Diagnosis of ASF means the identification of animals that are, or have previously been, infected with ASFV. An appropriate diagnosis therefore involves the detection and identification of ASFV-specific antigens or DNA and antibodies, to obtain relevant information to support control and eradication programmes.
AFRICAN SWINE FEVER (ASF) ANTIBODY DETECTION.
ASF-specific antibody detection is recommended for subacute and chronic forms as well as for large-scale testing and ASF eradication programmes, for several reasons:
- Antibodies are rapidly produced in the infected pig. In these pigs antibodies are usually detectable in serum samples from seven to ten days after infection;
- No vaccines are available against ASF. This means that ASF-specific antibodies are only induced by ASF virus infection;
- The long-lasting antibodies response. In pigs that have recovered from the disease, specific antibodies can be detected at high levels for many months or even for the lifetime of some of these pigs.
Specific ASF antibodies of maternal origin can be detected in piglets during the first weeks of life. The half-life of maternal antibodies in piglets is about three weeks. If antibodies are found in piglets older than three months, ASF antibodies are very unlikely to be of maternal origin.
Current ASFV antibody-based tests approved by the OIE involve the use of an ELISA for antibody screening, backed up by Immunoblotting (IB), Indirect Immunofluorescence (IIF) or the Indirect immunoperoxidase tests (IPT) as confirmatory tests (Gallardo et al., 2015; OIE 2019).
1. ASFV ANTIBODY DETECTION BY ELISA TEST.
Detection of specific antibodies against ASFV by ELISA is the OIE prescribed test for international trade so far. Currently there is a number of ASF ELISA variants including recombinant ELISAs (Gallardo et al., 2006, Gallardo et al., 2009; Pérez-Filgueira et al., 2006), and several (OIE) “in house” versions of the test based on the use of live virus as antigen. Three commercial ELISA kits are also available and validated for the detection of ASF antibodies based on the most antigenic proteins so far described such as p72, p32, pp62 and p54 (INGENASA, IDVET and SVANOVIR), of which the INGEZIM PPA COMPAC, K3 from INGENASA is the most widely used at EU level (EURL data gained from the annual interlaboratory comparison tests).
A cheaper alternative is to prepare a soluble antigen for use in an indirect ELISA, and procedure using this soluble antigen described in the World Organization for Animal Health in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Chapter 3.8.1 (OIE, 2019) and in this section. The OIE indirect ELISA is based on the use of semipurified cytoplasm soluble antigen obtained after the infection of cell cultures with ASFV. The Spanish strain of ASFV isolated in 1970 (E70) and adapted to grow in a monkey stable cell line (MS) is the virus used for antigen production. The antigen is fixed in the plate. Samples with antibodies against ASF will recognize antigen so an antigen-antibody complex will be formed. After that, the conjugate is added and fix with the antigen-antibody complex. With several washing steps, all material not fixed is removed. Adding substrate we can obtain de result of the technique: develop of color in wells, indicates ASF antibody presence.
The OIE indirect ELISA test for ASF has high specificity and sensitivity to allow confident diagnosis of ASF independent of the viral genotypes circulating in a particular region (Gallardo et al., 2013).
This indirect ELISA procedure has been improved and validated and has higher sensitivity than that obtained with the previous procedure for serum samples collected at earlier stages of infection, by adjusting the incubation time, incubation temperatures, buffers, concentrations of the antigen and the samples, as well as the type and concentration of the conjugate and substrate. These modifications are available at the OIE Manual for ASF diagnosis, 2019 edition.
2. asfv ANTIBODY DETECTION BY THE IMMUNOBLOTTING (IB) TEST.
Immunoblotting (IB) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization, electrophoretic separation, and transferring of proteins onto membranes (usually nitrocellulose). The membrane is overlaid with a primary antibody for a specific target and then with a secondary antibody labeled.
For the preparation of the ASF IB strips, the ASFV viral proteins, electrophoretically separated in SDS-PAGE gels, are transferred with a constant current intensity to the nitrocellulose filter. The filter is then cut into strips, which are blocked to sature the remaining protein binding sites. After blocking, the serum is added to allow the antibodies to react with the antigen strip. In the case of specific antibodies against ASF are present in the serum sample, the resulting inmunocomplexes will be visualized by addition of an A-peroxidase conjugate protein, and 4-chloro-1-naphtol as substrate.
The ASF-IB it is the recommended test by the World Organization for Animal Health (OIE, 2019) in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals for the confirmation of positive and doubtful samples by ELISA tests and also in case of sera incorrectly handled or bad preserved (inadequate storage or transportation) when simple analysis by ELISA may yield up to a 20% false-negative results. The technique has been fully validated over time with a sensitivity and specificity values of 98% and provides and increased ability to detect infected animals without clinical symptoms (carriers).
The OIE IB test for ASF test has high specificity and sensitivity to allow confident diagnosis of ASF independent of the viral genotypes circulating in a particular region (Gallardo et al., 2013).
Despite this method being highly sensitive, in ASF-endemic areas, where chronically infected animals of subclinical infections are present, non-specific characteristic pattern could be visualized, hindering the interpretation of the results. In this situation, an accurate evaluation of the results should be performed taking into consideration alternative confirmatory serological diagnostic tests such as IIF or IPT.
3. ASFV ANTIBODY DETECTION BY INDIRECT IMMUNOPEROXIDASE TEST (IPT).
ASFV naturally infected immune-system cells, monocytes-macrophages. The persistence of ASFV experimentally induced in Vero and MS (monkey stable) cells has been described through the action of CINH4 and 5-iodo-2’-desoxiuridina. The ASFV multiplies in the cytoplasm of the cell requiring the cell nucleus to do it. The entry in the cell is by endocytosis with the formation of vesicles in which several virions are enclosed fused their envelopes with the membrane endosome releasing to the cytoplasm cell. In Vero cell cultures this step seems to be associated with a receptor. The complete intracellular virus migrates to the membrane cell and it is released from the cell with a new cell envelope with viral proteins.
The immunoperoxidase technique (IPT) is an immune-cytochemistry technique on fixed cells to determine the antibody-antigen complex formation through the action of the peroxidase enzyme. In this procedure, Vero or MS cells are infected with ASFV adapted isolates to these cell cultures. The infected cells are fixed and are used as antigens to determine the presence of the specific antibodies against ASF in serum samples.
The IPT has been proved as the best test for ASF serological diagnosis due its superior sensitivity, but moreover, its performance to test any kind of porcine material such as blood, exudate tissues or body fluids (Gallardo et al., 2015). This is particularly relevant for wild boar surveillance and control programs. Currently the IPT technique is the selected confirmatory test at the EU NRLs (Nieto R., personal communication 2018), and is the recommended test by the World Organization for Animal Health (OIE, 2019) in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals for the confirmation of positive and doubtful samples by ELISA tests.
4. ASFV ANTIBODY DETECTION BY INDIRECT IMMUNOFLUORESCENCE TEST (IFI).
The IFI for ASF is a common serological diagnostic method, where the sensitivity of the histological techniques and the specificity of immunological techniques are combined.
This method uses fluorescein isothiocyanate conjugated against the immunoglobulins present in a sample serum, showing either immunological or fluorescent activity.
AFRICAN SWINE FEVER VIRUS (ASFV) DETECTION.
1. ASF VIRUS ISOLATION IN PORCINE PRIMARY CELL CULTURES..
Malmquist and Hay made one of the most important advances in the study of African swine fever virus (ASFV) in 1960 demonstrating that ASFV was able to infect and replicates in primary leukocyte cultures from pig peripheralblood. Virus isolation is based on the inoculation of sample material on susceptible primary cell cultures of porcine origin, monocytes and macrophages cells. If the ASF virus is present in the sample, it will replicate in the cells and the cytopathic effect (C.P.E) will be produced in the infected cells.
When the virus replicates in these cultures, the majority of the ASFV strains produced the haemadsorption reaction (HAD) due to adsorption of pig red blood cells on ASFV infected leukocytes. Cell lysis and C.P.E. usually occurs after 48-49 hours of haemadsorption. The importance of this finding relies on its specificity because none of the other pig viruses are capable of haemoadsorbing in leukocyte cultures.
Virus isolation and identification by HAD are recommended as a reference test for the confirmation of positive results of a prior antigen ELISA, Polymerase chain reaction (PCR) or Direct immunofluorescence tests (DIFT). They are also recommended when ASF has already been confirmed by other methods, particularly in case of a primary outbreak or case of ASF.
2. ASFV GENOME DETECTION BY THE POLYMERASE CHAIN REACTION (PCR).
Currently, the PCR is considered the ‘gold standard’ test for early detection of the disease due to its superior sensitivity, specificity, robustness and high-throughput application to detect the ASFV genome in any kind of clinical samples from domestic pigs, wild boar and ticks (Gallardo et al., 2015; Oura et al., 2013).
Over the last twenty years, a variety of PCR tests, including both conventional and real time (rPCR), have been developed and validated to detect a wide range of ASF isolates belonging to different known virus genotypes, non-haemadsorbing strains, and diverse virulence (Agüero et al, 2003; Fernández-Pinero et al, 2013; King et al, 2003; Tignon et al, 2011; Zsak et al, 2005). All of them have been designed in the VP72-coding region, a highly conserved gene coding the major viral protein, assuring the (potential) detection of any ASFV isolate. The OIE rPCR (King et al., 2003; OIE 2019) and the Universal probe library (UPL)OIE- rPCR (Fernández-Pinero et al., 2013; OIE 2019) are the most widely used for routine diagnosis at the EU´s national reference laboratories (NRLs) level (Nieto R., personal communication 2018). Both methods are able to provide a confident ASF diagnosis, although the UPL-PCR has greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease even when the typical clinical signs are not yet evident (Fernández-Pinero et al., 2013; Gallardo et al., 2015; Gallardo et al., 2019).
With regards to the the OIE-conventional PCR test (Agüero et al., 2003) it is important to point out that this PCR shows lower sensitivity than expected in the detection of the circulating ASFV genotype II strains responsibles of the current epidemic outbreaks in Asia and in Europe. This is due to a nucleotide mismatch close to the 3’ end of the reverse primer existing in the circulating viruses (Gallardo et al., 2015; Gallardo et al., 2019).
3. ASF ANTIGEN DETECTION BY DIRECT IMMUNOFLUORESCENCE TEST (DIF).
The DIF can be used to detect ASFV antigen in tissues of suspect pigs. The principle of the test is the microscopic detection of viral antigens on impression smears or thin cryosections of organ material from pigs suspected of being infected with ASFV. Intracellular antigens are detected using FITC-conjugated specific antibodies. Fluorescent inclusion bodies or granules appear in the cytoplasm of infected cells. DIF can also be used to detect ASFV antigen in leucocyte cultures in which no HAD is observed, and can thus identify non haemadsorbing strains of virus. It also distinguishes between the cytopathic effect (CPE) produced by ASFV and that produced by other viruses, such as Aujeszky’s disease virus or a cytotoxic inoculum.
This is a highly sensitive test for cases of peracute and acute ASF. However it is important to note that in subacute and chronic disease, the DIF has a significantly decreased sensitivity (40 %), related to the presence of antigen-antibody complexes in the tissues of infected pigs, which block the reaction with the ASF-conjugated antibody.
4. ASF ANTIGEN DETECTION BY ANTIGEN ELISA TEST.
A number of “in house” antigen ELISAs, including direct, indirect and sandwich ELISA formats, which employ monoclonal and polyclonal antibodies, were developed in the past although are not currently in use (Hutchings and Ferris, 2006; Vidal et al., 1997; Wardley et al., 1979). A commercially produced antigen ELISA kit is the only currently available (ELISA INgezim PPA DAS, Ingenasa, Spain), which allows the use of tissue and serum samples for the analysis. It is a rapid test and easy to scale up. However, comparative testing of field-derived samples showed a poor sensitivity (77.2%) of the commercially antigen ELISA test when compare to the real time PCR test even when there was a high virus load (Gallardo et al., 2015; Gallardo et al., 2019; Oura et al, 2013: Sastre et al., 2016). Consequently, the use of antigen ELISA is only recommended as a herd assay and should be combined with some other virological and serological tests.
AFRICAN SWINE FEVER VIRUS (ASFV) GENOTYPING.
The molecular epidemiology of the disease is investigated by sequencing of the 3’ terminal end of the B646L open reading frame encoding the p72 protein major capsid protein (Bastos et al., 2003), which differentiates up to 24 distinct genotypes (Achenbach et al., 2017; Boshoff et al., 2007; Quembo et al. 2018). To distinguish subgroups among closely related ASFV, sequence analysis of the tandem repeat sequences (TRS), located in the central variable region (CVR) within the B602L gene (Gallardo et al., 2009; Lubisi et al., 2005; Nix et al., 2006) and in the intergenic region between the I73R and I329L genes, at the right end of the genome (Gallardo et al., 2014), is undertaken. Several other gene regions such as the E183L encoding p54 protein, the CP204L encoding p30 protein, and the protein encoded by the EP402R gene (CD2v), have been proved as useful tools to analyse ASFVs from different locations and hence track virus spread.