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.
1. AFRICAN SWINE FEVER ANTIBODY DETECTION BY INDIRECT ELISA TEST.
The enzyme immunoassay ELISA test is a very useful technique widely used for the serological diagnosis of different animal diseases. Some of the most outstanding characteristics of this method are high sensitivity, specificity indexes, high speed and low cost. Large populations can be studied in short periods of time, thanks to the automatic equipment that today is available. This technique also provides easy interpretation of the results.
The technique more used for specific antibody detection of ASF is the ELISA, which is based in the use of antibodies or antigens labeled with an enzyme, so the resulting conjugates have enzymatic and immunologic activity. Being one of the components (antigen or antibody) labeled with an enzyme and insolubilized, the antibody-antigen reaction will be immobilized and it is easy develop by addition of specific substrate that could be read in a spectrophotometer.
The indirect ELISA is the recommended test for ASF antibody detection by the World Organization for Animal Health (OIE, 2012) in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The technique has been fully validated over time with sensitivity and specificity values of 95.8% and 97.3% respectively. Large number of samples can be tested in a short time with this method.
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.
Recent studies (Gallardo et al., 2013) have demonstrated that 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.
2. AFRICAN SWINE FEVER 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, 2012) 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).
Recent studies (Gallardo et al., 2013) have demonstrated that the OIE IB test for ASF has high specificity and sensitivity to allow confident diagnosis of ASF independent of the viral genotypes circulating in a particular region.
3. AFRICAN SWINE FEVER 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 fully validated at the EURL as alternative confirmatory test for antibody detection to ASF infection either in serum and exudate tissue samples or in a large-scale survey of ASF. The sensitivity and specificity values of 98.20% and 98.95%, respectively are comparable to Inmunoblotting (IB) and Indirect immunofluorescence assays (IFI).
4. AFRICAN SWINE FEVER 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. AFRICAN SWINE FEVER (ASF) VIRUS ISOLATION.
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. AFRICAN SWINE FEVER (ASF) VIRUS GENOME DETECTION BY THE POLYMERASE CHAIN REACTION (PCR).
The PCR is applied to detect the ASFV genome in blood, serum, organ samples and ticks. Small fragments of viral DNA are amplified by PCR to detectable quantities. The PCR provides a sensitive, specific and rapid alternative to virus isolation for the detection of ASFV, allowing the viral detection even before the appearance of clinical symptoms in the infected animals and provides much higher sensitivity and specificity than alternative methods for antigen detection, such as antigen ELISA and DIF assays. PCR enables the diagnosis of ASF to be made within hours of sample receipt so that control measures and restrictions can be implemented or lifted in a much shorter time scale than if virus isolation was employed as the sole diagnostic method.
Conventional (Aguero et al., 2003) and real time (King et al., 2003) PCRs recommended by the World Organization for Animal Health (OIE, 2012) in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals have been fully validated over time and are useful tools for routine diagnosis of the disease. Recently, new developed real time PCR procedures (Fernández-Pinero et al., 2012 and Tignon et al., 2011) have proved to provide higher sensitivity than OIE prescribed real time PCR method in ASFV genome detection in carriers’ animals. Primer sets and probes used in these molecular techniques are repeatedly designed within the VP72 coding region, a well-characterized and highly conserved region of the ASFV genome. A wide range of isolates belonging to all the 22 known p72 virus genotypes including both non-hemadsorbing and low virulent virus isolates can be detected with these PCR assays, even in inactivated or degraded samples.
The PCR is the tool of choice in case of hyperacute, acute or subacute infection with ASF. Furthermore, since the PCR detects viral genome it may be positive even when no infectious virus is detected by virus isolation so is a very useful tool for the detection of ASFV DNA in pigs infected with low or moderately virulent strains.
3. AFRICAN SWINE FEVER (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. AFRICAN SWINE FEVER (ASF) ANTIGEN DETECTION BY ANTIGEN ELISA TEST.
Viral antigens can also be detected using ELISA, but it is only recommended for acute forms of disease. As in the case of the DIF, that in subacute and chronic disease, the antigen ELISA has a significantly decreased sensitivity. This is likely to be due to antigen–antibody complexes in the tissues of infected pigs blocking the interaction between the ASFV antigen and ASF conjugate. It is therefore recommended to use the antigen ELISA only as a ‘herd’ tests and in conjunction with other virological tests.
AFRICAN SWINE FEVER VIRUS (ASFV) GENOTYPING.
The current approach for ASFV genotyping is based on the analysis of three independent regions located at the conserved central area of the ASFV genome comprising; i) partially sequence of the C-terminal end of the gene B646L encoding the major protein p72 (Bastos et al., 2003) which allow us to classify the ASFV in 22 major genotypes (Boshoff et al., 2007), ii) to sequence the full E183L-gene encoding the p54 protein (Gallardo et al., 2009) as a valuable additional genotyping method for molecular epidemiological studies of p72 genotype I viruses, particularly in West Africa where this genotype predominates, and iii) the sequencing of the central variable region within B602L-gene (CVR) characterized by the presence of amino acid tandem repeats (Nix et al., 2006; Gallardo et al., 2011). The CVR remains the genome target of choice when attempting to determine the origin and map the spread of closely related virus.