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.

Meat Exudate for Detection of African Swine Fever Virus Genomic Material and Anti-ASFV Antibodies.

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible.

Swift and Reliable "Easy Lab" Methods for the Sensitive Molecular Detection of African Swine Fever Virus.

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this "easy lab" workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the "easy lab" strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the "easy lab" concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the "easy lab" strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.

Rapid and highly sensitive portable detection of African swine fever virus.

African swine fever (ASF) continues to spread across Asia, devastating pig populations. The disease is nearly 100% fatal in pigs, and currently, there is no effective vaccine available. Therefore, early detection of ASF is critical for effective disease control. The testing process usually requires samples to be shipped to a central laboratory, which may take many hours of travel or shipping time, delaying the results needed for a rapid response. The ability to confirm ASFV-infected animals on-site or in a regional laboratory that has limited technical capacity and/or infrastructure should eliminate these issues. This study describes the successful transfer of a highly sensitive and specific laboratory-validated real-time PCR assay to a portable pen-side thermocycler, which can be operated in the field for rapid detection of ASFV following a quick manual nucleic acid extraction from a wide array of clinical samples including aggregate samples such as oral fluids. The performance of the portable assay was comparable to the laboratory-based assay. The true portability of the assay was evaluated in seven ASF-suspected farms in Vietnam by testing eighty-nine freshly collected whole blood samples on-site. The results obtained on-site were in agreement with the laboratory data obtained the following day. Availability of this field-deployable molecular assay would eliminate the need to ship samples to a central laboratory, when rapid laboratory results are required, ultimately improving the response time.

Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay.

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.

Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses.

BACKGROUND: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used. RESULTS: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent. CONCLUSION: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

Stability of African swine fever virus on heat-treated field crops.

African swine fever (ASF) is an infectious disease of pigs and represents a massive threat to animal health and the pig industry worldwide. The ASF virus (ASFV) is efficiently transmitted via blood and meat from infected animals and can be highly stable in the environment. There is therefore great concern about the potential role of contaminated raw materials used for feed or bedding in the spread of ASFV. Especially crops and derived products originating from areas with ASF in wild boar and thus with high environmental ASFV contamination may be a risk for virus introduction into domestic pig herds. However, little is known about the stability of ASFV on contaminated crops and possible inactivation methods. In this study, we tested the effect of drying and heat treatment on the inactivation of ASFV on six different types of field crops, namely wheat, barley, rye, triticale, corn, and peas, contaminated with infectious blood. Samples were analysed for the presence of viral DNA and infectious virus after 2 hr drying at room temperature or after drying and 1 hr exposure to moderate heat at a specific temperature between 40°C and 75°C. ASFV genome was detected in all samples by real-time polymerase chain reaction (PCR), including samples that had been dried for 2 hr and incubated for 1 hr at 75°C. On the other hand, no infectious virus could be detected after 2 hr drying using virus isolation in porcine macrophages in combination with the detection of ASFV by the haemadsorption test (HAT). We therefore conclude that the risk of ASFV transmission via contaminated crops is most likely low, if they are incubated for at least 2 hr minimum at room temperature. Nonetheless, to minimize the risk of transmission as much as possible crops from ASF-affected zones should not be used for pig feed.

Vertical transmission of anti-ASFV antibodies as one of potential causes of seropositive results among young wild boar population in Poland.

The present study attempted to elucidate possible routes leading to the achievement of sero- positive results, among young (aged ≤1 year) wild boar population. In the years 2017-2018, the National Reference Laboratory (NRL) for African swine fever (ASF) in Poland examined nearly 27-thousand wild boar blood samples, collected during an active surveillance of ASF risk zones, for the presence of viral DNA and anti-ASFV antibodies. Out of all the examined samples, 420 were positive. However, in more than half of them (292 samples) antibodies against African swine fever virus (ASFV) were detected, while ASFV DNA was not detected in blood. Out of all 292 seropositive/PCR-negative samples, 126 belonged to young wild boars (aged ≤1 year). For this reason, the NRL in Poland has examined 10 selected seropositive wild boar carcasses to confirm or exclude post-mortem lesions for ASF as well as to investigate the presence of viral DNA in the internal organs. Neither pathological lesions for ASF nor the presence of genetic material of ASFV were found in the examined wild boars. To elucidate this outcomes, following hypotheses about possible reasons of the obtained results were drawn: the presence of convalescent animals, infection of low-virulent ASFV isolate and the vertical transmission of antibodies through the colostrum.

Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder.

Since 2014, African swine fever virus (ASFV) has been spreading within Eastern Europe. Within affected regions, the virus has infected some farms with high biosecurity and a marked seasonality of outbreaks in domestic pigs has been observed. ASFV transmission from stable flies, Stomoxys calcitrans, has previously been shown both mechanically and via ingestion of whole flies. Hence, blood-feeding flies may offer one explanation for the introductions into high biosecurity farms and for the observed seasonality. The aim of this study was to further elucidate the potential role of stable flies in ASFV transmission. Different parts of flies were analyzed for the presence of viral DNA and infectious virus at different time points following in vitro feeding of the flies on blood from an ASFV-infected pig. Using qPCR, ASFV DNA was detectable in mouth parts of flies for at least 12 h and remained in head and body samples from the flies for up to three days following feeding. Infectious virus was detected in fly body samples prepared at 3 h and 12 h after feeding. The presence of infectious ASFV in stable flies following feeding on viremic blood means that such flies are capable of transporting infectious virus. The detection of ASFV DNA in the flies for up to three days following feeding suggests that qPCR analysis of blood-feeding flies during ASFV outbreaks could be a useful method to elucidate the role of these flies in ASFV transmission under field conditions.

Comparison of loop-mediated isothermal amplification (LAMP) and cross-priming amplification (CPA) for detection of African swine fever virus.

The reliable and rapid diagnosis of infectious animal diseases presents an exceptionally im- portant aspect when considering their control and prevention. The paper describes the compara- tive evaluation of two rapid isothermal amplification methods for diagnosis of African swine fever (ASF). The robustness of loop-mediated isothermal amplification (LAMP) and the cross-priming amplification (CPA) were compared using samples obtained from ASF confirmed animals. Both assays were evaluated in order to define their diagnostic capabilities in terms of ASF diagnosis and reproducibility of the results. Investigations showed no cross-reactivity for other pig patho- gens and no significant differences in the specificity of both assays. The sensitivity of LAMP reached 90%, while that of CPA was 70%. In conclusion, both methods are suitable for imple- mentation in preliminary ASF diagnosis but further improvements are required to enhance their diagnostic sensitivity.

Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses.

African swine fever virus (ASFV) is a macrophage-tropic virus responsible for ASF, a transboundary disease that threatens swine production world-wide. Since there are no vaccines available to control ASF after an outbreak, obtaining an understanding of the virus-host interaction is important for developing new intervention strategies. In this study, a whole transcriptomic RNA-Seq method was used to characterize differentially expressed genes in pigs infected with a low pathogenic ASFV isolate, OUR T88/3 (OURT), or the highly pathogenic Georgia 2007/1 (GRG). After infection, pigs infected with OURT showed no or few clinical signs; whereas, GRG produced clinical signs consistent with acute ASF. RNA-Seq detected the expression of ASFV genes from the whole blood of the GRG, but not the OURT pigs, consistent with the pathotypes of these strains and the replication of GRG in circulating monocytes. Even though GRG and OURT possess different pathogenic properties, there was significant overlap in the most upregulated host genes. A small number of differentially expressed microRNAs were also detected in GRG and OURT pigs. These data confirm previous studies describing the response of macrophages and lymphocytes to ASFV infection, as well as reveal unique gene pathways upregulated in response to infection with GRG.

African and classical swine fever situation in Ivory-Coast and neighboring countries, 2008-2013.

This study was conducted from 2008 to 2013 to determine the animal health status of Ivory Coast and neighboring countries (Burkina Faso, Ghana, Togo and Benin) for African swine fever (ASF) and classical swine fever (CSF), and to assess the risk factors for ASF introduction in Ivory Coast. Ivory Coast had probably been free from ASF from 1998 to 2014 when it was re-introduced in this country. However, the ASF virus was found in all neighboring countries. In contrast, no evidence of CSF infection was found so far in Ivory Coast and neighboring countries. To assess the risk of ASF reintroduction in Ivory Coast, we surveyed 59 modern pig farms, and 169 pig owners in 19 villages and in two towns. For the village livestock, the major risk factor was the high frequency of pig exchanges with Burkinabe villages. In the commercial sector, many inadequate management practices were observed with respect to ASF. Their identification should enable farmers and other stakeholders to implement a training and prevention program to reduce the introduction risk of ASF in their farms.

Evidence for the presence of African swine fever virus in an endemic region of Western Kenya in the absence of any reported outbreak.

BACKGROUND: African swine fever (ASF), caused by African swine fever virus (ASFV), is a severe haemorrhagic disease of pigs, outbreaks of which can have a devastating impact upon commercial and small-holder pig production. Pig production in western Kenya is characterised by low-input, free-range systems practised by poor farmers keeping between two and ten pigs. These farmers are particularly vulnerable to the catastrophic loss of livestock assets experienced in an ASF outbreak. This study wished to expand our understanding of ASFV epidemiology during a period when no outbreaks were reported. RESULTS: Two hundred and seventy six whole blood samples were analysed using two independent conventional and real time PCR assays to detect ASFV. Despite no recorded outbreak of clinical ASF during this time, virus was detected in 90/277 samples analysed by conventional PCR and 142/209 samples analysed by qPCR. Genotyping of a sub-set of these samples indicated that the viruses associated with the positive samples were classified within genotype IX and that these strains were therefore genetically similar to the virus associated with the 2006/2007 ASF outbreaks in Kenya. CONCLUSION: The detection of ASFV viral DNA in a relatively high number of pigs delivered for slaughter during a period with no reported outbreaks provides support for two hypotheses, which are not mutually exclusive: (1) that virus prevalence may be over-estimated by slaughter-slab sampling, relative to that prevailing in the wider pig population; (2) that sub-clinical, chronically infected or recovered pigs may be responsible for persistence of the virus in endemic areas.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Development of a novel lateral flow assay for detection of African swine fever in blood.

BACKGROUND: African swine fever (ASF) is a viral infectious disease of domestic and wild suids of all breeds and ages, causing a wide range of hemorrhagic syndromes and frequently characterized by high mortality. The disease is endemic in Sub-Saharan Africa and Sardinia. Since 2007, it has also been present in different countries of Eastern Europe, where control measures have not been effective so far. The continued spread poses a serious threat to the swine industry worldwide. In the absence of vaccine, early detection of infected animals is of paramount importance for control of the outbreak, to prevent the transmission of the virus to healthy animals and subsequent spreading of the disease. Current laboratory diagnosis is mainly based on virological methods (antigen and genome detection) and serodiagnosis. RESULTS: In the present work, a Lateral Flow Assay (LFA) for antigen detection has been developed and evaluated. The test is based on the use of a MAb against VP72 protein of ASFV, the major viral capsid protein and highly immunogenic. First experiments using VP72 viral and recombinant protein or inactivated culture virus showed promising results with a sensitivity similar to that of a commercially available Antigen-ELISA. Moreover, these strips were tested with blood from experimentally infected pigs and field animals and the results compared with those of PCR and Antigen-ELISA. For the experimentally infected samples, there was an excellent correlation between the LFA and the ELISA, while the PCR always showed to be more sensitive (38 % positive samples by PCR versus 27 % by LFA). The LFA was demonstrated to be positive for animals with circulating virus levels exceeding 10(4) HAU. With the field samples, once again, the PCR detected more positives than either the Antigen-ELISA or LFA, although here the number of positive samples scored by the LFA exceeded the values obtained with the Antigen-ELISA, showing 60 % positivity vs 48 % for the ELISA. For the two groups of sera, the specificity was close to 100 % indicating that hardly any false positive samples were found. CONCLUSIONS: The newly developed LFA allows rapid and reliable detection of ASFV, at field and laboratory level, providing a new useful tool for control programs and in situations where laboratory support and skilled personnel are limited.

Sero-prevalence and risk factors associated with African swine fever on pig farms in southwest Nigeria.

BACKGROUND: African swine fever (ASF) is one of the major setbacks to development of the pig industry in Nigeria. It is enzootic in southwest Nigeria. We determined the sero-prevalence and factors associated with ASF among-herd seropositivity in 144 pig farms in six States from southwest Nigeria during the dry and rainy seasons using indirect Enzyme Linked Immunosorbent Assay (ELISA) for ASF IgG antibodies. An interviewer-administered questionnaire was used to collect information on demography, environmental and management factors. We performed descriptive statistics, and univariate and multivariable analyses to determine the among-herd sero-prevalence of ASF and its associated factors. RESULTS: The overall herd sero-prevalence of ASF was 28% (95% Confidence interval (95% CI) 21-36); it was significantly higher (P <0.05) in the dry season (54%; 95% CI 37-70) than the rainy season (18%; 95% CI 11-27). In the univariate analysis, having a quarantine/ isolation unit within 100 m radius of a regular pig pen (OR = 3.3; 95% CI 1.3-8.9), external source of replacement stock (OR = 3.2; 95% CI 1.3-8.3) and dry season (OR = 5.3; 95% CI 2.2-12.7) were risk factors for ASF among-herd seropositivity. In the multivariable logistic regression, there was interaction between season and herd size. Our final model included season, source of replacement stock, herd size and interaction between herd size and season. Herds with an external source of replacement always had higher ASF sero-prevalence compared with herds with an internal source. The herd size effect varied between seasons. CONCLUSIONS: The ASF herd level sero-prevalence in southwest Nigeria was higher in pig herds with an external source of replacement stock and in the dry season. The effect of season of the year the samples were taken on ASF seropositivity was modified by herd size. We encourage strict compliance with biosecurity measures, especially using an internal source of replacement stock and measures that minimize movement on pig farms in southwest Nigeria, in order to enhance ASF free farms.

A longitudinal survey of African swine fever in Uganda reveals high apparent disease incidence rates in domestic pigs, but absence of detectable persistent virus infections in blood and serum.

BACKGROUND: African swine fever (ASF) is a fatal, haemorrhagic disease of domestic pigs, that poses a serious threat to pig farmers and is currently endemic in domestic pigs in most of sub-Saharan Africa. To obtain insight into the factors related to ASF outbreaks at the farm-level, a longitudinal study was performed in one of the major pig producing areas in central Uganda. Potential risk factors associated with outbreaks of ASF were investigated including the possible presence of apparently healthy ASF-virus (ASFV) infected pigs, which could act as long-term carriers of the virus. Blood and serum were sampled from 715 pigs (241 farms) and 649 pigs (233 farms) to investigate presence of ASFV and antibodies, during the periods of June-October 2010 and March-June 2011, respectively. To determine the potential contribution of different risks to ASF spread, a questionnaire-based survey was administered to farmers to assess the association between ASF outbreaks during the study period and the risk factors. RESULTS: Fifty-one (21 %) and 13 (5.6 %) farms reported an ASF outbreak on their farms in the previous one to two years and during the study period, respectively. The incidence rate for ASF prior to the study period was estimated at 14.1 per 100 pig farm-years and 5.6 per 100 pig farm-years during the study. Three pigs tested positive for ASFV using real-time PCR, but none tested positive for ASFV specific antibodies using two different commercial ELISA tests. CONCLUSIONS: There was no evidence for existence of pigs that were long-term carriers for the virus based on the analysis of blood and serum as there were no seropositive pigs and the only three ASFV DNA positive pigs were acutely infected and were linked to outbreaks reported by farmers during the study. Potential ASF risk factors were present on both small and medium-scale pig farms, although small scale farms exhibited a higher proportion with multiple potential risk factors (like borrowing boars for sows mating, buying replacement from neighboring farms without ascertaining health status, etc) and did not implement any biosecurity measures. However, no risk factors were significantly associated with ASF reports during the study.

Exposure of extensively farmed wild boars (Sus scrofa scrofa) to selected pig pathogens in Greece.

BACKGROUND: Increased density and distribution of wild boar populations are likely to promote interactions and transmission of certain pathogens, not only among wild boar but also from wild boar to livestock or humans and vice versa. OBJECTIVE: The purpose of this study was to determine seroprevalence against seven selected pathogens in wild boar living in four different areas in Greece. ANIMALS AND METHODS: In total, 359 serum samples were collected from extensively farmed wild boar (Sus scrofa scrofa) originating from four distinct geographical areas throughout Greece from April 2012 to August 2013. Samples were tested for antibodies to Actinobacillus pleuropneumoniae, African swine fever virus (ASFV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), Erysipelothrix rhusiopathiae, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). Prevalence was compared among the four regions using Fisher's exact test. RESULTS: Low overall seropositivities of 2.4% and 5.6% were detected for E. rhusiopathiae and PRRSV, respectively, higher ones for ADV (32.0%) and the highest (72.5% and 90.5%) for M. hyopneumoniae and A. pleuropneumoniae, respectively. All sera tested were found negative for antibodies directed against CSFV and ASFV. CONCLUSIONS: This is the first report of exposure of wild boars to selected pig pathogens in Greece. These results are indicative of the circulation of these pathogens in Greece with the exception of CSFV and ASFV and suggestive of the potential role of wild boars on their maintenance and transmission to their domestic counterparts and vice versa.

Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests.

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs.