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

Abstract

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.

Assessment of abattoir based monitoring of PRRSV using oral fluids

“…Additionally, abattoir-based surveillance could be implemented to screen for antibodies of endemic and/or foreign diseases for which oral fluid antibody detection tests available. These include African swine fever virus (Mur et al, 2013;Giménez-Lirola et al, 2016), Erysipelothrix rhusiopathiae (Giménez-Lirola et al, Panyasing et al, 2014;Ciacci-Zanella et al, 2015;Hughes et al, 2015;Panyasing et al, 2016), porcine circovirus type 2 (Prickett et al, 2011), and porcine epidemic diarrhea virus (Ouyang et al, 2015;Bjustrom-Kraft et al, 2016). Further studies are needed to characterize herd sensitivity and specificity of oral fluids-based testing at the abattoir for each pathogen.…”

Gaps in African swine fever: Analysis and priorities

“…At the same time, non‐invasive sampling methods are lacking, which are especially important for ASF control in northern Europe. Samples obtained through non‐invasive sampling methods such as oral fluid and faeces allow ASFV and anti‐ASFV antibodies detection (Davies et al., ; De Carvalho Ferreira, Weesendorp, Quak, Stegeman, & Loeffen, ; Giménez‐Lirola et al., ; Mur et al., ; Nieto‐Pelegrín, Rivera‐Arroyo, & Sánchez‐Vizcaíno, ). Commercial tests based on oral fluid are already available for porcine reproductive and respiratory syndrome as well as sampling guidelines for oral fluid‐based survey on grouped‐housed animals (Rotolo et al., ).…”

“…Since then, ASF has spread from the Caucasus region (Georgia, Azerbaijan and Armenia) to the Russian Federation (2007), Ukraine (2012), Belarus (2013), Estonia (2014), Latvia (2014), Lithuania (2014), Poland (2014) and Moldova (2016), where it has affected domestic pigs and wild boar EFSA, 2015;Gallardo, Nieto, et al, 2015; S anchez-Vizca ıno, Mur, & Mart ınez-L opez, 2013; World Organisation for Animal Health, Wahid Database (OIE WAHID) Interface, 2017). The disease is currently endemic in some parts of eastern Europe (Gogin, Gerasimov, Malogolovkin, & Kolbasov, 2013). Transboundary movement of this disease has been historically related to the single introduction of contaminated pork or pork products used to pig feed (S anchez-Vizca ıno & .…”

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

“…The entire virus isolation detection process takes 48-72 hours, which is not ideal for the high-throughput and rapid detection in resource-limited settings. Enzymelinked immunosorbent assay (ELISA), a technique of recognizing viral antigens, has also been widely used for ASFV detection (Cubillos et al, 2013;Giménez-Lirola et al, 2016). ELISA does not require expensive instruments and complicated amplification processes (Giménez-Lirola et al, 2016).…”

“…Enzymelinked immunosorbent assay (ELISA), a technique of recognizing viral antigens, has also been widely used for ASFV detection (Cubillos et al, 2013;Giménez-Lirola et al, 2016). ELISA does not require expensive instruments and complicated amplification processes (Giménez-Lirola et al, 2016). However, ELISA has poor sensitivity and relies on fragile temperaturesensitive reagents (Gallardo et al, 2015).…”