African swine fever (ASF) is one of the most important viral diseases of domestic pigs and wild boar. Apart from endemic cycles in Africa, ASF is now continuously spreading in Europe and Asia. As ASF leads to severe but unspecific clinical signs and high lethality, early pathogen detection is of utmost importance. Recently, “point-of-care” (POC) tests have been intensively discussed for the use in remote areas but also in the context of on-farm epidemiological investigations and wild boar carcass screening. Along these lines, the INGEZIM ASFV CROM Ag lateral flow assay (Eurofins Technologies Ingenasa) promises virus antigen detection under field conditions within minutes. In the present study, we evaluated the performance of the assay with selected high-quality reference blood samples, and also with real field samples from wild boar carcasses in different stages of decay from the ongoing ASF outbreak in Germany. While we observed a sensitivity of roughly 77% in freeze-thawed matrices of close to ideal quality, our approach to simulate field conditions in direct carcass testing without any modification resulted in a drastically reduced sensitivity of only 12.5%. Freeze thawing increased the sensitivity to roughly 44% which mirrored the overall sensitivity of 49% in the total data set of carcass samples. A diagnostic specificity of 100% was observed. However, most of the German ASF cases in wild boar would have been missed using the lateral flow assay (LFA) alone. Therefore, the antigen-specific LFA should not be regarded as a substitute for any OIE listed diagnostic method and has very limited use for carcass testing at the point of care. For optimized LFA antigen tests, the sensitivity with field samples must be significantly increased. An improved sensitivity is seen with freeze-thawed samples, which may indicate problems in the accessibility of ASFV antigen.
Livestock industry supports the livelihood of around 1.3 billion people in the world, with swine industry contributing with 30 % of total livestock production worldwide. To maintain and guarantee this production, a pivotal point according to the OIE is addressing potential biohazards. To control them, permanent sero-surveillance is crucial to achieve more focused veterinary public health intervention and prevention strategies, to break the chains of transmission, and to enable fast responses against outbreaks. Within this context, multiplex assays are powerful tools with the potential to simplify surveillance programs, since they reduce time, labour, and variability within analysis. In the present work, we developed a multiplex bead-based assay for the detection of specific antibodies to six relevant pathogens affecting swine: ASFV, CSFV, PRRSV, SIV, TB, and HEV. The most immunogenic target antigen of each pathogen was selected as the target protein to coat different microsphere regions in order to develop this multiplex assay. A total of 1544 serum samples from experimental infections as well as field samples were included in the analysis. The 6plex assay exhibited credible diagnostic parameters with sensitivities ranging from 87.0 % to 97.5 % and specificities ranging from 87.9 % to 100.0 %, demonstrating it to be a potential high throughput tool for surveillance of infectious diseases in swine.
Infection with African swine fever virus (ASFV) causes a highly lethal hemorrhagic disease in domestic and Eurasian wild pigs. Thus, it is a major threat to pig populations worldwide and a cause of substantial economic losses. Recently, less virulent ASFV strains emerged naturally, which showed higher experimental virulence in wild boar than in domestic pigs. The reason for this difference in disease progression and outcome is unclear but likely involves different immunological responses. Unfortunately, besides the importance of CD8α+ lymphocytes, little is known about the immune responses against ASFV in suids. Against this background, we used a multicolor flow cytometry platform to investigate the T-cell responses in wild boar and domestic pigs after infection with the moderately virulent ASFV strain “Estonia2014” in two independent trials. CD4–/CD8α+ and CD4+/CD8α+ αβ T-cell frequencies increased in both subspecies in various tissues, but CD8α+ γδ T cells differentiated and responded in wild boar only. Proliferation in CD8α+ T cells was found 10 days post infectionem only. Frequencies of T-bet+ T cells increased in wild boar but not in domestic pigs. Of note, we found a considerable loss of perforin expression in cytotoxic T cells, 5 and 7 dpi. Both subspecies established a regulatory T-cell response 10 dpi. In domestic pigs, we show increasing levels of ICOS+ and CD8α+ invariant Natural Killer T cells. These disparities in T-cell responses might explain some of the differences in disease progression in wild boar and domestic pigs and should pave the way for future studies.
African swine fever (ASF) is a viral disease that affects members of the Suidae family. The notifiable disease is considered a major threat to the pig industry, animal health, and food security worldwide. According to the European Food Safety Authority, ASF virus (ASFV) survival and transmission in feed and feed materials is a major research gap. Against this background, the objective of this study was to determine the survival of ASFV on re-contaminated spray dried porcine plasma (SDPP) when stored at two different temperatures. To this means, commercial SDPP granules were contaminated with high titers of ASFV in a worst-case re-contamination scenario. Three samples per time point and temperature condition were subjected to blind passaging on macrophage cultures and subsequent haemadsorption test to determine residual infectivity. In addition, viral genome was detected by real-time PCR. The results indicate that heavily re-contaminated SDPP stored at 4°C remains infectious for at least five weeks. In contrast, contaminated SDPP stored at room temperature displayed a distinct ASFV titer reduction after one week and complete inactivation after two weeks. In conclusion, the residual risk of ASFV transmission through re-contaminated SDPP is low, if SDPP is stored at room temperature for a period of at least two weeks before feeding.
African swine fever (ASF) has spread across many countries in Europe since the introduction into Georgia in 2007. We report here on the first cases of ASF in wild boar detected in Germany close to the border with Poland. In addition to the constant risk of ASF virus (ASFV) spread through human activities, movements of infected wild boar also represent a route of introduction. Since ASF emerged in Western Poland in November 2019, surveillance efforts, in particular examination of wild boar found dead, were intensified in the regions of Germany bordering with Poland. The first case of ASF in wild boar in Germany was therefore detected by passive surveillance and confirmed on 10th September 2020. By 24th September 2020, 32 cases were recorded. Testing of samples from tissues of carcasses in different stages of decomposition yielded cycle threshold values from 18 to 36 in the OIE-recommended PCR which were comparable between the regional and national reference laboratory. Blood swabs yielded reliable results, indicating that the method is suitable also under outbreak conditions. Phylogenetic analysis of the ASFV whole-genome sequence generated from material of the first carcass detected in Germany, revealed that it groups with ASFV genotype II including all sequences from Eastern Europe, Asia and Belgium. However, some genetic markers including a 14 bp tandem repeat duplication in the O174L gene were confirmed that have so far been detected only in sequences from Poland (including Western Poland). Epidemiological investigations that include estimated postmortem intervals of wild boar carcasses of infected animals suggest that ASFV had been introduced into Germany in the first half of July 2020 or even earlier.
Understanding African swine fever virus (ASFV) transmission in a population is essential for strategies to minimize virus spread during an outbreak. ASFV can survive for extended periods of time in animal products, carcasses, and the environment. Recent studies have shown that wild boar demonstrate interest in carcasses at an advanced stage of decay and in the soil where the remains of wild boar once were. While ASFV nucleic acids have been found in the environment around infected farms, data on the survival of the virus in soil are scarce. We investigated different soil matrices spiked with ASFV-positive blood from infected wild boar to see if ASFV can remain viable in the soil beneath infected carcasses. Moreover, we tried different mitigation strategies that could be used in affected regions. As expected, ASFV genome detection was reliably possible over the full range of sampling days. Soil pH, structure, and ambient temperature played a significant role for the stability of infectious ASFV. Infectious ASFV was demonstrated in specimens originating from sterile sand for at least three weeks, and from ordinary beach sand for up to two weeks. In yard soil, infectious ASFV was demonstrated for one week, and in soil from a swampy area for three days. Virus was not recovered from two acidic forest soils. All risk mitigation experiments with citric acid or calcium hydroxide resulted in complete inactivation in our experimental setup. In conclusion, stability of infectious ASFV is almost non-existent in forest soils but rather high in sandy soils. However, given the high variability, treatment of carcass collection points with disinfectants should be considered for additional risk reduction. In this respect, biocidal nature and occupational safety have to be considered.