Sigade Aafrika katku epidemioloogia Eestis ja ühe viirustüve iseloomustus

A Thesis for applying for the degree of Doctor of Philosophy in Veterinary Science.African swine fever (ASF) is an OIE-listed, dangerous viral disease of pigs, which has a devastating impact on animal health and the pig industry in many countries worldwide. During the period 2014–2020, ASF has been the main animal health issue in Estonia. This dissertation consists of three independent studies, which have been conducted with the aim to analyse the epidemiology of ASF and the course of the epidemic in the Estonian wild boar population, as well as in domestic pigs. In Study I, wild boar surveillance data (n = 7,015) collected from two distinct study areas from September 2014 to September 2016 were analysed. A statistically significant difference between the two areas in the temporal course of ASF seroprevalence was found. These findings indicate that ASF might have been introduced to the north-east of Estonia earlier then to the south of the country. The probability of detecting an ASF-positive animal was higher in young animals (< 1 year). Within wild boar found dead, there was a higher probability of finding an ASF-positive result compared to hunted animals. In Study II, the biological characteristics of the ASF virus strain (Est 14/WB) circulating in the wild boar population of north-east Estonia in 2014 were evaluated. Oronasal inoculation of ten young wild boar led to an acute and severe course of the disease in all infected animals. Nine animals died and one animal recovered completely from the disease. In conclusion, the ASFV strain was still found to be highly virulent. In Study III, the epidemiology of ASF in all 26 domestic pig outbreak herds that occurred in Estonia during the period 2015–2017 was retrospectively analysed. On most of the farms, the first clinical signs were mild and not ASF-specific despite the high virulence of the circulating virus. The highest mortality (29.7%) was seen on backyard farms (1–9 pigs) and the lowest (0.7%) on large commercial farms (> 1000 pigs). The spread of the virus within farms was slow and the contagiousness of the virus was relatively low. Farms of all sizes and types have been at risk. 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 domestic 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.Sigade Aafrika katk (SAK) on ohtlik sigade viirushaigus, mis põhjustab tõsiseid tagajärgi nii loomade tervisele kui ka majanduslikku kahju sektorile. Ajavahemikul 2014-2020 on SAK olnud peamine loomatervise probleem Eestis. Käesolev väitekiri koosneb kolmest eraldiseisvast uuringust, mis on viidud läbi eesmärgiga analüüsida SAK-i epidemioloogiat ning epideemia arengut Eesti metssigade populatsioonis ja kodusigadel. Esimeses uuringus analüüsiti kahest eraldiasuvast metssigade populatsioonist ajavahemikul septembrist 2014 kuni septembrini 2016 kogutud metssigade SAK andmeid (n=7015). Ilmnes, et SAK-i epidemioloogia oli neis kahes populatsioonis erinev. Seroloogiliste tulemuste ajaline analüüs näitas, et mediaan-aja mõju levimusele oli uuritavates piirkondades oluliselt erinev. Sellest tulenevalt võib eeldada, et SAK-i viirus ringles Kirde-Eestis enne, kui see Eestis ja piirkonnas ametlikult diagnoositi. Veel selgus, et tõenäosus leida SAK-positiivne metssiga oli kõrgem noorte loomade rühmas (vanus < 1 aasta) ning surnuna leitud loomade seas. Teises uuringus selgitati Kirde-Eestis 2014. aastal ringelnud SAK-i viirustüve (Est 14/WB) bioloogilisi omadusi. Katses nakatati oronasaalselt kümme umbes nelja kuu vanust metssiga. Kõigil loomadel tekkis äge haigestumine, neist üheksa looma surid ning üks tervenes haigusest täielikult. Katse tulemusel ei tuvastatud uuritud viirustüve virulentsuse langust. Kolmandas uuringus selgitati SAK-i epidemioloogiat kodusigadel. Selleks viidi epidemioloogiline uuring läbi kõigis (n = 26) seafarmides, kus ajavahemikul 2015–2017 diagnoositi SAK-i puhangud. Enamikus nakatunud karjades olid SAK-i esimesed kliinilised tunnused vaatamata haigustekitaja kõrgele virulentsusele leebed ning ebatüüpilised. Kõige kõrgemat suremust (29,7%) võis täheldada kodumajapidamistes (1-9 siga) ning kõige madalamat (0,7%) suurtes tootmisfarmides (>1000 siga). Viiruse levik nakatunud farmides oli aeglane ning nakkavus väike. Risk nakatuda tuvastati kõigis farmide suurusgruppides ning pidamisviiside juures. Uuringu tulemusel ilmnes, et SAKV-i levik kodusigade farmide ümbruskonna metssigade populatsioonis oli peamine riskitegur kodusigade nakatumiseks; 88% juhtudest leiti SAK-positiivseid metssigu puhangufarmist kuni 15 km kaugusel.The publication of this dissertation is supported by the Estonian University of Life Sciences

Review: African swine fever (Pestis Africana Suum)

African swine fever (ASF) is one of the most devastating diseases that affect domestic pig and wild boar. The causative agent of ASF is African swine fever virus (ASFV) that is the sole member of the genus Asfivirus and the family Asfarviridae. The virus is a large icosahedral double-stranded DNA virus in length 170-193 kb depending on the isolate. Both clinical signs and pathological changes may vary considerably depending on strain virulence, virus dose and host factors. Epidemiology of ASF is very complex especially in Africa and Southern Europe, where soft tick and wild pig species are involved to the transmission cycles. In Eastern Europe ASF was first diagnosed in Georgia in April 2007, after what it spread fast to several neighbouring countries in Transcaucasia region and the Russian Federation, where it is still circulating and is now endemic. In 2012, ASFV was reported in Ukraine, in 2013 in Belarus and in 2014, in European Union countries, including Poland, Lithuania, Latvia and Estonia. The spread of the virus among wild boar in Estonia in period from September 2014 to March 2016 has been comprehensive including 12 counties out of 15. During the same period in total 18 outbreaks has been occurred in domestic pigs. For ASF no vaccine or treatment is available, therefore control of the disease based on early warning, early detection, effective control surveillance programs and stamping out in case of the outbreak. Currently is ASF a major threat to the pig industry in Europe

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.This study was supported by the INIA (Projects RTA2015-00033-C02-01, AT2015-002) and the European Union Reference laboratory for ASF (Grant no.: UE- LR PPA/03). We would like to thank all the staff at the INIA-CISA who worked in the animal facilities department.Peer reviewe

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

IntroductionAfrican 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 methodsTo 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.ResultsThe 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.DiscussionThese 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

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

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

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

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