Simultaneous Detection of Antigen and Antibodies of African Swine Fever in a Novel Combo Lateral Flow Assay.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

First insight into strongylid nematode diversity and anthelmintic treatment effectiveness in beef cattle in the Czech Republic explored by HTS metagenomics.

Parasitic diseases and mitigation of their effects play an important role in the health management of grazing livestock worldwide, with gastrointestinal strongylid nematodes being of prominent importance. These helminths typically occur in complex communities, often composed of species from numerous strongylid genera. Detecting the full diversity of strongylid species in non-invasively collected faecal samples is nearly impossible using conventional methods. In contrast, high-throughput amplicon sequencing (HTS) can effectively identify co-occurring species. During the four-year project, we collected and analysed faecal samples from beef cattle on >120 farms throughout the Czech Republic. Strongylids were the predominant nematodes, detected in 56% of the samples, but at a low level of infection. The apparent limitations in identifying strongylid taxa prompted this pilot study on a representative group of samples testing positive for strongylids using ITS-2 metabarcoding. The most widespread genera parasitizing Czech cattle were Ostertagia (O. ostertagi) and Oesophagostomum spp., followed by Trichostrongylus and Cooperia, while Bunostomum, Nematodirus and Chabertia were present only in a minority. As comparative material, 21 samples of cattle from the Danube Delta in Romania were used, which, in contrast, were dominated by Haemonchus placei. Finally, the effect of ivermectin treatment was tested at two Czech farms. After treatment with the anthelmintic, there was a shift in the strongylid communities, with a dominance of Cooperia and Ostertagia.

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.

Experimental Evidence of the Long-Term Survival of Infective African Swine Fever Virus Strain Ba71V in Soil under Different Conditions.

The survival of African swine fever virus (ASFV) on different matrices and its infectivity in wild as well as domestic swine is still a matter of interest. ASFV is resistant to environmental effects; this fact is enhanced by the presence of organic material. Therefore, the aim of this work was to determine the ability of laboratory ASFV to survive in soil at different temperatures (4 and 22 °C) and with and without the presence of blood using culture procedures. The suitability of the procedure for determining the viability and titre of the ASFV field strain by the hemadsorption method was also verified, when a higher decrease in virus infectivity in the case of clay compared with peat was demonstrated. The stability of the virus was clearly temperature-dependent, the infectious virus was detected after 112 days, and the viral DNA was still detected in the matrix 210 days after inoculation in a relatively high and stable concentration (between 106 and 107 genome equivalents/mL). Based on this knowledge, soil and other environmental samples could provide rapid and reliable information on the disease outbreak and serve as indicators of the risk posed by the affected locality.

The winner takes it all: dominance of Calicophoron daubneyi (Digenea: Paramphistomidae) among flukes in Central European beef cattle.

In Europe, paramphistomosis caused by Paramphistomum spp. was historically regarded as being of minor importance. However, Calicophoron daubneyi has recently been recognized as an emerging pathogen in Europe due to its increasing prevalence and negative impact on livestock production. In search for paramphistomid flukes, 5573 beef cattle fecal samples from 115 farms across the whole Czech Republic were examined from March 2019 to June 2021. The eggs of paramphistomid flukes were identified in 29.9% of samples. Internal transcribed spacer 2 sequences from 90 adult flukes and 125 fecal samples collected across Czech Republic confirmed C. daubneyi infection in the Czech beef cattle. Ninety mitochondrial DNA sequences obtained from adult C. daubneyi specimens revealed 13 individual haplotypes, two of them recorded for the first time. Although C. daubneyi is a new parasite in beef cattle herds in the Czechia, it clearly dominates the parasitological findings in the country's beef cattle. The common occurrence of C. daubneyi in most of the beef cattle herds indicates environmental conditions suitable also for the life cycle of Fasciola hepatica and risk of its emergence.

Whole-Genome Sequence of an African Swine Fever Virus Isolate from the Czech Republic.

Between June 2017 and April 2018, an outbreak of African swine fever (ASF) affected wild boar in the southeast of the Czech Republic. Here, we present the whole-genome sequence of the causative ASF virus. It belongs to genotype II and shows very high identity with other strains from Eastern Europe.

Wild boar deathbed choice in relation to ASF: Are there any differences between positive and negative carcasses?

African swine fever (ASF) is a fatal, infectious disease affecting wild boars and domestic pigs, mostly resulting in their deaths. Previous studies showed that carcasses of infected wild boars pose a serious threat for ASF virus transmission and leaving of dead bodies in the environment enables persistence of the disease in the given affected area. Therefore, the prompt finding and removal of the carcasses is crucial for effective ASF control. This study reveals habitat preferences of ASF-positive wild boars for their deathbeds, which could greatly improve the effectivity in the search for infected carcasses. The vast majority (71%) of carcasses were found in forests (although forests occupy only 26.6% of the high-risk area - Zlin region, Czech Republic), especially in young forest stands; 91.3% of infected wild boar carcasses, which were found in forests, were in stands of up to 40 years of age, where infected individuals search for calm and quiet places. The preference of younger forest stands is significantly higher for infected individuals (p < 0.001). On meadows, infected individuals preferred a higher herb layer (p = 0.002) compared to non-infected individuals. A higher preference of places more distant from roads and forest edges was observed for the infected individuals as well (p < 0.001 in both cases). No differences in deathbed habitat preference were observed between selected sex-age categories. The distance between carcasses and water source was observed to be dependent on current mean temperature. Carcasses were found closer to the water sources at higher mean temperature. Because of the comparable character of the landscape, presented models are applicable across Central Europe and have the potential to greatly facilitate the search for infected carcasses.

Confirmed cannibalism in wild boar and its possible role in African swine fever transmission.

For the monitoring of wild boar interactions with conspecific carcasses, seven wild boar carcasses were placed in different sites in the Czech Republic during winter season. Data were collected by camera traps. In total, 732 visits were recorded of wild boars at the carcass sites. Direct contact with the carcass was detected in 81% of the records. Cannibalism was observed in 9.8% of all recorded visits. The first direct contact was observed on average 30 days after the carcass had been placed in its respective site. Cannibalism was observed on average after 70 days. The effect of sex-age class on direct contact was not proven in our study. The presented findings show that cannibalism in wild boar can play a substantial role in the ASF epidemic. These results highlighted the importance of the removal of infected carcasses from the habitat, which is crucial for African swine fever eradication.