Assessing pig farm biosecurity measures for the control of Salmonella on European farms.

Salmonella spp. is a common zoonotic pathogen, causing gastrointestinal infections in people. Pigs and pig meat are a major source of infection. Although farm biosecurity is believed to be important for controlling Salmonella transmission, robust evidence is lacking on which measures are most effective. This study enrolled 250 pig farms across nine European countries. From each farm, 20 pooled faecal samples (or similar information) were collected and analysed for Salmonella presence. Based on the proportion of positive results, farms were categorised as at higher or lower Salmonella risk, and associations with variables from a comprehensive questionnaire investigated. Multivariable analysis indicated that farms were less likely to be in the higher-risk category if they had '<400 sows'; used rodent baits close to pig enclosures; isolated stay-behind (sick) pigs; did not answer that the hygiene lock/ anteroom was easy to clean; did not have a full perimeter fence; did apply downtime of at least 3 days between farrowing batches; and had fully slatted flooring in all fattener buildings. A principal components analysis assessed the sources of variation between farms, and correlation between variables. The study results suggest simple control measures that could be prioritised on European pig farms to control Salmonella.

Wild boar (Sus scrofa) carcasses as an attraction for scavengers and a potential source for soil contamination with the African swine fever virus.

The wild boar (Sus scrofa) is a social animal species native to Eurasia. During the last decade, the wild boar population in Estonia has been severely affected by the African swine fever virus (ASFV), which has also affected domestic pig farming. The potential transmission routes of ASFV remain unclear and are currently under intensive investigation. This pilot study aimed to clarify the frequency and characteristics of contacts between living wild boars and the carcasses of their conspecifics, which could play a role in the transmission of ASFV. Wild animals' contact and scavenging behavior on wild boar carcasses were studied using trail cameras in an experimental setting on Hiiumaa, Western Estonia. Four legally hunted carcasses were used in the present study. This study aimed to determine whether intraspecies scavenging occurs in wild boars. The persistence of ASFV DNA in soil contaminated with infected wild boar carcasses was investigated separately. Among the 17 identified wildlife species that visited wild boar carcasses, the common raven (Corvus corax) was the most frequent one (37.26%), followed by raccoon dogs (Nyctereutes procyonoides; 4.25%), carcass conspecific/wild boars (3.16%), and red foxes (Vulpes vulpes; 2.14%). Regarding the direct contact with the carcass, the same species ranking was detected: common raven (74.95%), raccoon dogs (9.94%), wild boars (4.21%), and red foxes (4.21%). No clear signs of cannibalism were noted among the wild boars, although brief physical contact with the carcasses was evident. The persistence of ASFV DNA in soil contaminated by infected wild boar carcasses was investigated separately. This study revealed that ASFV DNA from infected carcasses could be detected in forest soil for prolonged periods, even after removing the carcasses. Hence, the carcasses of infected wild boars may play an important role in spreading the African swine fever virus in wild boar populations; thus, prompt removal and disinfection of the soil could be considered necessary to limit the spread of the infection.

The Effectiveness of Protection and Surveillance Zones in Detecting Further African Swine Fever Outbreaks in Domestic Pigs-Experience of the Baltic States.

In the event of an outbreak of African swine fever (ASF) in pig farms, the European Union (EU) legislation requires the establishment of a restricted zone, consisting of a protection zone with a radius of at least 3 km and a surveillance zone with a radius of at least 10 km around the outbreak. The main purpose of the restricted zone is to stop the spread of the disease by detecting further outbreaks. We evaluated the effectiveness and necessity of the restricted zone in the Baltic States by looking at how many secondary outbreaks were detected inside and outside the protection and surveillance zones and by what means. Secondary outbreaks are outbreaks with an epidemiological link to a primary outbreak while a primary outbreak is an outbreak that is not epidemiologically linked to any previous outbreak. From 2014 to 2023, a total of 272 outbreaks in domestic pigs were confirmed, where 263 (96.7%) were primary outbreaks and 9 (3.3%) were secondary outbreaks. Eight of the secondary outbreaks were detected by epidemiological enquiry and one by passive surveillance. Epidemiological enquiries are legally required investigations on an outbreak farm to find out when and how the virus entered the farm and to obtain information on contact farms where the ASF virus may have been spread. Of the eight secondary outbreaks detected by epidemiological investigations, six were within the protection zone, one was within the surveillance zone and one outside the restricted zone. Epidemiological investigations were therefore the most effective means of detecting secondary outbreaks, whether inside or outside the restricted zones, while active surveillance was not effective. Active surveillance are legally prescribed activities carried out by the competent authorities in the restricted zones. Furthermore, as ASF is no longer a rare and exotic disease in the EU, it could be listed as a "Category B" disease, which in turn would allow for more flexibility and "tailor-made" control measures, e.g., regarding the size of the restricted zone.

High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Differentiation of African Swine Fever Virus Strains Isolated in Estonia by Multiple Genetic Markers.

The African swine fever virus (ASFV) was first detected in Estonia, in September 2014. In the subsequent three years, the virus spread explosively all over the country. Only one county, the island of Hiiumaa, remained free of the disease. Due to the drastic decrease in the wild boar population in the period of 2015-2018, the number of ASFV-positive cases among wild boar decreased substantially. From the beginning of 2019 to the autumn of 2020, no ASFV-positive wild boar or domestic pigs were detected in Estonia. A new occurrence of ASFV was detected in August 2020, and by the end of 2022, ASFV had been confirmed in seven counties in Estonia. Investigations into proven molecular markers, such as IGR I73R/I329L, MGF505-5R, K145R, O174L, and B602L, were performed with the aim of clarifying whether these cases of ASFV were new entries or remnants of previous epidemics. The sequences from the period of 2014-2022 were compared to the Georgia 2007/1 reference sequence and the variant strains present in Europe. The results indicated that not all the molecular markers of the virus successfully used in other geographical regions were suitable for tracing the spread of ASFV in Estonia. Only the B602L-gene analysis enabled us to place the ASFV isolates spreading in 2020-2022 into two epidemiologically different clusters.

A multi gene-approach genotyping method identifies 24 genetic clusters within the genotype II-European African swine fever viruses circulating from 2007 to 2022.

Introduction: African swine fever (ASF) is a contagious viral disease of pigs and wild boar that poses a major threat to the global swine industry. The genotype II African swine fever virus (ASFV) entered the European Union (EU) in 2014 and since then fourteen countries have been affected, Italy and North Macedonia being the last in 2022. While whole genome sequencing remains the gold standard for the identification of new genetic markers, sequencing of multiple loci with significant variations could be used as a rapid and cost-effective alternative to track outbreaks and study disease evolution in endemic areas. Materials and methods: To further our understanding of the epidemiology and spread of ASFV in Europe, 382 isolates collected during 2007 to 2022 were sequenced. The study was initially performed by sequencing the central variable region (CVR), the intergenic region (IGR) between the I73R and I329L genes and the O174L and K145R genes. For further discrimination, two new PCRs were designed to amplify the IGR between the 9R and 10R genes of the multigene family 505 (MGF505) and the IGR between the I329L and I215L genes. The sequences obtained were compared with genotype II isolates from Europe and Asia. Results: The combination of the results obtained by sequencing these variable regions allowed to differentiate the European II-ASFV genotypes into 24 different groups. In addition, the SNP identified in the IGR I329L-I215L region, not previously described, grouped the viruses from North Macedonia that caused the 2022 outbreaks with viruses from Romania, Bulgaria, Serbia and Greece, differentiating from other genotype II isolates present in Europe and Asia. Furthermore, tandem repeat sequence (TRS) within the 9R-10R genes of the multigene family 505 (MGF505) revealed eight different variants circulating. Discussion: These findings describe a new multi-gene approach sequencing method that can be used in routine genotyping to determine the origin of new introductions in ASF-free areas and track infection dynamics in endemic areas.

Eight Years of African Swine Fever in the Baltic States: Epidemiological Reflections.

African swine fever (ASF) was first detected in Lithuania, Latvia, and Estonia in 2014 and has since been circulating in the Baltic States with a similar epidemiological course characterized by persistence of the disease in the wild boar population and occasional spill-over infections in domestic pigs. The aim of the present study was to evaluate surveillance data on ASF in wild boar from the three countries to improve our understanding of the course of the disease. ASF surveillance and wild boar population data of the countries were analyzed. In all three countries, a decrease in the prevalence of ASF virus-positive wild boar was observed over time. Although somewhat delayed, an increase in the seroprevalence was seen. At the same time, the wild boar population density decreased significantly. Towards the end of the study period, the wild boar population recovered, and the prevalence of ASF virus-positive wild boar increased again, whereas the seroprevalence decreased. The decreasing virus prevalence has obviously led to virus circulation at a very low level. Together with the decreasing wild boar population density, the detection of ASF-infected wild boar and thus ASF control has become increasingly difficult. The course of ASF and its continuous spread clearly demonstrate the necessity to scrutinize current ASF surveillance and control strategies fundamentally and to consider new transdisciplinary approaches.

African Swine Fever Re-Emerging in Estonia: The Role of Seropositive Wild Boar from an Epidemiological Perspective.

African swine fever (ASF) emerged in Estonia in 2014. From February 2019 to August 2020, no pigs or wild boar tested positive for ASF virus (ASFV), only ASFV-specific antibodies could be detected in shot wild boar. However, ASF recently re-emerged in wild boar. We tested three hypotheses that might explain the current situation: (i) ASFV may have been present throughout, but at a prevalence below the detection limit; (ii) seropositive wild boar may have remained infectious (i.e., virus-carriers) and kept the epidemic going; or (iii) ASF was gone for 1.5 years, but was recently re-introduced. Using Estonian surveillance data, the sensitivity of the surveillance system and the confidence in freedom from ASF were estimated. Furthermore, the detection probability was determined and cluster analyses were performed to investigate the role of serological positive wild boar. The results suggest that the surveillance system was not able to detect virus circulation at a design prevalence below 1%. With respect to the confidence in freedom from ASF, the results indicate that circulating virus should have been detected over time, if the prevalence was ≥2%. However, the decreasing wild boar population density and ongoing surveillance activities made ASFV circulation at a low prevalence unlikely. Cluster analyses provided no evidence for a significant accumulation of serologically positive wild boar in temporal connection to the re-emergence of ASFV. Further targeted research, such as long-term experimental studies and molecular epidemiology, is necessary to improve our knowledge on the epidemiology of ASF and to control the disease more effectively.

Dynamics of African swine fever virus (ASFV) infection in domestic pigs infected with virulent, moderate virulent and attenuated genotype II ASFV European isolates.

This study aimed to compare the infection dynamics of three genotype II African swine fever viruses (ASFV) circulating in Europe. Eighteen domestic pigs divided into three groups were infected intramuscularly or by direct contact with two haemadsorbent ASFVs (HAD) from Poland (Pol16/DP/ OUT21) and Estonia (Est16/WB/Viru8), and with the Latvian non-HAD ASFV (Lv17/WB/Rie1). Parameters, such as symptoms, pathogenicity, and distribution of the virus in tissues, humoral immune response, and dissemination of the virus by blood, oropharyngeal and rectal routes, were investigated. The Polish ASFV caused a case of rapidly developing fatal acute disease, while the Estonian ASFV caused acute to sub-acute infections and two animals survived. In contrast, animals infected with the ASFV from Latvia developed a more subtle, mild, or even subclinical disease. Oral excretion was sporadic or even absent in the attenuated group, whereas in animals that developed an acute or sub-acute form of ASF, oral excretion began at the same time the ASFV was detected in the blood, or even 3 days earlier, and persisted up to 22 days. Regardless of virulence, blood was the main route of transmission of ASFV and infectious virus was isolated from persistently infected animals for at least 19 days in the attenuated group and up to 44 days in the group of moderate virulence. Rectal excretion was limited to the acute phase of infection. In terms of diagnostics, the ASFV genome was detected in contact pigs from oropharyngeal samples earlier than in blood, independently of virulence. Together with blood, both samples could allow to detect ASFV infection for a longer period. The results presented here provide quantitative data on the spread and excretion of ASFV strains of different virulence among domestic pigs that can help to better focus surveillance activities and, thus, increase the ability to detect ASF introductions earlier.

Molecular Characterization of African Swine Fever Virus Isolates in Estonia in 2014-2019.

After the extensive spread of the African swine fever virus (ASFV) genotype II in Eastern Europe, the first case of African swine fever (ASF) in Estonia was diagnosed in September 2014. By the end of 2019, 3971 ASFV-positive wild boars were found, and 27 domestic pig outbreaks were reported. A selection of ASFV isolates from wild boar and domestic pigs (during the period of September 2014-2019) was molecularly characterized using standardized genotyping procedures. One of the proven markers to characterize this virus is the central variable region (CVR) within the B602L gene. In summer 2015, a new ASFV genotype II CVR variant 2 (GII-CVR2) was confirmed in Estonia. The results suggest that the GII-CVR2 variant was only confirmed in wild boar from a limited area in southern Estonia in 2015 and 2016. In addition to GII-CVR2, a single nucleotide polymorphism (SNP) that resulted in amino acid change was identified within the genotype II CVR variant 1 (GII-CVR1). The GII-CVR1/SNP1 strain was isolated in Estonia in November 2016. Additional GII-CVR1/SNP1 cases were confirmed in two neighbouring counties, as well as in one outbreak farm in June 2017. Based on the available data, no GII-CVR2 and GII-CVR1/SNP1 have been reported by other affected European countries. The spread of variant strains in Estonia has been limited over time, and restricted to a relatively small area.

Epidemiological analysis of the 2015-2017 African swine fever outbreaks in Estonia.

African swine fever (ASF) was first detected in the Estonian wild boar population in September 2014, while the first domestic pig farm was affected in July 2015. In the present study, we aimed to analyse, retrospectively, the epidemiology of the disease in all 26 outbreaks in domestic pig herds that occurred in Estonia during the period 2015-2017. Formal interviews were conducted to estimate the high-risk period for every farm, and to identify the possible origin of the ASF virus and the mode of virus introduction. Furthermore, the clinical manifestation of the disease as well as the course of the disease within the farm were investigated. Survival analysis was used to calculate herd incidence and to estimate outbreak risk. A hierarchical Bayesian space-time model was used to analyse the associations between outbreaks and ASF occurrence in wild boar. The spatial and temporal distribution of outbreaks was analysed to characterise the ASF epidemic in the Estonian domestic pig population from 2015 to 2017. The estimated high-risk period varied from seven to 20 days with a median of 11 days. On most of the affected farms, the first clinical signs were mild and not specific to ASF despite the high virulence of the circulating virus. Morbidity and mortality were often limited to a single pen or unit of the farm. The highest mortality (29.7%) was seen on backyard farms with 1-10 pigs and the lowest (0.7%) on large commercial farms (>1000 pigs). The spread of the virus within affected farms has been slow and the contagiousness of the virus has been relatively low. Farms of all sizes and types have been at risk, including large commercial farms operating at a high biosecurity level. In none of the affected farms could the specific route of introduction be verified. However, the findings suggested that virus introduction occurred via indirect transmission routes due to insufficient biosecurity. The total herd incidence of outbreaks was similar across all three years, being 2.4% in 2015 and 2016, and 2.0% in 2017. All outbreaks occurred from June to September, during the warmest period of the year. The results suggest that the increase in ASF cases in local wild boar populations is the main risk factor leading to the infection of farms; 88% of outbreaks occurred in areas where ASF virus was detected in wild boar prior to the outbreak, within a radius of 15 km from the outbreak farm.

Hunters' Acceptance of Measures against African Swine Fever in Wild Boar in Estonia.

African swine fever (ASF) was first identified in Estonia in 2014, initially detected in wild boar and spreading to affect almost the whole country from late 2016 onwards. Passive surveillance and the control measures applied in Estonia are the main actions in the attempt to control the wild boar population and therefore limit the spread of ASF. Implementation and success of both activities depend mainly on the involvement and commitment of the executing force: the Estonian hunters. Thus, their acceptance of the measures is of utmost importance and with the help of participatory methods, their acceptability can be assessed. Participatory epidemiology allows the involvement of key stakeholders in planning control measures and surveillance strategies and gathering information otherwise inaccessible. By conducting focus group discussions and utilizing participatory tools, this study aimed to assess the acceptance of ASF control measures by hunters in Estonia. Furthermore, the study aimed to detect means to improve the motivation of hunters to support passive surveillance. Among hunters, the results ranked the trust in lower authorities (e.g. local official veterinarians) towards implementing control measures as high (in contrast to higher officials e.g. 'Ministry of Rural Affairs'), while perceiving themselves as the most trustworthy group among those implementing ASF control measures. Hunting and every measure supporting increased hunting, for example selective hunting, bait feeding and incentives for hunting wild boar, were deemed favourable for hunters. These measures also received the highest trust for controlling ASF. All measures hindering hunting and the movement of wildlife, for example fencing or involvement of the army in ASF control, were described as unpleasant or even unethical and trust in these measures to control the disease successfully was lacking. When assessing the perceived consequences for hunters of finding a dead wild boar, arising financial costs, additional workload and time consumption were highlighted. In line with these results, the two tools with the strongest motivational effect for taking part in passive surveillance were: (1) higher monetary incentives as compensation for the hunters' work, and (2) the reduction of the negative consequences by limiting the hunters' duties to solely reporting found dead wild boar. In conclusion, participatory methods can be used as a highly suitable tool for the evaluation of acceptance of measures and surveillance systems. Potentially, the results can help to improve control and passive surveillance in Estonia, as well as functioning as an example for other countries battling or awaiting ASF.

To sample or not to sample? Detection of African swine fever in wild boar killed in road traffic accidents.

African swine fever (ASF) in wild boar remains a threat for the global pig industry. Therefore, surveillance is of utmost importance, not only to control the disease but also to detect new introductions as early as possible. Passive surveillance is regarded as the method of choice for an effective detection of ASF in wild boar populations. However, the relevance of wild boar killed through road traffic accidents (RTA) for passive surveillance seems to be unclear. Using comprehensive ASF wild boar surveillance data from Estonia and Latvia, the prevalence of ASF-infected wild boar was calculated and the probability of infection as measured by PCR compared for animals that were hunted, found dead, shot sick or killed in a RTA. The number of samples originating from wild boar killed in a RTA was low and so was the ASF prevalence in these animals. However, the reasons for this low number of RTA animals remain unknown. Therefore, we recommend to sample wild boar killed in a RTA to a greater extent, also to explore, if this approach can increase the detection probability, and to avoid missing disease introduction.

How to Demonstrate Freedom from African Swine Fever in Wild Boar-Estonia as an Example.

Estonia has been combatting African swine fever (ASF) for six years now. Since October 2017, the disease has only been detected in the wild boar population, but trade restrictions had to remain in place due to international regulations. Yet, the epidemiological course of the disease has changed within the last few years. The prevalence of ASF virus (ASFV)-positive wild boar decreased steadily towards 0%. In February 2019, the last ASFV-positive wild boar was detected. Since then, positive wild boar samples have exclusively been positive for ASFV-specific antibodies, suggesting the possible absence of circulating ASFV in the Estonian wild boar population. However, as the role of seropositive animals is controversially discussed and the presence of antibody-carriers is regarded as an indication of virus circulation at EU and OIE level, Estonia remains under trade restrictions. To make the disease status of a country reliable for trading partners and to facilitate the process of declaration of disease freedom, we suggest to monitor the prevalence of seropositive wild boar in absence of ASFV-positive animals. The possibility to include ASF in the list of diseases, for which an official pathway for recognition of disease status is defined by the OIE should be evaluated.

Analysis of Estonian surveillance in wild boar suggests a decline in the incidence of African swine fever.

African swine fever (ASF) in wild boar populations is difficult to control. In affected areas, samples from all wild boar shot and found dead are investigated. The use of laboratory tests allows estimating the duration of the infection in affected animals. The study aimed to test the hypothesis that the stage of the epidemic in different areas of Estonia can be assessed on the basis of prevalence estimates. ASF surveillance data of Estonian wild boar were used to estimate prevalences and compare them between the East and West of Estonia. The temporal trend of the estimated prevalence of ASF virus positive animals and of the estimated seroprevalence of wild boar showing antibodies against ASFV was analyzed. Due to the potential influence of population density on the course of ASF in wild boar, also population density data (number of wild boar/km2) were used to investigate the relationship with laboratory test results. In areas, where the epidemic had already lasted for a long time, a small number of new cases emerged recently. The prevalence of samples that were only seropositive was significantly higher in these regions as compared to areas, where the epidemic is in full progress. The observed course of the disease could be the beginning of an ASF endemicity in this region. However, the results may also indicate that ASF has started to subside in the areas that were first affected in Estonia.

Deletion at the 5'-end of Estonian ASFV strains associated with an attenuated phenotype.

African swine fever (ASF) was introduced into the Eastern European Union in 2014 and led to considerable mortality among wild boar. In contrast, unexpected high antibody prevalence was reported in hunted wild boar in north-eastern Estonia. One of the causative virus strains was recently characterized. While it still showed rather high virulence in the majority of experimentally infected animals, one animal survived and recovered completely. Here, we report on the follow-up characterization of the isolate obtained from the survivor in the acute phase of infection. As a first step, three in vivo experiments were performed with different types of pigs: twelve minipigs (trial A), five domestic pigs (trial B), and five wild boar (trial C) were inoculated. 75% of the minipigs and all domestic pigs recovered after an acute course of disease. However, all wild boar succumbed to infection within 17 days. Representative samples were sequenced using NGS-technologies, and whole-genomes were compared to ASFV "Georgia 2007/1". The alignments indicated a deletion of 14560 base pairs at the 5' end, and genome reorganization by duplication. The characteristic deletion was confirmed in all trial samples and local field samples. In conclusion, an ASFV variant was found in Estonia that showed reduced virulence.

Development of African swine fever epidemic among wild boar in Estonia - two different areas in the epidemiological focus.

African swine fever (ASF) in wild boar emerged in Estonia for the first time in September 2014. The first affected region was located in the South of Estonia close to the border with Latvia. It was considered to be epidemiologically connected to the outbreaks in the North of Latvia. About two weeks later, cases were detected in the North of Estonia, close to the Russian border. In the present study, we aimed to investigate the epidemiological courses of the disease in the South and in the North of Estonia. Potential associations between risk factors and the laboratory test results for ASF were examined. A hierarchical Bayesian space-time model was used to analyze the temporal trend of the ASF seroprevalence in the two areas. Young wild boar were statistically significant more likely to be ASF-positive by both, serology and virus detection, than older animals. A statistically significant difference between the two areas in the temporal course of the seroprevalence was found. While the seroprevalence clearly increased in the South, it remained relatively constant in the North. These findings led to the hypothesis that ASF might have been introduced earlier into the North of Estonia then into the South of the country.