BioSecurity Report by dr. Sandor Kecskeméti

Report on the Hungarian molecular biological studies based on samples taken from wild boars and food Prepared by: dr. Kecskeméti Sándor


Report on the Hungarian molecular biological studies based on samples taken from wild boars and food

African swine fever is an acute disease of domestic pigs and European wild boars, characterized by fever, haemorrhages, and in many cases, results in high mortality. It was first described in Kenya in 1921 and from there, it spread throughout Africa. From Africa, it was imported multiple times to Europe, Central and South America, and then to Georgia in 2007, from where it started and has caused the biggest pandemic in recent times. The African swine fever virus belongs to the Asfarviridae family, within the Asfivirus genus. It has a complex structure, large, multi-layered, with icosahedral symmetry, and is enveloped. Its natural reservoirs are the Ornithodoros ticks and wild pigs living in Africa.

The virus is resistant to environmental effects. From the perspective of spreading the infection, particularly important are raw meat, frozen meat, raw smoked, salted, marinated meat products, undercooked or poorly cooked foods, swill, food leftovers, and kitchen waste. The virus strains can be classified based on their virulence, genetic properties, and serological behaviour.

In Hungary, the first case was identified in 2018 in a dead wild boar in Heves County. The isolate showed 99-100% similarity to the Georgia 2007 strain. The virus gradually spread among wild boars, reaching as far as Fejér County. Since the case in Fejér County, the infection has not spread to other counties, so the risk classification was downgraded, and other areas were exempted from restrictions. African swine fever has not been identified in domestic pigs in Hungary.

The hosts of the virus are the warthog (Phacochoerus africanus), the giant forest hog (Hylochoerus meinertzhageni), the bushpig (Potamochoerus sp), the Ornithodoros ticks, the European wild boar, and the domestic pig. Infection among animals and herds can occur through direct contact with an infected, sick animal, or indirectly. The organs contain the virus at high titers; 1 gram of spleen tissue can contain up to 1012 viruses, and 1 ml of blood can contain 108 viruses. The epidemiology of the infection varies by region, and different infection cycles (African cycle, tick-domestic pig cycle, domestic pig cycle) are distinguished. Since 2014, in Eastern Europe, a cycle associated with European wild boar habitats that is different from the previous ones has been observed. In a "normal case", the epidemic spreads slowly among wild boars (1-2 km/month). Among domestic pigs, or from wild boars to domestic pigs, it spreads with human intervention, by violating epidemic protection rules. The antibodies produced as a result of the infection do not have a sufficient virus-neutralizing effect. Despite intensive vaccine development experiments, an effective and safe commercial vaccine is not yet available.

High virulence strains cause the hyper acute and acute forms of the disease, medium virulence strains lead to the acute and sub acute forms, while low virulence strains result in the chronic form of the disease. In the acute form, animals lose their appetite, become lethargic, and their body temperature reaches 40-42°C. The skin becomes flushed, turns cyanotic (bluish), and small necrotic areas and subcutaneous hemorrhages can be seen. Animals suffering from the acute form have a 90-100% mortality rate within a week of the first symptoms appearing. A hyperemic enlargement of the spleen is very characteristic of the disease. Hemorrhages can occur in the lymph nodes, the renal cortex, and the mucous membrane of the urinary bladder. The low virulence strains cause a chronic form of the disease with nonspecific symptoms.

African swine fever cannot be definitively diagnosed based solely on clinical and/or pathological examination. Therefore, laboratory tests are essential for accurate disease diagnosis and for making successful control measures. Direct methods are available for the detection of the causative agent of the disease, while indirect methods are used to detect the antibodies produced. From the perspective of controlling the disease, detecting the virus is of greater significance a few days after infection at the onset of viremia, while detecting antibodies becomes more important in the later stages of the infection. Virus positivity with antibody negativity indicates a current, fresh infection; virus positivity with antibody positivity indicates an ongoing infection, whereas antibody positivity with virus negativity indicates a past infection.

For the determination/exclusion of African swine fever, samples recommended by the European Union's African Swine Fever Reference Laboratory and the WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2021 Chapter 3.9.1 should be examined. For diagnosis, the laboratory examination of the tonsils, submandibular, retropharyngeal, preauricular lymph nodes, mesenteric lymph node, spleen, kidney, lung, ileum, tubular bone, sternum, native blood sample, and EDTA-anticoagulated blood sample is required. For domestic pigs, it is recommended to remove the spleen or a part of it through a cut made along the left rib arc, while for wild boars, sampling using a swab from the gunshot channel is also suggested. Superficial inguinal lymph nodes, dried blood samples on Whatmann 903 filter paper or "dried blood spot sampling", FTA cards (Flinders Technology Associates), and dried swab samples can also be suitable for examinations. African swine fever has no public health implications; therefore, the examination of food samples is primarily of scientific importance.

Methods for virus detection

The causative Asfivirus can be isolated. The virus can be detected from the organs of the infected animal using immunofluorescence, its antigens can be identified using antigen-detection ELISA rapid tests (pen-side test, lateral flow devices), and its nucleic acid can be detected using real-time polymerase chain reaction (real-time PCR) and gel-based PCR reaction. Hemadsorption or immunofluorescence staining supplemented virus isolation is a sensitive confirmatory method for detecting infectious virus, but it is unsuitable for a large number of routine examinations. In everyday laboratory diagnostics, due to its high sensitivity and specificity, the potential for automation, and mass testing capabilities, real-time PCR has become widespread.

Detection of the ASF virus using the real-time PCR method

The PCR test can be completed within a few hours (a maximum of one working day), allowing for immediate implementation of epidemic control measures. Virus cultivation is not necessary, but the procedure requires separate rooms, a laboratory equipped with special instruments, and trained personnel. PCR can detect strains belonging to all 24 genotypes. PCR can still yield positive results even when infectious virus can no longer be detected through virus isolation. The costs of the tests can be reduced by pooling samples to some extent without significantly decreasing the sensitivity of the reaction.

Due to the high sensitivity of PCR, special attention must be paid to both false positive reactions and false negative results. False positivity can arise from an ASF virus-containing sample, cross-contamination from nucleic acid extraction control or the positive control, and contamination from reagents and equipment in the laboratory. To avoid false positives during PCR, different stages of the test should be conducted in well-separated rooms, and workflows should be organized in one direction in accordance with the increasing level of DNA exposure. Disposable gloves, sterile single-use filtered pipette tips, and generally sterile single-use instruments should be used. In the room where the master mix is prepared, clean protective clothing (not used for other workflows) must be worn. Instruments, equipment, and surfaces must be continuously disinfected and decontaminated. Mistakes during sample collection, especially when done in large numbers on-site, can lead to false positive results. False results (but not false PCR reactions!) can be caused by contamination of the collector's clothing or gloves during sample collection, failure to decontaminate the used tools (knife, scissors, scalpel), and incorrect sample collection techniques.

Detection of the infection using serological methods

In animals that have been infected, antibodies appear which can be detected for a long time, even for years. In Europe, due to the absence of a vaccine and vaccination, seropositivity clearly indicates past exposure. Antibodies can be detected using ELISA, immunoblotting techniques, indirect fluorescent antibody testing, indirect immunoperoxidase tests, and rapid antibody tests. Infections caused by the virulent genotype II virus result in acute disease, so during the current outbreak, animals typically die before the appearance of antibodies. In practice, due to their simplicity, speed, and automation capabilities, ELISA methods have become the most widespread.

The study details the most important steps of the examinations and disinfection procedures used in 5 annexes. (Annex 1: Sample preparation, homogenization, inactivation, heated-off board lysis nucleic acid extraction (IndiMag Pathogen kit for KingFisher heated OFF-BOARD lysis nucleic acid extraction), Annex 2: Detection of the ASF virus using real-time PCR method (Virotype ASFV 2.0 PCR Kit), Annex 3: Detection of antibodies against the African swine fever virus using ELISA (Ingezim PPA Compac K3 ELISA test), Annex 4: Detection of antibodies against the African swine fever virus using ELISA (ID Screen® African Swine Fever Indirect Confirmation Test), Annex 5: Disinfection at Debrecen Immunological, Virological, and TSE Laboratory).

The report contains a detailed presentation of work organization and biological safety rules pertaining to laboratories dealing with the examination of African swine fever (including the laboratory layout and equipment, disinfection, access to the laboratories, and special rules concerning the operation of the ASF laboratory). For the reliable inactivation of the pathogen, the sample is heat-treated in a thermo-block at 72°C for 30 minutes, and then it is submerged in a disinfectant solution for 15 minutes in the transfer window before being removed.

The study outlines the EU and Hungarian regulations, instructions, and guidelines related to African swine fever, its diagnosis, and laboratory examinations.



2023. 11. 22.

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