African swine fever

[See: OIE reports on African swine fever for an overview of the latest situation.]

ASF is widespread in Africa, particularly south of the Sahara, where the disease is mostly endemic. Thirty-four countries in sub-Saharan Africa have experienced at least one confirmed ASF outbreak (Penrith, 2020).

In Europe, ASF is endemic in Sardinia (Italy). In 2007, ASF entered Eastern Europe. ASF was detected in Georgia, near the port of Poti (Beltrán-Alcrudo et al., 2008) and from there, the disease quickly spread to neighbouring countries (Armenia, Azerbaijan and Russian Federation) and subsequently to Ukraine, Belarus and Moldova. ASF spread into four EU member countries in 2014, namely Lithuania, Poland, Latvia, and Estonia; and in 2017, ASF was reported for the first time in Czech Republic and Romania (Jurado et al., 2018). In 2018, ASF was detected in wild boar in Hungary, Belgium and Bulgaria. In 2019, the disease was reported in Slovakia and Serbia and in February 2020 it was reported in Greece (Penrith, 2020). Germany reported ASF in wild boar in September 2020 (for more information, see OIE’s World Animal Health Information Database (WAHIS) Interface).

The first case of ASF in China was reported in August 2018 and the disease has spread rapidly (Li and Tian, 2018). ASF was first reported in Mongolia in January 2019, Vietnam in February 2019, Cambodia in April 2019, Democratic People’s Republic of Korea in May 2019, Hong Kong in May 2019 and Laos in June 2019. The disease has also spread to the Philippines, Myanmar, Indonesia, Papua New Guinea and Timor-Leste (Penrith, 2020). Deaths among village pigs in north-eastern India commenced in the Arunachal Pradesh district in late January 2020, with outbreaks occurring in 11 villages in Arunachal Pradesh and Assam between January and April, with the deaths of 3701 pigs. The cause of the deaths was confirmed to be a genotype II ASF virus on 18 May 2020 (Penrith, 2020).

Introduction

Due to the great similarity of the clinical signs and lesions of ASF and those of other haemorrhagic pig diseases, laboratory diagnosis is an essential prerequisite for correct diagnosis (Sánchez-Vizcaíno, 2006).

Clinical Diagnosis

The clinical presentation varies depending on the virulence of the virus, the route of exposure, dose of virus and the species of pig infected, normally wild boar are more resistant (Sánchez-Vizcaíno, 2006).

Three forms of ASF have been described: acute, sub-acute and chronic.

The isolate currently circulating in the Russian Federation and Caucasus region belongs to genotype II (Rowlands et al., 2008) and only induces acute forms of the disease. Other forms of the disease can be observed in Sardinia (Italy) where ASF virus genotype I is circulating or in Africa where all the 22 ASF virus genotypes are circulating (Sánchez-Vizcaíno, 2006).

Acute form

Acute infections are characterised by high mortality (90-100%), fever (40-42ºC), leucopaenia and thrombocytopaenia. Reddening of the skin at the tips of ears, and chest and abdominal areas are frequently observed in white pigs. Vomiting and haemorrhagic diarrhoea may be observed. Abortion in pregnant sows is frequently described. One or two days before dying, the affected animals usually present anorexia, listlessness, cyanosis and incoordination (Mebus et al., 1983).

Sub-acute form

Sub-acute infections are produced by moderately virulent virus isolates, which present similar but less intense symptoms than those in the acute form. The mortality of the sub-acute form is approximately 30-70%, depending on the virus isolate. Illness and abortion is frequently observed.

Chronic form

Chronic infections result in low mortality (2-10%). Symptoms include weight loss, respiratory problems, arthritis, chronic skin ulcers or necrosis.

Lesions

See Pathology.

Differential Diagnosis

Differential diagnosis should consider the following diseases:

• classical swine fever or hog cholera


• erysipelas


• salmonellosis


• pasteurellosis

Laboratory Diagnosis

The samples that should be collected for ASF laboratory diagnosis are lymph nodes, kidneys, spleen, lung, blood and serum. Tissues are used for virus isolation (HA test) and viral antigen detection (PCR techniques and DIF test), while blood is used for virus isolation and PCR. Tissue exudates and serum are used for antibody detection by: IIF, ELISA or IB.

A wide variety of laboratory tests are available for ASF viral DNA and antibody detection (Sánchez-Vizcaíno, 2006). The most convenient, safe and specific tests for virus detection are: PCR techniques (both real time and conventional) (King et al., 2003; Agüero et al., 2004), direct immunofluorescence (DIF) (Bool et al., 1969) and haemadsortion (Malmquist and Hay, 1960).

The detection of the ASF virus genome by PCR has been developed with the use of sets of primers from a highly conserved region of the viral DNA, to detect all range of ASF isolates belonging to all the known virus genotypes including both non-haemadsorbing viruses and low virulence ones. This test is particularly useful for viral DNA identification from tissues which are poorly conserved, even if they have undergone putrefaction or the virus has been inactivated. It is an excellent and relatively rapid technique (results could be obtained in 5 hours) for ASF diagnosis, and is the most frequently technique used in worldwide laboratories for ASF virus detection. It needs good training and good laboratory practices to avoid contaminations and false positive results . Two types of PCR are validated by OIE for ASF diagnosis, conventional PCR (Agüero et al., 2003) and real-time PCR (King et al., 2003).

Direct immunofluorescence (DIF) is based on the detection of viral antigen in impression smears or frozen tissue sections with a fluorescein labelled immunoglobulin directed against the ASF virus. It is a very rapid (one hour) and economic test with high sensitivity to the acute form of ASF. However, for sub-acute or chronic infections, the DIF test has a sensitivity of only 40%. This decrease in sensitivity seems to be related to the formation of antigen-antibody complexes, which do not facilitate the reaction with the ASF conjugate (Sánchez-Vizcaíno, 1986). The use of DIF together with an indirect immunofluorescence test (IIF) makes it possible to detect 85 to 95% of all ASF cases (acute, sub-acute and chronic) in less than three hours (Sánchez-Vizcaíno, 1986).

The haemadsorption test (HA) is a technique used as the gold standard test for ASF virus identification due to its sensitivity and specificity. This test is usually only performed in ASF reference laboratories in order to confirm any new outbreak and when other tests have yielded negatives. HA is based on the haemadsorption characteristics that most ASF virus isolates induce when pig macrophages are infected in the presence of erythrocytes. A characteristic rosette around the infected macrophages develops before the cytopathic effect appears. A small number of field strains have shown cytopathic effect without producing the haemadsorption phenomenon (Sanchez Botija, 1982); these strains are identified using the DIF test on the sediments of these cell cultures. HA is relatively economical but access to ASF-free pigs and sterile facilities are also needed.

The lack of an effective vaccine against ASF virus and the long duration of the specific ASF-IgG reaction in infected pigs (detectable in blood on days 6-10 post-inoculation and subsequently for protracted periods, even years) has made the study of ASF antibody reactions an important prerequisite for the detection of sub-acute and chronic forms of ASF, and for ASF eradication programs (Arias and Sánchez-Vizcaíno, 2002). Several techniques have been adapted for ASF antibody detection, but the most common, practical and inexpensive tests are ELISA (Sánchez-Vizcaíno et al.,1986), immunoblotting (IB) (Pastor et al., 1987) and IIF (Bool et al., 1969).

The IIF test is a fast technique and has high sensitivity and specificity for the detection of ASF antibodies from either sera or tissue exudates (Sanchez Botija et al., 1970). It is based on the detection of ASF antibodies that bind to a monolayer of cell lines (by MS) infected with an adapted ASF virus. The antibody-antigen reaction is detected by a fluorescein labelled protein A. ELISA testing is the most useful method for large-scale serological studies. It is highly sensitive and specific, simple, rapid and economic and commerical kits are available. It is based on the detection of ASF antibodies bound to the viral proteins which are attached to a solid phase by addition of protein A-conjugated with an enzyme that produces a visible colour reaction when it reacts with the appropriate substrate.

The IB test is a highly specific, sensitive and easy to interpret technique which has been successfully used as a confirmatory method to IIF for low or doubtful ELISA sera (Sánchez-Vizcaíno, 2006).

Immunology

The immune response to ASF virus infection is still poorly understood. The main difficulty encountered has been the lack of neutralising antibodies and the great variability of the virus isolates. These characteristics made the production of an effective vaccine impossible to date.

Monocytes and macrophages are the main target cells for ASF virus replication; no evidence of virus replication in T or B lymphocytes has been observed (Minguez et al., 1988; Gomez-Villamandos et al., 1995). However, a lymphopenia, due to apoptosis of lymphocytes, mainly in the T area of the lymphatic organs, have been described (Carrasco et al., 1996).

Another characteristic of the response to ASFV is the lack of viral neutralisation. ASF virus-specific neutralised antibodies have never been demonstrated to entirely fulfil the classic definition of antibody neutralisation. It has proved impossible to neutralise 100% of the homologous virus, even with sera from recovered animals infected with attenuated ASF isolate; in this case a 10% fraction of non-neutralising virus is observed to persist (Ruiz et al., 1986). In contrast, animals that have recovered from ASFV infections can produce neutralised antibodies to foot and mouth virus (De Boer, 1967), and T cytotoxic lymphocytes (CD 8+) from recovered pigs are able to destroy macrophages infected with ASFV (Martin and Leitao, 1994).

ASF virus is very antigenic, inducing antibodies to a great number of viral proteins. IgM is detectable in animal sera at 4-6 days post inoculation and IgG is detectable at 6-9 days post inoculation with low or medium virulent ASF isolates (Sánchez-Vizcaíno, 2006).

Humoral and cell-mediated immunity does not seem to be affected in pigs infected with ASF virus.

As no vaccine for ASF is available, the control of this disease is based on rapid laboratory diagnosis and the enforcement of strict sanitary measures. Depending on the epidemiological status of disease in a particular region, different measures are recommended.

Epizootiological studies have shown that the most frequent source of ASF contamination in infection-free countries is refuse from international airports or ports. All leftover food from aeroplanes and ships should be routinely incinerated or efficiently sterilised. Import policy for animals and animal products should consider the disease status of the exporting nation. In infected European areas such as Sardinia (Italy) where the disease is enzootic and where mild or non-apparent clinical signs can be observed, the most important aspects of ASF prevention are the control of animal movement and the use of extensive serological surveys to detect carrier pigs. In endemic areas of Africa, the most important factor is to control the natural tick vectors and wild pig reservoirs, and/or limit their contact with domestic pigs.

During disease outbreaks, the rapid and efficient slaughtering of all pigs and the proper disposal of carcases and all waste material is critical. Other important aspects to consider are the cleaning and disinfecting of affected farms, designation of the infected area and increased control of animal movements. Serological surveys should be undertaken in the surrounding area. In the presence of any suspicious haemorrhagic pig disease, a differential laboratory diagnosis should be undertaken; as low virulence ASF strains do not produce significant lesions.