vaccine against F4 + enterotoxigenic E. coli , it is necessary to determine the conservation of the adhesin subunit. Hereto, the faeG sequence was determined of 21 F4ac + E. coli field isolates from piglets with diarrhoea and subsequently compared with these of the reference strain GIS26 and previously reported FaeG sequences from F4ab, F4ac and F4ad antigenic variant strains. The FaeG amino acid sequence was 96–100% homologous within each F4 serotype, but only 92 and 88% when the F4ab and F4ad antigenic variants were compared with the F4ac antigenic variant. Furthermore, the conserved regions of the adhesin suggest a donor strand mechanism in F4 fimbriae assembly as reported for type 1 and P pili. In conclusion, the results of the reported experiments support the usefulness FaeG in an oral subunit vaccine against F4 + E. coli infections or as a mucosal carrier since the adhesin is conserved among F4 + E. coli field isolates.

Fimbriae often play a prominent role in anchoring bacterial cells to host tissue and mediate the first step in pathogenesis. As a consequence, there is a continuous development of new strategies to block the binding of fimbriae to their specific receptor on host cells. The present study demonstrates the specific interaction of F4 (K88) fimbriae and porcine enterocytes using a real-time biomolecular interaction analysis system (BIAcore® 3000), based on the principles of surface plasmon resonance (SPR). This method offers new opportunities to screen therapeutics for prevention of adhesion and subsequent disease without receptor purification.

Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.

An overwhelming number of infectious diseases in both humans and animals are initiated by bacterial adhesion to carbohydrate structures on a mucosal surface. Most bacterial pathogens mediate this adhesion by fimbriae or pili which contain an adhesive lectin subunit. The importance of fimbriae as virulence factors led to research elucidating the regulation of fimbrial expression and their molecular assembly process. This review provides an overview of the current knowledge of induction, expression and assembly of F4 (K88) fimbriae and discusses its unique as well as its identical characteristics compared to other intensively studied fimbriae or pili expressed by Escherichia coli.

Only a few vaccines are commercially available against intestinal infections since the induction of a protective intestinal immune response is difficult to achieve. For instance, oral administration of most proteins results in oral tolerance instead of an antigen-specific immune response. We have shown before that as a result of oral immunization of piglets with F4 fimbriae purified from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune response. F4 fimbriae are very stable polymeric structures composed of some minor subunits and a major subunit FaeG that is also the fimbrial adhesin. In the present study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201 (I to V) as determinants for F4 polymeric stability. The interaction between the FaeG subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae behaved identically in F4R binding and showed equal stability in the gastro-intestinal lumen. Oral immunization experiments indicated that a higher degree of polymerisation of the fimbriae in the intestine was correlated with a better F4-specific mucosal immunogenicity. These data suggest that the mucosal immunogenicity of soluble virulence factors can be increased by the construction of stable polymeric structures and therefore help in the development of effective mucosal vaccines.

F18(+)Escherichia coli infections causing post-weaning diarrhoea and/or oedema disease are a major cause of economic losses in pig industry. To date, no preventive strategy can protect pigs from F18(+)E. coli infections. One of the most attractive approaches to eliminate F18(+)E. coli infections is the selection for pigs that are resistant to F18(+)E. coli infections. However, this strategy was not believed to be favourable because of reports of genetic association with the stress-susceptibility gene in the Swiss Landrace. To investigate this potential association more thoroughly, 131 randomly selected Belgian hybrid pigs were genotyped for both the F18(+)E. coli resistance alleles (FUT1(A)) and the stress-susceptibility alleles (RYR1(T)) and their association was investigated by determining the linkage disequilibrium. This linkage disequilibrium (LD=-0.0149) is close to zero and does not differ significantly from 0 (likelihood ratio test chi(1)(2)=1.123, P=0.29), demonstrating no association between the FUT1(A) and RYR1(T) alleles. Furthermore, only a small fraction (4.6%) of the Belgian pigs was found to be resistant to F18(+)E. coli infections. Our results suggest that selection for F18(+)E. coli resistant pigs might be an attractive approach to prevent pigs from F18(+)E. coli infections, unlike to what has previously been postulated.

Post-weaning diarrhoea and oedema disease in weaned piglets are caused by infection with F4+ or F18+ Escherichia coli strains. There is no commercial vaccine available, but it is shown that oral immunization of weaned piglets with purified F4 fimbriae induces a protective mucosal immune response. In the present study, piglets were orally and nasally immunized with purified F18 fimbriae in the presence of the mucosal adjuvant LT(R192G) or CTA1-DD, respectively. This immunization could not lead to protection against F18+ E. coli infection. The induced F18-specific immune response was directed towards the major subunit FedA and weakly towards the adhesive subunit FedF. The results of these experiments demonstrate that it is difficult to induce protective immunity against F18+ E. coli using the whole fimbriae due to the low response against the adhesin.

The present study analysed quantitatively the mucin 4 polymorphism for determing the F4ac/ab receptor status of a total of 63 pigs by comparing it with the invitro villous adhesion assay. The probability of a susceptible genotype for the mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250 mm villi (P = 0.029 for F4ab, P = 0.030 for F4ac), with the odds ratio for each unit increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004–1.069]) and 1.018 (95% CI [1.002– 1.034]). In the phenotypic in vitro villous adhesion test, a cut-off value of 5 bacteria was chosen as a criteria for the distinction between an F4R positive and F4R negative pig. The sensitivity and specificity for the in vitro villous adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and 24%, respectively, for F4ab as well as F4ac. Absence of adhesion of F4ac and F4ab ETEC to the villous brush borders was not associated with genotypic resistance suggesting that there is at least one other receptor for F4ab/ac Escherichia coli. As a consequence, not only mucin 4 gene polymorphism but also expression of these other receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pigs. # 2007 Published by Elsevier B.V.

In developing veterinary mucosal vaccines and vaccination strategies, mucosal adjuvants are one of the key players for inducing protective immune responses. Most of the mucosal adjuvants seem to exert their effect via binding to a receptor/or target cells and these properties were used to classify the mucosal adjuvants reviewed in the present paper: (1) ganglioside receptor-binding toxins ( cholera toxin, LT enterotoxin, their B subunits and mutants); ( 2) surface immunoglobulin binding complex CTA1-DD; (3) TLR4 binding lipopolysaccharide; (4) TLR2-binding muramyl dipeptide; (5) Mannose receptor-binding mannan; (6) Dectin-1-binding beta 1,3/1,6 glucans; (7) TLR9-binding CpG-oligodeoxynucleotides; (8) Cytokines and chemokines; (9) Antigen-presenting cell targeting ISCOMATRIX and ISCOM. In addition, attention is given to two adjuvants able to prime the mucosal immune system following a systemic immunization, namely 1 alpha, 25(OH)(2)D-3 and cholera toxin. ispartof: Veterinary research vol:37 issue:3 pages:511-539 ispartof: location:England status: published

Transgenic plants are attractive bioreactors to large-scale production of recombinant proteins because of their relatively low cost. This study reports for the first time the use of transgenic plants to reduce enterotoxigenic Escherichia coli (ETEC) excretion in its natural host species. The DNA sequence encoding the major subunit and adhesin FaeG of F4+ ETEC was transformed into edible alfalfa plants. Targeting of FaeG production to chloroplasts led to FaeG levels of up to 1% of the total soluble protein fraction of the transgenic alfalfa. Recombinant plant-produced FaeG (pFaeG) remained stable for 2 years when the plant material was dried and stored at room temperature. Intragastric immunization of piglets with pFaeG induced a weak F4-specific humoral response. Co-administration of pFaeG and the mucosal adjuvant cholera toxin (CT) enhanced the immune response against FaeG, reflected a better induction of an F4-specific immune response. In addition, the intragastric co-administration of CT with pFaeG significantly reduced F4+ E. coli excretion following F4+ ETEC challenge as compared with pigs that had received nontransgenic plant material. In conclusion, transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against F4+ ETEC infections.

Receptor-mediated uptake of orally administered antigen can lead to an antigen-specific immune response, whereas oral administration of most other non-replicating soluble antigens results in the induction of oral tolerance. In the present study, it is shown that fimbriae purified from an F4(K88)(+) enterotoxigenic Escherichia coli strain can function as a mucosal carrier molecule for the model antigen human serum albumin (HSA). Glutaraldehyde-coupled F4/HSA conjugates were able to bind F4 receptor positive (F4R(+)) enterocytes, but not to F4R(-) enterocytes. Moreover, oral immunization of F4R(+) pigs with F4/HSA conjugates induced a HSA-specific immune response, whereas oral immunization with HSA/HSA conjugates did not. This mucosal carrier function of F4 fimbriae was improved following oral co-administration of the F4/HSA conjugates with the mucosal adjuvant cholera toxin (CT) to F4R(+) pigs, since both humoral and cellular HSA-specific responses were significantly increased. In comparison with F4R(+) pigs, the HSA-specific response was reduced following oral F4/HSA+CT immunization of F4R(-) pigs. This indicates that F4 fimbriae as mucosal carrier and CT as adjuvant synergistically improve the induction of a HSA-specific immune response following oral immunization of pigs. These results could open new perspectives in the development of vaccines against enteropathogens.

Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4 + E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4 + E. coli excretion following F4 + ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism.

Newly weaned piglets can be orally immunised against F4+ enterotoxigenic Escherichia coli (ETEC) infection with F4 fimbriae. However, to efficiently develop a vaccine against ETEC induced postweaning diarrhoea, knowledge of the stability of the F4 fimbriae to different pH and gastric digestion is needed. The gastrointestinal pH in suckling and recently weaned piglets was measured and the stability of F4 fimbriae to different pH and to pepsin was assessed in vitro. In the stomach the lowest pH was found in the fundus gland region. Gastric pH values below 2.5 were not found in suckling piglets or at the day of weaning, in contrast to piglets 1 and 2 weeks postweaning. Along the first half of the small intestine and in the caecum, a negative correlation was found between pH and age. The F4 fimbriae were stable to pH 1.5 and 2 for 2 h, whereas longer incubation periods resulted in conversion of the multimeric forms into monomers. The F4 fimbriae were partially degraded by incubation for 15-30 min in simulated gastric fluid at pH 1.5 and 2, and completely digested from 3 h onwards. At pH 3, the fimbriae maintained their antigenicity for at least 4h. The results demonstrate that gastric digestion will only have a limited impact on oral immunisation since liquid passes through the stomach relatively quickly (50% within 2 h). However, we previously demonstrated that the transit times are prolonged shortly after weaning. Shortly after weaning it could be necessary to protect the F4 fimbriae against gastric digestion to obtain efficient oral immunisation of the piglets.

Enterotoxigenic and verotoxigenic F18+ Escherichia coli colonising the pig small intestine, adhere to receptors on intestinal villous enterocytes by F18 fimbriae. The aim of the present study was to define the F18R nature. The knowledge on the nature of this receptor could be important for the development of receptor-based treatments against F18+ E. coli-induced disease. The adhesion of F18+ E. coli to pig intestinal villous enterocytes was analysed in an in vitro assay. The adhesion of F18+ E. coli but not of F4ac+ E. coli was strongly inhibited by monoclonal antibodies (mAb) with blood group H-2 specificity. Conversely, blood group H-1 specific mAb could not inhibit the adhesion of F18+ E. coli nor F4ac+ E. coli. Moreover, the blood group H-2 trisaccharide strongly inhibited the adhesion of F18+ E. coli, but only partially the adhesion of F4ac+ E. coli. These data demonstrate that the F18 receptor contains the blood group antigen H-2 (alpha-fuc-(1-2)-beta-Gal-(1-4)-GlcNAc) as major carbohydrate.

In this study it was analysed whether intramuscular (IM) immunisation of piglets with F4 during the suckling period could protect against oral challenge with F4 + - Escherichia coli and whether addition of 1α,25(OH) 2 D 3 or CpG-ODN could improve this protection. F4-seronegative F4-receptor positive pigs were divided into four groups of five pigs each. The pigs were intramuscularly injected with F4 fimbriae only or supplemented with 1α,25(OH) 2 D 3 (D 3 -group) or CpG-ODN (CpG-group). The control group received PBS in IFA. Seven days after the second immunisation, all pigs were intragastrically inoculated with 1×10 10 CFU of F4 + - E. coli . All F4-injected groups, showed a reduced faecal excretion of F4 + - E. coli . However, this reduction was only statistically significant in the D 3 -group 2 days post challenge. Pigs in the latter group showed a secondary antibody response upon challenge, indicating that F4-primed memory B-cells were present in the gut-associated lymphoid tissues at that moment. CpG-ODN, on the other hand, did not enhance the F4-specific antibody response. However, CpG-ODN significantly increased the F4-specific as well as mitogen-induced proliferation of pheripheral blood monomorphonuclear cells indicating a direct or indirect overall effect on T-lymphocytes. In conclusion, supplementation with 1α,25(OH) 2 D 3 or CpG-ODN improved protection against an F4 + - E. coli infection. This protection was most obvious for 1α,25(OH) 2 D 3 and indicates its potential use in veterinary vaccines against enteropathogens.

To develop a vaccine against Escherichia coli -induced post-weaning diarrhea and edema disease, insights in the induction of the protective immune response following infection with these pathogenic E. coli is needed. Therefore, the fimbriae-specific antibody response of newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-IIv) producing F18 + verotoxigenic E. coli (VTEC) (strain 107/86) was compared with the response following an infection with LT producing F4 + enterotoxigenic E. coli (ETEC) (strain GIS 26). F4 + ETEC were able to colonize the gut very soon after infection, as peak excretion of F4 + E. coli bacteria was seen 2 days post-infection (dpi), but had already disappeared 7 dpi. On the other hand, F18 + VTEC infection resulted in a slower colonization of the gut as the peak excretion of F18 + E. coli was observed between 3 and 5 dpi, but this colonization remained longer as F18 + E. coli were detected till 9 dpi in feces. Furthermore, this fast colonization pattern of F4 + ETEC is accompanied with the presence of F4-specific antibodies in mucosal tissues and serum from 4 dpi onward, with maximal amounts of F4-specific IgA in the jejunal lamina propria and serum 7 dpi. In contrast, F18-specific IgA was only readily detected in the jejunal lamina propria 15 dpi and showed a maximum serum titer 21 dpi. Besides this faster induction and higher antibody response, the switch from IgM to IgA and IgG was also earlier following the F4 + ETEC infection.

The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively. Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens. A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)). Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection. In F4R(-) pigs such an intestinal mucosal immune response does not occur. However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen. The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.

Between 16 November 2017 and 15 February 2018, one highly pathogenic avian influenza (HPAI) A(H5N6) and five HPAI A(H5N8) outbreaks in poultry holdings, two HPAI A(H5N6) outbreaks in captive birds and 22 HPAI A(H5N6) wild bird events were reported within Europe. There is a lower incursion of HPAI A(H5N6) in poultry compared to HPAI A(H5N8). There is no evidence to date that HPAI A(H5N6) viruses circulating in Europe are associated with clades infecting humans. Clinical signs in ducks infected with HPAI A(H5N8) seemed to be decreasing, based on reports from Bulgaria. However, HPAI A(H5N8) is still present in Europe and is widespread in neighbouring areas. The majority of mortality events of wild birds from HPAIV A(H5) in this three-month period involved single birds. This indicates that the investigation of events involving single dead birds of target species is important for comprehensive passive surveillance for HPAI A(H5). Moreover, 20 low pathogenic avian influenza (LPAI) outbreaks were reported in three Member States. The risk of zoonotic transmission to the general public in Europe is considered to be very low. The first human case due to avian influenza A(H7N4) was notified in China underlining the threat that newly emerging avian influenza viruses pose for transmission to humans. Close monitoring is required of the situation in Africa and the Middle East with regards to HPAI A(H5N1) and A(H5N8). Uncontrolled spread of virus and subsequent further genetic evolution in regions geographically connected to Europe may increase uncertainty and risk for further dissemination of virus. The risk of HPAI introduction from Third countries via migratory wild birds to Europe is still considered much lower for wild birds crossing the southern borders compared to birds crossing the north-eastern borders, whereas the introduction via trade is still very to extremely unlikely.