Evaluation of seven commercial African swine fever virus detection kits and three Taq polymerases on 300 well-characterized field samples

Highlights

• African swine fever virus is a major threat to the global pork industry.

• Diagnosis is an important and efficient weapon to contain ASFV’s spreading.

• A validation is necessary in order to evaluate the quality of the results and guarantee harmonized diagnostics.

• Evaluation of the sensitivity and specificity of seven commercial ASFV real-time PCR detection kits.

• The seven commercial kits and most Taq polymerases provided sensitive and reliable ASFV diagnosis.

Abstract

African swine fever virus (ASFV) is a complex double stranded DNA virus, responsible for a highly infectious and fatal disease in pigs and boars and for important deterioration of animal welfare. Over the last decade, the disease spread to several European and Asian countries causing unprecedented dramatic economic losses in pig industry. In the absence of a vaccine, affected countries rely on trustful diagnostic tests and adapted testing policies to set up control programs to fight against the disease. In this study, we evaluated the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases on 300 well-characterized wild boar samples collected in Belgium during the 2018–2019 outbreak. This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results.

Introduction

African swine fever virus (ASFV) is a complex double stranded DNA virus, 170–190 kbp in size, sole virus of the Asfarviridae family, genus Asfivirus (King et al., 2012). It is the causative agent of African swine fever (ASF), a disease affecting exclusively Suidea. ASF is a highly infectious and fatal disease, with mortality rate ranging up to 100 %, for domestic pig and boar belonging to the Sus Scrofa species (Dixon et al., 2004).

ASFV originates from Africa where it subclinically infects African wild pigs, such as bushpig and warthog (Phacochoerus and Potamochoerus spp.) in which it can persist for months or even years. Spreading of the disease can be very swift as transmission occurs either through direct contact with infected animals or indirect contact with contaminated fomites or ingestion of infected pork products. Furthermore, soft ticks of the Ornithodoros genus are an important biological vector, in the regions where they are present, such as Africa (Dixon et al., 2004). In the last decade, the global situation alarmingly deteriorated with some 50 countries worldwide affected by the disease, putting at risk the pig industry. (World Organisation for animal Health, 2019a).

The introduction of ASFV genotype II in Georgia in 2007 initiated a new epidemic situation in Eurasia (Rowlands et al., 2008). The disease rapidly spread through the Caucasus, the Russian Federation and several countries of Eastern Europe. After the Czech Republic in 2017 (Depner et al., 2017), the ASFV was introduced via an unknown source of infection into the wild boar population of Belgium during the summer 2018 (Linden et al., 2019). The situation worsened substantially in August 2018, when the Republic of China reported the first outbreak of ASF, which rapidly spread throughout the country and Southeastern Asia (FAO, 2020). The contamination from wildlife is not sufficient to explain the spread of the virus throughout the world. International trade and transport of contaminated animals, pork products or waste, as well as poor biosecurity measures in the pig production sector are arguably the underlying causes of ASFV’s expansion (Chenais et al., 2019).

In the absence of a vaccine, the implementation of adapted policy instruments, such as biosecurity regulations, surveillance strategies and outbreak response policies, are important tools to prevent ASFV’s dissemination in order to protect animal health. These instruments are however only effective when they rely on solid diagnosis tests for ASFV detection (Dixon et al., 2020).

Haemadsorption test, virus isolation, antigen detection by FAT (fluorescent antibody test) or enzyme-linked immunosorbent assay (ELISA), as well as conventional and real-time polymerase chain reaction (real-time PCR) are the most widely used methods for ASFV diagnosis. Haemadsorption and virus isolation are sensitive and reliable confirmatory methods for the detection of infectious virus but are also laborious and not suitable for a rapid routine diagnostic. In addition, antigen detection methods are not sensitive enough in animals with low viremia levels and can be impaired in presence of antibodies (Sánchez‐Vizcaíno et al., 2019). Real-time PCR is currently preferred to the gel-based conventional PCR which is more time-consuming. Indeed, real-time PCR has been recognized to be the most rapid, sensitive and reliable method by the World Organisation for animal Health (2019b). This method relies on the amplification of conserved ASFV genome regions using primers and the detection of this amplification through the fluorescent emission of a specific probe. When working with poor-quality samples (i.e. field samples from dead wildlife), false negative results, due to damaged nucleic acids or the presence of inhibitors, are avoided by controls such as the amplification of housekeeping genes (Belák and Thorén, 2001).

Several conventional and real-time PCR methods for the detection of ASFV have been described in the literature (Agüero et al., 2003; Fernández-Pinero et al., 2013; King et al., 2003) and recommended by the OIE (World Organisation for animal Health, 2019b). Tignon et al. (2011) developed a sensitive and specific real-time TaqMan PCR assay avoiding false negative results by the inclusion of an internal endogenous extraction control amplifying the swine beta-actin gene. In addition, a previous assessment of ASFV diagnostic techniques (Gallardo et al., 2015) indicated the high sensitivity of the Universal Probe Library PCR (Fernández-Pinero et al., 2013).

In parallel, the recent ASFV introduction in Eastern Europe favored to the development of numerous commercial kits easily available on the European market.

Given the existence of such a variety of commercial diagnostic tools, there is a need for validation in order to provide confidence in these tools and assure the quality of the results. The aim of the present evaluation was to estimate the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases regarding the ASFV Belgium 2018/1 strain.