Classic swine fever. How African swine fever spreads and how to deal with it African swine fever quarantine

African swine fever is a viral disease characterized by high contagiousness and acute course. It can cause the imminent death of the entire population of pigs. Initially, the disease affected wild boars, but subsequently the virus began to spread to domestic pigs.

General characteristics of the disease

African plague is also known as Montgomery's disease - after the name of the researcher who proved its viral nature. This is an infectious process in which inflammatory processes develop, fever occurs, and the blood supply to the internal organs stops.

The disease-causing DNA-containing virus of the Asfarviridae family spreads to the entire population, regardless of the age of the pigs.

In individuals who died from this disease, the following pathological changes are observed in the body:

• multiple lesions of the connective tissue;
• multiple sources of hemorrhage;
• severe pulmonary edema;
• an increase in the size of the spleen, kidneys, liver gland;
• serous-hemorrhagic fluid in the respiratory system and in the stomach;
• content of blood clots in the lymph.

The virus that causes this severe disease is resistant to external conditions. It survives temperature extremes, multiplies when it dries, crystallizes and rots. Also, the virus is resistant to formalin and alkaline environments, but is sensitive to acids.

In pickles and smoked meats, this virus can persist for several weeks or months. In the feces, it remains active for about 160 days, in the urine - up to 60 days. In soil, the virus can survive for 180 days, in bricks and wood - from 120 to 180 days. In meat, it remains for about 5-6 months, in the bone marrow - up to 6-7 months.

The first time a case of this formidable disease was registered in 1903 in South Africa. The infectious process spread to wild boars. Subsequently, the disease spread to many African countries in the southern part of the Sahara.

In the middle of the twentieth century, a case of African plague was registered in Portugal. This happened after meat products from Angola were brought into the country. Later, the infectious process spread to the territories of Spain, Cuba, France, Holland, and Malta.

In Russia, as well as Ukraine, Georgia, Armenia and Abkhazia, African swine fever was first detected in 2007.

The statistics of outbreaks of African plague by year is as follows:

• Kenya - 1921;
• Portugal - 1957 and also 1999;
• Spain - 1960;
• France - 1964, as well as 1967 and 1974;
• Italy - 1967, 1969, 1978-1984 and 1993;
• Cuba - 1971;
• Malta - 1978;
• Dominican Republic - 1978;
• Brazil - 1978;
• Belgium - 1985;
• Holland - 1986;
• Russia - 2007;
• Georgia - 2007;
• Armenia - 2007.

Analyzing the reasons for the rapid spread of infection, the researchers concluded that in most cases this is facilitated by contaminated food waste.

The plague was brought to Russia from Georgia. In turn, this virus spread in Georgia due to the misuse of waste from international ships that transported contaminated meat and products from it. The media covered information that the corpses of animals that died in this country were found in ordinary landfills, river banks and on the sea coast.

In areas that are considered stationary for African swine fever, there is a frequency of outbreaks: in Africa, this viral process occurs every 2-4 years, in Europe - every 5-6 years.

At the moment, this infectious disease of pigs is registered in 24 countries of the world.

Methods of transmission of the virus

The source of the virus is a sick pig. Also, African plague is transmitted from virus carriers, which can be people, insects, birds and animals.

This disease that affects domestic pigs is transmitted in the following ways:

• as a result of close contact of a sick animal with a healthy one: infection occurs through the oral cavity, skin, mucous membranes of the eyes;
• through contaminated food waste, as well as equipment intended for slaughtering pigs;
• from pets, birds, rodents, insects and people who were in the infected area - a slaughterhouse or a warehouse;
• through the bite of a tick-carrier of the virus;
• through vehicles that have been contaminated while transporting sick pets;
• through food waste that is added to the feed of pigs without having previously processed them appropriately.

The duration of the incubation period of the disease is about 5-10 days.

For the human body, this disease does not pose a danger, since it is not sensitive to the virus of this type. However, a person is able to act as a virus carrier and infect pigs through contact with them.

Symptoms of African swine fever

The disease can occur in three forms:

• Lightning. In this case, the disease develops in 2-3 days and inevitably ends in the death of the infected animal.
• Acute. This form of the disease is characterized by severe clinical manifestations.
• Chronic. This form is poorly manifested, it is very rare. Most often, this kind of African plague is observed among wild boars.

This pathology is characterized by the following manifestations:

• an increase in body temperature to 42 degrees, such indicators are kept until the moment of death of the animal;
• general depression;
• weakness;
• cough;
• serous conjunctivitis;
• increased thirst;
• lack of appetite;
• discharge of purulent masses from the nose and eyes;
• severe shortness of breath;
• paresis of the hind limbs;
• vomit;
• fever;
• swollen lymph nodes;
• exhaustion;
• discoloration of the skin on the abdomen and under the breasts to red or dark purple;
• constipation or bloody diarrhea;
• dysmotility;
• pinpoint hemorrhages in the lower abdomen, neck, ears.

Sick individuals are hammered into the far corner of the barn, constantly lying on their side. The tail of infected pigs unwinds. If African plague strikes pregnant sows, they spontaneously abort.

Some individuals can survive, but they remain carriers of the virus for a long time, therefore they threaten other animals. Immunity in this case is not developed: pigs that have had African plague become ill with it again.

Diagnostic methods

African swine fever can be identified by the characteristic symptoms of this infectious process, which manifest themselves externally.

The diagnosis is made in a complex manner, based on laboratory data, as well as the results of a pathoanatomical study. In the diagnostic center, samples of the lungs, spleen, lymph nodes, blood and its serum are examined.

To identify the pathogen, PCR, hemadsorption, and fluorescent antibodies are used.

Ways to solve the problem

The African swine fever virus is spreading rapidly. It is forbidden to carry out therapeutic measures, the only way out is the complete destruction of infected individuals. An adequate method of treating pigs that are sick with African swine fever does not currently exist.

When the infectious process spreads, it is necessary first of all to determine the boundaries of the focus of the spread of infection and declare a quarantine regime.

All individuals infected with African plague must be destroyed by a bloodless method. The territory where the slaughter of animals affected by the virus is planned must be isolated.

The bodies of dead and destroyed pigs, as well as their waste products, feed residues and inventory are burned. The same must be done with feeders, partitions, dilapidated premises. The resulting ash must be mixed with lime and buried in the ground. The depth must be at least 1 m.

All rooms in which animals stayed must be treated with special solutions. This should be done 3 times, with an interval of 3-5 days. For disinfection use a solution of bleach, sodium hypochlorite.

All pig farms within 25 km of the infected area are slaughtered, even if the pigs are healthy.

Quarantine after detection of African swine fever lasts at least 40 days. During this period, it is prohibited to export any products derived from animals (even if they are not derived from pigs) outside the zone. Within six months after the outbreak of infection, the export and sale of any agricultural plant products is prohibited.

Measures related to the elimination of the epidemic of African swine fever must be provided by veterinary services.

Prevention

Currently, there is no vaccine that could protect livestock from African swine fever. Work in this direction is underway, but they are experimental in nature. Scientists note that in the next 10 years, a vaccine against this viral disease will not be invented.

There are preventive measures that can minimize the risk of an outbreak of African swine fever. These include:

If an outbreak of African swine fever is suspected among the pig population, it is necessary to immediately report this to the relevant authorities - the sanitary and epidemiological station.

Source: Guidelines for veterinarians prepared by the UN FAO

Within the global livestock sector, the pig sector plays a key role as a source of animal protein. Increasing global demand for meat has led to pork becoming an essential food product due to the rapid growth of pigs, efficient feed conversion, rapid turnover and fertility. Pork is the most consumed terrestrial meat, accounting for over 37% of global meat consumption, followed by chicken (35.2%) and beef (21.6%) (FAO, 2013).

Over the past decades, there has been a steady growth in the pig sector (Fig. 1), but in different countries of the world, the growth rate is not uniform. There are large populations in China and parts of Southeast Asia such as Vietnam, Western Europe, the central and eastern United States of America, Central America and southern Brazil. In Africa, where ASF is endemic, the number of pigs is constantly on the rise, indicating the spread of pig farming practices in a continent where ruminants are now the dominant domestic animal species. Religious and cultural factors greatly influence the distribution of pigs, for example, there are few or no pigs in Muslim areas (Fig. 2).

This sector is characterized by a deep gap between traditional, small-scale subsistence production on the one hand, and industrial pig production with increasing vertical integration on the other. Of course, there are a number of intermediate types of farms between them.

In recent decades, commercial pig production has undergone significant intensification. A large number of several of the most productive pig breeds are bred on a limited number of large farms, with a corresponding increase in livestock output. Large-scale production systems have achieved a high level of uniformity by relying on the same genetic material and thus being able to use similar feeds and infrastructure. Although large-scale production is able to meet an increasing share of global demand for pork, about 43 percent of pigs are still kept in small backyards, especially in developing countries (Robinson et al. 2011).

In developing countries, most pigs are still bred on traditional, small-scale, subsistence farms, where they serve as more than just a source of meat. In these low cost systems, pig farming generates added value by converting household waste into protein while providing manure to fertilize fields and fishponds. Therefore, pork contributes to nutrition and food security, while live animals are a financial safety net, playing a significant role in cultural traditions and providing additional funds for school fees, medical care and small investments.

These two very different production groups have different priorities in production practices or biosecurity investments to prevent and control swine disease. Indeed, backyards are characterized by low biosecurity, outdated farming practices and technology, and poor awareness of animal health regulations (outbreak reporting, traffic and transport management, certification, vaccination, etc.) which play an important role. in the introduction, spread and control of ASF and a number of other swine diseases.

ASF virus

The causative agent of ASF is a unique enveloped cytoplasmic DNA-containing arbovirus that is the only member of the Asfarviridae family (Figure 3). Although it was previously thought that there was only one ASFV serotype, recent studies have classified 32 ASFV isolates into eight different serogroups based on the haemadsorption delay test (HAd) (Malogolovkin et al., 2015). However, the genetic characterization of all ASF virus isolates known to date has demonstrated 23 genotypes associated with geographic locations, with numerous subgroups illustrating the complexity of ASF epidemiology (Figure 4). The genotype reflects the variability of the water gene and protein segment (\/P772) and is used primarily for phylogenetic and molecular epidemiological purposes (eg, to determine the source of outbreaks). To the best of our knowledge, it does not determine virulence or other disease parameters.

Animals that have been infected

In the natural forest cycle, Ornithodoros eyeless soft ticks (also known as South African poison mites) as well as African wild pigs are the reservoir and natural host of the ASF virus. Ticks transmit the virus through their bites.

All members of the pig family (Suidae) are susceptible to infection, but clinical disease is observed only in domestic and wild pigs, as well as in their close relative * wild European boar. Wild African pigs are asymptomatic carriers of ASFV and are a reservoir of the virus in parts of Africa (Figure 5). These include African wild boars (Phacochoerus africanus and P. aethiopicus), brush ears (Potamochoerus porcus and Potamochoerus larvatus) and large forest pigs (Hylochoerus meinertzhageni).

Geographic distribution of ASF

ASF is currently prevalent in sub-Saharan Africa, Eastern Europe, the Caucasus and the Italian island of Sardinia. With the increased circulation of ASF, there is growing concern that the virus will spread to other parts of the world. Any country with a pig sector is at risk. Experience shows that the disease can enter any country that is unaffected by the virus and located thousands of miles away, mainly through meat arriving on board aircraft and ships and then improperly disposed of meat or meat carried by individual passengers. Of particular concern is the possibility of the virus spreading to East Asia. In China, which is highly dependent on pork production and has almost half the world's domestic pig population, an ASF epidemic would spell catastrophic consequences for the production and trade of pig products, with serious implications for global food security.

Official information on the status and dates of ASF outbreaks can be obtained from the WAHIS Global Animal Health Information System hosted by the World Organization for Animal Health (OIE).

Africa

ASF is considered endemic in most of sub-Saharan Africa (Figure 6), and is also highly dynamic as it often occurs in new areas. This dynamic is mainly due to the huge growth of the swine sector in Africa‚ as in some countries (eg Madagascar, Namibia, Uganda) pig populations have doubled in less than a decade (FAOSTAT - http://www.fao.org/faostat/ ). Another important reason is the increase in the movement of people and goods. Growth in the pig sector continues despite disorganized and unsafe marketing systems that do not encourage producers to invest in improved pork production.

Most of the growth is observed in private backyards with a low level of biosecurity, which creates problems in terms of the spread of the disease. In addition, with the tools currently available, eradicating ASF in Africa is a very difficult task because there is no vaccine and no compensation mechanisms. Therefore, prevention and control efforts should focus on methods to improve livestock production, biosecurity, and protection of disease-free areas (through trade regulation and pig sector development programs that focus on educational and preventive measures). At the same time, it should be recalled that the dynamics of ASF differ from subregion to subregion.

East Africa

African swine fever was first detected in Kenya in 1909 after European domestic pigs were imported into the country (Montgomery, 1921). In East Africa, the virus persists in a forest cycle between African wild boars and Ornithodoros burrowing ticks. The first outbreaks occurred in pigs owned by European settlers, and it was found that erecting fences around the farm could eliminate African boar and ticks, and in this way pigs could be protected from infection. However, pig farming has since become very popular in the region, and a large number of the animals are in unsafe conditions or free-ranging. This has led to repeated outbreaks of ASF, mainly due to the movement and transport of pigs and pork, and not due to wildlife. The increase in suburban pig farming has led to outbreaks around big cities such as Kampala, Nairobi, Mombasa and Dar es Salaam. A cycle between domestic pigs and Ornithodoros ticks has also been found in Kenya (Gagliardo et al. 2011).

South Africa

The African wild boar forest cycle is present in the northern parts of the subregion (Botswana, Malawi, Mozambique, Namibia, Zambia, Zimbabwe and northeastern South Africa). In Malawi and Mozambique, a cycle involving domestic pigs and ticks is defined as "highly likely". Angola and Mozambique regularly report outbreaks, while other countries have seen sporadically outbreaks of ASF associated with the African wild boar. Zimbabwe in 2015, after a gap of more than 2 years, reported the first outbreak in free-range pigs. In the northeastern part of South Africa, where a significant proportion of African wild boars are infected with the ASF virus, a control zone has been established in which pig production is allowed only under strict biosecurity conditions. However, sporadic outbreaks still occur as a result of illegal activities. The rest of South Africa, Lesotho and Swaziland have historically remained ASF free, although in 2012 South Africa experienced the first out-of-control outbreak in fifty years due to the illegal movement of pigs into the area. The Indian Ocean islands remained free from ASF until 1997, when the virus was introduced into Madagascar, where it has since become endemic.

In 2007, Mauritius experienced an invasion of the virus, which was eradicated the following year. The subregion shows a high level of genetic variability (Figure 2) associated with the presence of the forest cycle.

Central Africa

The Democratic Republic of the Congo and the Republic of the Congo are historically endemic. It is likely that the forest cycle is at fault in at least some parts of these countries, as infected African boars have been reported in the Republic of the Congo (Plowright et al. 1994; Saliki et al. 1985).

Other srrans in the region have also reported outbreaks, especially Cameroon, which experienced its first invasion in 1982, shortly after the pig population doubled. In 1973, the island nation of Sao Tome and Principe experienced outbreaks that were quickly eradicated. In 2010, Chad reported the first outbreak in the south of the country, although there were sporadic reports of ASF in Chad in the 1980s (Plowright et al. 1994). Interestingly, ASF genotype IX, traditionally found in East Africa, as well as genotype I have recently been reported in this region (Figure 2).

West Africa

The first official OIE report on ASF in West Africa was from Senegal in 1978, but a 1959 virus isolate from Dakar confirms that the virus was introduced there at least two decades earlier. In West Africa, the disease appears to have affected southern Senegal and its neighbors (Guinea-Bissau, the Gambia and Cape Verde) until 1996, when Côte d'Ivoire experienced its first outbreak, followed by an epizootic affecting most countries in the region with significant pig production (Benin, Nigeria, Togo, Ghana and Burkina Faso). The disease has since become endemic in most of these countries, with the exception of Côte d'Ivoire, where it was eradicated within a year before a new invasion in 2014. Niger and Mali reported their first outbreaks in 2009 and 2016. It has been shown that the forest cycle involving feral pigs or ticks of the genus Ornithodoros is not involved in the maintenance of the virus. Only the I| genotype circulates, suggesting introduction rather than evolution of the virus in the region (Figure 2).

Eastern Europe and the Caucasus

In 2007, ASF appeared in Georgia. Genotype II ASF originated in South East Africa and was most likely introduced by ship as waste, either converted into pig feed or discarded in a location accessible to grazing pigs. The disease spread rapidly in the Caucasus (Armenia in 2007 and Azerbaijan in 2008) and the Russian Federation (2007). In the past few years, the disease has gradually spread westwards, first to Ukraine (2012) and Belarus (2013), then to the European Union (Lithuania, Poland, Latvia and Estonia, 2014) and Moldova (2016) (Figure 6).

One of the main routes of infection in Eastern Europe is through the pork marketing chain, when cheap contaminated pork and pork products from infected regions are imported. Waste feeding to pigs and improper disposal of carcasses cause infection in susceptible swine populations. The fact that ASFV remains contagious for weeks and even months in tissue and pork products allows it to persist in the environment (eg, animal carcasses) and in chilled and frozen meat and meat products.

In ASF-affected EU Member States, wild boars play a major role in the infection, spread and maintenance of ASF. How this happens is not entirely clear, but it is thought to be largely dependent on wild boar population density and their interaction with pigs in low biosecurity pig farms (free-range and grazing pigs). Carcasses of infected animals and food waste containing contaminated pork are also thought to play a role in this process.

To sum up, ASF is now firmly established, i.e. endemic in some regions of the Caucasus and Eastern Europe, where it not only causes serious problems in trade, but also causes significant damage to small-scale pig production.

Previous ASF incursions outside of Africa

In Europe, ASF first entered Portugal from West Africa in 1957. After the destruction of the disease, the genotype I of ASFV reappeared in the country in 1960, and then spread throughout Europe (in Italy - in 1967; in Spain - in 1969; in France - in 1977; in Malta - in 1978; in Belgium - in 1985 ; and in the Netherlands in 1986). It also hit the Caribbean (Cuba - 197171980; Dominican Republic - 1978; Haiti - 1979) and Brazil (1978). All countries managed to bring the situation under control, with the exception of Spain and Portugal, where the fight against the disease lasted several decades until the 90s of the last century, as well as the Italian Mediterranean island of Sardinia, where ASF became endemic from the time the virus invaded in 1978, circulating , mainly among free-range pigs and wild boars.

Transmission

The ASF virus has different cycles - traditionally there is a forest cycle, a tick-pig cycle and an internal cycle (pig-pig). More recently, the wild boar cycle has been described, which can sometimes occur along with the above cycles. The forest cycle occurs only in parts of Africa and includes the African wild boar and ticks Ornithodoros moubata complex. The mite-pig cycle includes pigs and ticks of the genus Ornithodoros spp., which are described as infesting parts of Africa and the Iberian Peninsula.

Transmission from the forest cycle (African wild pig) to the domestic cycle (pig farms) occurs through indirect tick transmission. This can happen when pigs and African boars come into contact, especially when African boars burrow on farms, or when ticks enter villages through the carcasses of African boars killed for food.

forest infection cycle

This cycle includes the natural hosts of ASFV, i.e. African wild boar and soft ticks Ornithodoros moubata complex, which act as biological vectors in South and East Africa. However, little information is available in relation to other African regions. In addition, the specific role of other wild African pigs, such as the bush pig, still needs to be clarified.

Transmission of ASFV is maintained by transmission of the virus from the tick to the African boar (Fig. 7). African wild boars become infected from the bites of the Ornithodoros tick in the first 68 weeks of life while they are in the burrow (Figure 8). Subsequently, they develop viremia and infect other ticks. After a short period of virus presence in their blood (23 weeks), young African boars recover and show no clinical signs.

In endemic areas, up to 100 percent of African wild boars may have antibodies to ASFV. The virus can usually be isolated from the lymph nodes of African boars of any age, although viremia sufficient to infect ticks has only been found in burrowed neonates. It is likely that African boars experience repeated reinfections when ticks attack them, with small amounts of the virus remaining latent in the lymph nodes.

Tick ​​populations can remain infected and contagious for a long time due to transstage, sexual and transovarial transmission of the virus in the population, allowing the virus to survive even in the absence of viraemic hosts. Infected ticks play an important role in the long-term maintenance of the disease, surviving for months in burrows and up to several years after being infected by an infected host.

Infection cycle between pig and tick

In the Iberian Peninsula, EASF easily found a suitable host - Ornithodoros erraticus, a native tick that lived in pig shelters. Ticks then became involved in maintaining ASF and transmitting it to pigs, despite the absence of wild African pigs. The cycle has also been described in parts of Africa and has been well documented in Madagascar, Malawi and Mozambique, although ticks do not appear to play a large role in virus transmission within swine populations (Haresnape and Mamu 1986; Kwembo et al. , 2015; Ravaiomanana et al., 2010).

Several species of Ornithodoros ticks have been shown to be competent vectors of ASFV in both field and experimental conditions (Table 1). However, what happens in the laboratory does not necessarily reflect what happens in the field. For Ornithodoros ticks to become competent vectors in the field, pigs must be the preferred hosts, and if these are not available, natural transmission of the virus is likely to remain limited. Vector competence can also vary greatly within a species or groups of closely related species, depending on the properties of a particular population. Although Ornithodoros ticks have been reported from the currently affected areas of the Caucasus and southern Eastern Europe, there is no indication that they are involved in the ASF epizootic cycle or that they can actually transmit the disease.

Infectious cycle of domestic pigs

In this cycle, most common in domestic pigs, the virus persists in pigs in the absence of wild boar and ticks (Figure 9). The virus can be spread through direct oronasal contact through contact with secretions from infected pigs, through ingested pork or other contaminated products, or indirectly through contaminated objects.

The virus is transmitted from one farm to another almost exclusively due to human intervention, such as transporting animals or equipment, feeding contaminated food, etc. This route of transmission requires large, constantly replenishing swine populations to keep the virus circulating. However, even in the absence of infected pigs, the virus sometimes persists in refrigerated or frozen meat, allowing it to persist for a long time and reappear when these meat products are fed to pigs.

Infectious cycle of wild boar

In Eastern Europe, the Caucasus and Sardinia, wild boar populations play an important role in keeping the virus circulating and infecting, especially where there is free range or pigs digging through the garbage. This is also possible due to other violations of biosecurity, such as dumping contaminated feed or leftover food, fences that allow nose-to-nose contact between animals, etc. The transport of wild boar to hunting grounds and/or for control purposes, as well as hunters, may also play a role (Figure 7).

The role of the wild boar in this process, however, is still not fully understood. In the Caucasus and the Russian Federation, where wild boar densities are relatively low, their infection did not last long, and was mainly maintained by virus spillovers from domestic pigs. However, as ASF moved westward into dense populations of wild boar in Poland and the Baltic countries (Figure 98), consistent transmission and continuous outbreaks were observed throughout the year. In these regions, the wild boar is considered to be the true epidemiological reservoir of this virus, with most cases occurring during the summer months.

In those parts of Eastern Europe where temperatures remain below 0°C for much of the winter, a new, previously unseen epidemiological scenario is unfolding. The virus present in infected carcasses in fields and forests remains infectious until spring, when wild boars (and possibly free-range pigs, although this is rare) can stumble upon such carcasses, eat them, and become infected (Fig. 9A) .

Human intervention, such as hunting, feeding, fencing, etc., has serious consequences for the development of epizootics in wild boar populations. Hunting can cause wild boar to escape hunters into other areas, spreading ASF, but it can also be very useful in controlling animal density (and thus transmission of the virus). Different types of hunting can also produce different effects, such as guided hunting or female hunting, etc. Similarly, feeding can increase transmission of the virus due to the large numbers of wild boars congregating at feeding grounds, but at the same time allowing more wild boars to survive harsh winter conditions.

ASF transmission and persistence of ASF

The incubation period is the period from the time of infection (i.e. when the virus enters the animal) to the onset of the disease (i.e. when the animal shows clinical signs). In the case of ASF, this period ranges from 4 to 19 days, depending on the virus, the susceptible host and the route of infection. Virus shedding may begin up to two days before clinical signs appear. The period a pig sheds the virus may vary depending on the virulence of the particular strain of ASFV: pigs infected with a less virulent strain of ASFV may be persistently infectious for more than 70 days after infection.

The virus is shed in saliva, tears, nasal secretions, urine, feces, and secretions from the genital tract. Blood, in particular, contains large amounts of the virus. Therefore, pigs can become infected through contact with many different infectious sources, mainly infected pigs, contaminated pork and other swine products (eg food waste) and objects (eg bedding). These infected animals and contaminated materials can be transported by vehicles and people over long distances.

Although ASF is associated with high mortality (most infected animals die), it is not as contagious as some other transboundary animal diseases such as foot-and-mouth disease. This means that ASF usually spreads slowly and some animals may not be infected with the virus.

In a suitable protein-rich environment, ASFV remains stable over a wide range of temperatures and pH levels for long periods of time, it is also resistant to autolysis and various disinfectants. Thus, neither putrefaction, nor the ripening process, nor the freezing of meat can inactivate it. Consequently, the virus survives in secretions, carcasses, fresh meat and some meat products for varying periods of time. It can remain infective for at least 11 days in faeces, 15 weeks in chilled meat (and probably longer in frozen meat), and months in bone marrow or smoked ham and sausage, unless cooked or smoked at high temperature (table 2). The method of preparation is very important for the spread of ASF. Undercooked, undercooked, cured, or salted meats, as well as blood, carcasses, or feed prepared from them, can be a source of infection if fed to pigs or disposed of with municipal waste in places where they can be eaten by pigs or wild boars. Cooking meat at 70°C for 30 minutes inactivates the virus (Figure 10).

The introduction of new pigs into a herd or pigsty often results in individuals attacking and biting each other. In the case of free-range or grazing pigs, infection can occur through contact with infected stray animals, wild boars, their carcasses or food debris. In addition, the virus can be transmitted by using the same needle to vaccinate or treat multiple pigs. Transmission of the virus by artificial insemination has not been proven, but the possibility is not ruled out.

Vector transmission is also possible through the bites of infected Ornithodoros ticks. Some blood-sucking insects, namely Stomoxys calcitrans, have been shown to be able to retain and transmit ASFV for at least 24 hours after contact with a diseased individual (Mellore et al. 1987), which is especially important in transmission within a herd .

Infection through large bodies of water, such as rivers and lakes, seems unlikely, since the concentration of the virus, once diluted with water, becomes less than infectious levels.

Clinical picture and autopsy data

As a rule, the disease is characterized by the sudden death of pigs, regardless of age or sex. Animals isolated from the rest of the herd, such as sows with young suckling piglets, can avoid infection due to the rather low contagiousness of ASF. The rate of spread of the disease within a herd (and the number of victims) can vary from a few days to a few weeks, depending on the type of pig farm, management and biosecurity measures. In fact, ASF, although highly lethal, is less common than some other transboundary animal diseases such as foot-and-mouth disease. In addition, some indigenous pig breeds in Africa have developed a degree of tolerance to ASF. Wild boars, by virtue of belonging to the same species as domestic pigs, show the same clinical picture.

Clinical signs associated with ASFV infection are highly variable (see Table 3) and depend on various factors: virus virulence, pig breed, route of transmission, infectious dose, and local endemicity.

According to their virulence, ASFVs are divided into three main groups: high-virulence, moderate-virulence, and low-virulence isolates (Figure 11). Clinical forms of ASF range from hyperacute (very acute) to asymptomatic (indistinguishable). As shown in Figure 11, highly virulent ASFV isolates cause hyperacute and acute forms of the disease, moderately virulent isolates cause acute and subacute forms. Low-virulence isolates have been described in endemic areas (in addition to circulating virulent viruses), are characterized by milder symptoms, and are sometimes associated with subclinical or chronic ASF. The incidence (ie the proportion of affected animals) will depend on the virus isolate and the route of transmission.

Although it is not known exactly, the incubation period for natural infection has been reported to vary from 4 to 19 days. The clinical course of the disease can be less than seven days after infection in the acute form, up to several weeks, or even months, in the chronic form. The mortality rate depends on the virulence of the isolate, it can be as high as 1007% in highly virulent strains affecting pigs of all ages, but can be less than 20% in the chronic form. In the latter case, the disease is often fatal in pregnant and young pigs that are sick with other diseases or are debilitated for another reason. Survival rates against highly virulent strains observed in some endemic areas may be higher, possibly due to pig adaptation to the virus.

Super sharp shape

It is characterized by high temperature (41-42°C), loss of appetite and lethargy. Sudden death may occur within 1-3 days before any clinical signs develop. Often there are no clinical signs or organ damage.

acute form

After an incubation period of 4-7 days (rarely up to 14 days), in animals with an acute form of ASF, the temperature rises to 40-42 ° C and appetite disappears; animals look sleepy and weak, huddle together and lie on the floor (Fig. 12), their breathing rate increases.

Death often occurs within 6-9 days for highly virulent strains, or within 11-15 days for moderately virulent isolates. In domestic pigs, mortality often reaches 90-100 percent. The same signs are observed in wild boars and wild pigs. The acute forms are easily confused with other diseases, mainly classical swine fever, swine erysipelas, poisoning, salmonellosis and other septicemic conditions (see next chapter on differential diagnosis). Infected pigs may show one or more of the following clinical signs:

• blue-violet areas and hemorrhages (punctate or dilated) on the ears, abdomen and/or hind legs (Fig. 12);
• discharge from the eyes and nose;
• redness of the skin of the chest, abdomen, perineum, tail and legs (Fig. 12);
• constipation or diarrhea that can go from mucosal to bloody (melena);
• vomit;
• abortion in pregnant sows at all stages of pregnancy;
• bloody foam from the mouth/nose and discharge from the eyes (Fig. 15);
• the area around the tail may be contaminated with bloody feces (Figure 12).

In wild boars, it is difficult to notice discoloration and bleeding on the skin due to the darker color of the skin and thick coat. The same applies to dark-skinned breeds of pigs.

Carcasses of pigs that die in the acute stage of the disease may remain in good condition, although they may show external clinical signs. The most recognizable autopsy findings (Figure 13): enlarged, edematous and completely hemorrhagic lymph nodes that look like blood clots (especially gastrointestinal and renal); enlarged, friable spleen dark red to black with rounded edges; and petechial (pinpoint) hemorrhages in the renal capsule.

At autopsy, the following phenomena are usually found:

1. Hemorrhages under the skin;
2. Excess fluid in the heart (hydropericardium - accumulation of yellowish fluid) and body cavities (hydrothorax, ascites) (Fig. 15);
3. Petechiae on the surface of the heart (epicardium), bladder and kidneys (in the cortical layer of the kidney and renal pelvis) (Fig. 14);
4. In the lungs, hyperemia and petechiae, foam in the trachea and bronchi, and severe alveolar and interstitial pulmonary edema are possible (Fig. 15);
5. Petechiae, ecchymosis (extensive hemorrhage) and excess clotted blood in the stomach and small and large intestines (Fig. 14);
6. Hepatic hyperemia and hemorrhage in the gallbladder.

Infected wild boars in Eastern Europe show the same autopsy features and have the same clinical signs, but
due to their thick, dark coat, outward clinical signs are less obvious (Figure 16).

Subacute form

The subacute form of the disease is caused by moderately virulent isolates and may occur in endemic regions. Pigs usually die within 7-20 days, with a mortality rate of 30-70 percent. The surviving pigs recover in a month. Clinical signs resemble (although they are usually less intense) those in the acute form of the disease, except that vascular changes are less pronounced with hemorrhages and edema.

A common symptom is intermittent fever, which is accompanied by depression and loss of appetite. Movement of the animals can be painful, and the joints are often swollen with accumulated fluid and fibrin. There may be signs of difficulty breathing and pneumonia. Pregnant sows may have an abortion. Serous pericarditis (fluid around the heart) often develops into more advanced forms of fibrinous pericarditis.

Chronic form

In the chronic form, often the mortality rate is less than 30%. This form has been described in countries where ASF has long been present, such as Spain, Portugal and Angola. The chronic form originates from naturally attenuated viruses, or from a vaccine virus released during vaccine field research, which is suspected to have occurred in the Iberian Peninsula in the 1960s. Clinical signs begin 14 to 21 days after infection with mild fever followed by mild respiratory distress and joint swelling (moderate to severe). This is often accompanied by reddening of the skin that swells and becomes necrotic (Figure 17). Further autopsy findings include pneumonia with caseous necrosis (sometimes with focal mineralization) in the lungs, fibrinous pericarditis, and edematous lymph nodes that may be partially hemorrhagic (mainly mediastinal lymph nodes) (Figure 17).

Differential Diagnosis

African swine fever does not always present with the full set of clinical signs described in the previous section. In the early stages of the disease, or when a small number of animals are involved, it can be difficult to make a clinical diagnosis. The diagnosis of ASF is often hypothetical and the symptoms can be confused with other diseases and/or conditions. In addition, a number of swine (and wild boar) diseases can have mortality rates similar to those seen in acute outbreaks of ASF. the diagnosis is not definitive until it is confirmed by a laboratory.

In addition to the major differential diagnoses listed in this chapter (Table 4), other generalized septicemias and hemorrhagic conditions may also be considered.

Classic swine fever

The most important differential diagnosis of ASF is classical swine fever, also known as swine cholera, which is caused by a Pestivirus of the Flaviviridae family. As with ASF, it comes in a variety of clinical manifestations or forms. Acute CSF has almost the same clinical signs and autopsy findings as acute ASF, and it also has a high mortality rate. Clinical signs may include high fever, anorexia, depression, hemorrhages (into the skin, kidneys, tonsils, and gallbladder), conjunctivitis, respiratory signs, weakness, crowded animals, blue skin, and death within 2 to 10 days. The only way to distinguish between the two is through laboratory confirmation. It would be unwise to attempt to vaccinate animals against CSF before confirming the diagnosis, as ASF can be spread by untrained personnel during vaccination.

Porcine reproductive and respiratory syndrome (PRRS)

PRRS, sometimes referred to as "blue ear disease", is characterized by pneumonia in growing and finishing pigs, and abortions in gestating sows. This is often accompanied by fever, flushing and, in particular, a bluish tint to the skin of the ears. Diarrhea is also characteristic. Although mortality from PRRS is generally not high, highly pathogenic PRRS viruses have decimated entire pig herds in China, Vietnam and Eastern Europe over the past few years, characterized by high mortality, high fever, lethargy, anorexia, cough, dyspnea, lameness, and cyanosis/blue (skin ears, limbs and perineum).

Autopsy findings include damage to the lungs (interstitial pneumonia) and lymphoid organs (thymus atrophy and edema and hemorrhage in the lymph nodes) and petechial hemorrhages in the kidneys.

Piglet dermatitis and nephropathy syndrome (PDNS)

This is one of the circovirus-2 related diseases in pigs. LDNP usually affects growing pigs and pigs in the final stage of fattening. Although the clinical signs speak for themselves, there are no specific diagnostic tests.

The syndrome is characterized by dark red to purple skin lesions that are usually most pronounced in the posterior trunk and perineum, although in severe cases the flank and iliac abdomen may also be affected. Lesions in the walls of blood vessels caused by necrotizing vasculitis (inflammation of the blood vessels) are microscopically easily distinguishable from lesions in ASF. The disease is also accompanied by anorexia, depression and severe nephrosis (inflammation of the kidney), which is usually the cause of death. Lymph nodes may also be enlarged. The incidence is generally low, but affected pigs die very frequently.

Pig erysipelas

This bacterial disease, caused by Erysipelothrix rhusiopathiae, affects pigs of all ages and can affect pigs in both small and extensive pig farms and commercial intensive systems. The disease can manifest itself in acute or subacute forms. The acute form, which usually occurs in young pigs, is characterized by sudden death, although mortality is usually much lower than in ASF.

Two or three days after infection, diseased pigs may develop very characteristic diamond-shaped skin lesions due to necrotizing vasculitis (inflammation of the blood vessels). In adult pigs, this is usually the only clinical manifestation of the disease. As with acute ASF, the spleen may be hyperemic AND visibly hardened. autopsy findings also include congestion of the lungs and peripheral lymph nodes, as well as hemorrhages in the cortex of the kidney, heart, and serosa of the stomach. Isolation of the bacterium can confirm the diagnosis, and pigs respond well to penicillin treatment. Microscopic changes are of a different nature than with ASF.

Aujeszky's disease

Aujeszky's disease, also known as pseudorabies, causes serious neurological and reproductive problems and is often fatal. Although almost all mammals can be infected, pigs are the most commonly affected and are the reservoir. Young animals are most affected, mortality during the first two weeks of life reaches 100%. Piglets usually develop a fever, stop eating, develop neurological signs (tremors, convulsions, paralysis), and often die within 24-36 hours.

Older pigs (over two months old) may develop similar symptoms, but they usually have respiratory signs and vomiting, and mortality is not as high. Sows and boars mostly show respiratory signs, but pregnant sows may abort or give birth to weak piglets with tremors. Focal necrotic and encephalomyelitis lesions may be in the brain, cerebellum, adrenal glands, and other internal organs such as the lungs, liver, or spleen. White spots in the liver in fetuses or very young piglets are very characteristic of this infection.

Salmonellosis (and other bacterial septicemias)

Salmonellosis usually affects young pigs. If treatment is started on time, animals respond well to antibiotic therapy. The diagnosis is confirmed by bacteriological culture. ASF-like signs include fever, loss of appetite, respiratory or gastrointestinal disturbances, and a flushed, inflamed carcass at the time of slaughter.

Animals can die 3-4 days after infection. Pigs that die from septic salmonellosis show cyanosis of the ears, legs, tail, and abdomen. Autopsy findings may include petechial hemorrhages in the kidneys and on the surface of the heart, an enlarged spleen (but with a normal color), swelling of the mesenteric lymph nodes, an enlarged liver, and pulmonary congestion.

Poisoning

When a large number of pigs suddenly die, the possibility of poisoning should be considered. Some toxic substances can cause the same kind of bleeding as in ASF. And while coumarin-based rat poison, such as warfarin, can cause massive bleeding, it is more likely to affect a few pigs than the whole herd.

Some fungal toxins in moldy feed, such as aflatoxin and stachybotryotoxin, can cause bleeding and serious mortality. Accidental or malicious pesticide poisoning can result in the death of pigs of all ages, but the death of all pigs within 24-48 hours, with few or no clinical signs, with no lesions found at autopsy, will help distinguish this outcome from ASF. Poisoning is unlikely to be accompanied by fever.

The sections in this chapter have been and are taken from the FAO Good Emergency Management Practice (GEMP): The Essentials (FAO, 2011), which can be consulted for more details.

It is wise to have an investigation kit ready at the local veterinary office at all times so that the veterinarian can get started as soon as possible with minimal delay. The equipment should ideally include a digital camera, an OCR and a means of rapid communication (mobile phone, but may include a radio), as well as all necessary equipment for sampling, proper packaging and transport of samples (GEMP, 2011).

Suspicion of ASF is usually reported by the farmers themselves or by a private veterinarian. When faced with a suspected outbreak of ASF on a farm/hold, the following steps should be taken immediately, prior to laboratory confirmation, based on the assumption of a field diagnosis of ASF:

• Collect farm and affected animal data (see Box 1).
• Infected and suspected farms must be quarantined immediately, i.e. no people, vehicles, animals or swine products should leave or be brought into the farm until the diagnosis has been confirmed.
• Establish disinfection points for people and vehicles at the entrances and exits of the building in which the pigs are kept. Employees and visitors must ensure that shoes, clothing and equipment are disinfected when leaving the farm. If the veterinarian or other employees must come into contact with sick animals or potentially contaminated materials, they must use personal protective equipment.
• Carry out inspections in each room of the farm, clinical examination of individual animals and post-mortem examination of dead (or slaughtered) animals. When conducting a clinical examination of suspicious animals, a systematic approach is required.
• It is also important to record your findings as you progress through the survey. The completed form will help you to carry out this task effectively. In the presence of a large number of animals, it is necessary to prioritize which animals to examine. First of all, it is necessary to conduct an examination of animals with obvious clinical signs.

• Appropriate samples should be taken as soon as possible and sent to a laboratory for diagnosis immediately (see Sampling section). In the event that many animals show clinical signs, specimens from five of them should be sufficient to establish a diagnosis.
• Conduct an outbreak investigation (also known as an epidemiological investigation).
• Neighboring farmers or those who have recently bought or sold animals from this farm, i.e. contact persons at risk must be notified so that they can test their animals (and report any symptoms found to the veterinary authorities) and stop the movement of pigs and products from and to these piggeries. Service providers who have recently visited these farms should also be notified.

• Even with proper cleaning and disinfection, employees involved in an outbreak investigation on a potentially infected farm should not visit other farms for at least 24 hours to prevent the possibility of accidental spread of the disease.
• When it comes to an outbreak in a free-range or grazing pig farm, the first step is to return all uncovered animals and keep them indoors, or at least tethered.

How to conduct an outbreak investigation

This section is taken from the EuFMD Online Tutorial.

An outbreak investigation, also known as an epidemiological investigation, should determine:

(a) How long does the illness last?

b) possible sources of the disease;

c) what movements of animals, people, vehicles or other objects could lead to the spread of the disease;

d) the magnitude of the problem by counting the number of cases, defining epidemiological units and assessing the population at risk. This information is critical when deciding on an effective control strategy and monitoring the implementation of the control strategy once these measures have already been taken.

The first step is to define an epidemiological unit (unit), which should include all pigs with a similar level of risk of infection. These will be all susceptible animals under the same management or biosecurity system, i.e. usually farms. However, this unit can expand to the level of a village if there are no real boundaries between farms. It is important to remember that geographically separated farm units may be in the same management system and be part of the same epidemiological unit.

Time-frame/graphing helps to determine when infection and transmission of the disease is suspected to have occurred, and provides an opportunity to guide outbreak investigations. This graph is used to determine windows of time when virus introduction (based on incubation period) and spread to other sites (based on virus isolation period) could occur.

Once the schedule has been created, the next step is to use it to trace the source of the virus and spread it further in order to establish contacts that could lead to transmission of the virus within the calculated time. Risk factors for the spread of the disease include:

• movement of animals or products of animal origin (eg pork);
• employees visiting the premises and in direct contact with animals on other farms, such as a veterinarian or other farmers;
• farm workers visiting other livestock holdings;
• movement of vehicles or equipment between livestock holdings;
• direct contact of animals at farm boundaries;
• wild pigs or their products.

Once possible sources of infection have been identified, it is important to prioritize further epidemiological investigations. This allows a quick investigation and identification of all contacts that may contribute to the further spread of the disease. Priority should be given to those contacts that occurred during the period of time when infection was possible.

This scheduling is especially important when staff and resources are limited, as is often the case. Contact types are also important. Priority should be given to:

• large farms where more animals are present;
• "intersections" where animals from multiple locations meet, including livestock markets and slaughterhouses;
• farms where there is a regular movement of animals, for example, from livestock traders;
• direct contact with animals, for example, when buying animals;
• adjacent rooms where there are pigs.

There are various ways to investigate possible contacts:

Interview

Interviewing effectively requires certain skills, especially in a situation where the farmer is likely to be under severe stress. Farmers are often fearful of strangers and especially government officials. It is extremely important to take the time to build trust with the interviewee. Also, don't plan to visit more than one farm per day. We offer you some ideas that you can find in box 2.

Other sources of information

Review livestock and personnel movement records. Veterinary records, diaries, bills of lading and invoices or receipts from deliveries can also provide valuable information. Remember that the farmer at such times can be very upset, it is difficult for him to remember and convey all the details, and therefore the notes become an even more valuable source of information.

In addition to interviewing the farmer, you should carefully inspect the premises. It is necessary to go around the premises around the outer perimeter to determine if there is any contact with neighboring pigs or feral pigs. It is sometimes useful to make a sketch of the area, indicating the place where animals are kept, groups of animals, entry and exit, and its boundaries.

For epidemiological investigation and tracking purposes, it may be useful to contact other visitors to the premises, such as veterinarians, milk collectors or artificial insemination technicians.

Ensuring biosecurity when visiting a farm

This section has used material from the EuFMD online training course. A detailed video demonstrating the steps below can be viewed at: https://www.youtube.com/watch?v=ljS-53r0FJk&feature=youtu.be

Before leaving:

• Be sure to remove all unnecessary equipment from the vehicle.
• Arrange "clean" and "dirty" areas in the back seat and trunk of your car, lined with plastic sheeting.
• Make sure you bring all the necessary equipment with you. To do this, it makes sense to draw up a checklist (see Box 3). It is useful to have a standard list of equipment needed to set up a disinfection point. Such a list may be in your emergency plan or in your manual.

On arrival

• The car must not enter the territory (leave it at the entrance to the farm).
• Choose a suitable location on a clean and dry surface (preferably concrete) for your disinfection point, clearly delineating the clean and dirty sides (gates).
• Remove all unnecessary clothing and items (e.g. jacket, tie, watch) and take everything out of pockets.
• Electronic equipment (such as a mobile phone) needed on the farm should be placed in a sealed plastic bag to facilitate subsequent cleaning and disinfection. The phone on the farm should never be taken out of the bag, it can only be used if it is in a plastic bag at the same time.
• Take from the car all the items necessary for disinfection to be taken to the farm.
• You may need to bring your own water for making detergents and disinfectants.

Training

• Place a plastic sheet on the clean side of the disinfection point.
• Place items you will bring with you to the farm on the dirty side of the disinfection point (such as black plastic bags and sample containers).
• Mix the water you brought with you with the detergent in one bucket and the disinfectant in two buckets. Two buckets - one with detergent and one with disinfectant - will remain on the dirty side, you will use them to remove the dirt that you "collected" on the farm. Another disinfectant bucket with its own brush will be on the clean side.
• Often the disinfectant is specific, intended for use in the case of a particular disease. The concentration and time of exposure should be carefully monitored.

Dressing (on the clean side)

• Take off your shoes and leave them on the plastic sheet.
• The disposable protective suit is put on first, and then it is tucked into the boots. Gloves must be attached with adhesive tape.
• Waterproof overalls (if weather conditions require) should cover the boots. He has his own layers of disposable gloves that can be replaced when they get dirty.
• Shoe covers must cover at least the sole and bottom of rubber boots.
• Put on a protective hood and double-check the list before you step off the plastic sheet and head to the farm.

Stripping (on the dirty side)

• Before leaving the premises, use the products available on the farm to clean very dirty areas.
• Wash the sample container with detergent and brush before dipping it in the disinfectant for the required period of time, and then place it in the sample bag on the clean side.
• Wash and disinfect the bag containing the phone and other similar items that you took to the farm.
• Remove the shoe covers and place them on the dirty side in plastic bags. Roll up the waterproof coverall (if you are wearing one) to the top of the boots before cleaning the boots with detergent and brush, especially the bottom (possibly using a screwdriver to clean the soles). Then use the detergent to wash the entire suit, including the hood.
• Remove the second pair of gloves (outer) and place them in the bag on the dirty side before the unwashed waterproof coverall is removed and placed in the disinfectant solution. After being in the solution for the required time, it must be placed in a bag on the clean side.
• Boots, if necessary, can be quickly washed again and properly disinfected.
• The first pair of gloves (the inner) should be removed and placed in the bag on the dirty side before removing the inner suit (legs should be pulled out of the boots when removing the suit, and then you can put your boots back on). The suit must be placed in the bag on the dirty side.

On the clean side

• Take your feet out of the boots and step on the clean side of the sheet before taking the boots and disinfecting them on the clean side (you need another bucket to disinfect). Lastly, place them in the bag on the clean side. Here it is also necessary to disinfect hands and glasses, as well as the face (with disinfectant wipes).
• Reusable equipment and specimens should be double bagged and kept closed.

You can put on your normal shoes again.

• If the buckets on the dirty side are your own, they need to be disinfected, placed in two bags, and only then they can be taken away. Any buckets from the farm should be left on the dirty side.
• The bags need to be placed on the dirty area in the car.
• Ask the farmer to take the garbage for processing, if necessary.
• After leaving the farms, samples/equipment should be sent in for diagnostics immediately.
• If you don't have pigs nearby, you can go home, then take a shower and wash your hair thoroughly. All clothes that were on you that day should be put in a disinfectant for 30 minutes and washed at a temperature above 60 ° C. If there are pigs where you live, do it all elsewhere.
• Do not visit any place where pigs are kept for at least three days.

Along with the self-disinfection procedures, it is also necessary to wash and disinfect the vehicle. Before starting the visit, make sure that the car is free of unnecessary items and that it is clean. Place a piece of plastic sheet in the parts of the vehicle where the equipment is stored and divide it into two parts: clean and dirty. Remember to follow local vehicle disinfection regulations.

You should, if possible, wash and disinfect the outside of the vehicle before you leave the affected area, and then repeat this procedure inside and outside the vehicle after you return to your base.

• Remove any plastic sheets that have been draped over the car and dispose of them properly.
• Wash the outside of the car using a washer or hose and a disposable sponge to remove any visible dirt. Don't forget to clean hidden places such as wheel arches, tire tread and the bottom of the car.
• After all the dirt has been removed, spray the disinfectant on the outside of the machine.
• Get rid of debris inside the machine, remove all dirt (take care of proper waste disposal).
• Wipe down steering wheel, pedals, shifter, handbrake, etc. cloth soaked in disinfectant.

If ASF is suspected in wild boar

First, it is very important to have a clear definition of a suspected case of ASF in wild boar. Such definitions are likely to vary depending on the epidemiological situation in the region/country, and may become more stringent as risk increases. The definition generally covers any wild boar with clinical signs or abnormal behavior, or any harvested animal with lesions (found after autopsy), or any wild boar found dead or killed in traffic incidents (especially in high-risk areas).

The suspicion that wild boars may be infected is usually reported by hunters, although foresters, hikers, mushroom pickers, etc. can also report it. It varies by country, but hunters can play a very prominent role in detecting the disease. To enlist their cooperation, you will need some motivation, for example, money. It is important that every hunter in a risk area is trained to recognize the clinical signs of ASF in order to know what type of specimen to take and how, to notify the authorities in a timely manner, and to know how to dispose of the carcass. Hunters must also ensure that any hunted wild boar is butchered in a designated area and that the offal or by-products are properly disposed of, such as placed in designated containers or pits.

If the health of the animal is suspected, hunters may have to store the entire carcass in a refrigerator (usually a hunting lodge) until laboratory results are available.

Suspicious carcasses found in the forest should, if possible, be picked up and transported (by car, sled, etc.) to a safe place for incineration or disposal. In addition, they can be destroyed on site by incineration or landfill.

If clinically suspected, the following measures should be taken immediately:

• Collect data on affected animals (number, age, sex, pathological lesions, location, etc.).
• Ensure that all who have been in contact with the animal carcass, their shoes, clothing and equipment are disinfected. If the veterinarian and other employees come into contact with sick/dead animals or potentially contaminated materials, they must use personal protective equipment.
• Conduct clinical examination and post-mortem examination of animals.
• Collect appropriate samples and bring them to a laboratory for diagnosis as soon as possible (see section ASF Laboratory Diagnosis, page 39). In some cases, especially if carcasses are in remote locations, hunters must take samples themselves.
• Conduct an outbreak investigation (epidemiological investigation).
• Notify neighboring farmers of the event so they can check for clinical signs in their animals and close them.
• Even after proper cleaning and disinfection, employees who are involved in the investigation of a potentially infected wild boar due to a disease outbreak should not visit farms for at least 48 hours to avoid inadvertent spread of the disease.

When conducting an epidemiological investigation involving wild animals, the protocols will differ from those used on farms, taking into account the different characteristics of the population. The interviewees will not be animal owners, but people who regularly visit the forest, such as the leader or members of the local hunting club, local forest rangers, etc. Questions may include:

• Who hunted in the area - both local and visiting hunters?
• Have there been driven hunts (with beaters) in the last month or two?
• What are the geographic boundaries of the reserve?
• What is the management practice in the reserve?
• What are the biosecurity measures?
• What is hunting hygiene?
• Are there any populations of domestic pigs in the area?
• Immediate actions at the farm level in the event of a suspected outbreak

Standard Operating Procedures (SOP) (GEMP, 2011)

SOPs are critical to ensure that suspicious cases are properly investigated. They should include:

• safety notes for investigators and pet owners;
• a list of equipment to be taken, including sampling equipment;
• criteria for establishing the degree of contamination of the area and, on the basis of this, a biologically safe entry point;
• taking biosecurity precautions when entering and leaving the location;
• arrival restrictions on the movement of livestock, food, personnel, vehicles and equipment;
• necessary examinations (number and type of animals); taking samples from animals with similar characteristics;
• sample handling;
• the procedure for sending samples for testing; and - a procedure for communicating interim findings to the appropriate authorities.

Specialized Diagnostic Team (GEMP, 2011)

It is recommended that a specialized diagnostic team (or teams) be assigned and can be mobilized immediately. Team members must be equipped and ready to travel on short notice. For this mission, the team needs to take with them all the equipment needed to investigate the outbreak, collect and transport diagnostic specimens, and communicate quickly. The team must travel to the outbreak site accompanied by local veterinary staff, including the local veterinarian. The team must conduct a clinical examination, collect a history, make a preliminary epidemiological investigation, trace the movement of suspicious animals and collect a wide range of diagnostic samples, both for the suspected disease and for any other endemic or exotic diseases that can be included in the differential diagnosis. The team must transport these samples to the laboratory. It must also take the necessary immediate action to control the disease at the site of the outbreak and must have the legal authority to do so. In addition, it must have the authority to issue immediate instructions to local animal health officials. The team should immediately report to the oblast/regional veterinarian and the chief veterinarian an assessment of the situation, including the steps taken to confirm the diagnosis and recommendations for a further disease control strategy, including the establishment of infected and surveillance areas. The composition of the diagnostic team may vary depending on the circumstances, but may include:

• veterinary pathologist from the central or regional veterinary diagnostic laboratory;
• a specialist epidemiologist, preferably with experience or professional training in the field of transboundary and emerging diseases, and especially in the field of the suspected disease;
• a veterinarian with extensive experience in endemic diseases;
• any specialist required for a specific examination.

Sampling, packaging and transport of samples

This practical guide is intended for field and laboratory teams.

Sample selection

The starting point for any laboratory investigation of ASF is sampling. An important consideration here is the purpose of the investigation, such as disease diagnosis, disease surveillance or health certification. Which animals should be sampled will depend on the purpose of the sampling.

For example, when investigating an outbreak (passive surveillance), the target group is sick and dead animals, but if you want to find out if animals are susceptible to disease (active surveillance), then the oldest animals should be sampled.

Those staff who take samples (and conduct clinical examinations) should be trained in how to immobilize pigs (during clinical examination and sampling).

The sampling team should bring sufficient supplies and equipment to take samples (see Box 4) from a certain number of animals, with a reserve in case the materials/equipment become unusable (e.g. leaking vacutainers, etc.) .). Also, be sure to bring everything you need for data collection, personal protection/biosecurity, and sample transport (see section “Sample Transport Materials” in Box 4).

It is recommended to use the field sampling form in order to collect all the necessary samples and information on site. If samples are to be sent to a regional/international reference laboratory, it is recommended that samples be taken in duplicate so that one can be sent and the other can be stored, thus avoiding the need to thaw and aliquot/separate samples for shipment.

Samples should be taken from the community using the correct methods to avoid undue stress and injury to the animal or self harm. Samples should be taken under sterile conditions to avoid cross-contamination, and new needles should always be used for each individual animal to avoid disease transmission. All specimens awaiting testing should be considered infected and handled accordingly. All materials used for on-farm sampling should be disposed of in accordance with national regulations, eg bagged and transported back to the laboratory for autoclaving/proper disposal.

Diagnostic laboratories require specimens to be delivered to the laboratory in good condition and clearly and permanently labelled.

Sample types

a. Whole blood

Collect whole blood from the jugular vein, inferior vena cava, or ear vein using sterile tubes (vacutainers) with an anticoagulant (EDTA - purple stopper). If the animal has already died, blood can be taken from the heart, but this must be done immediately. Avoid the use of heparin (green plug) because it may inhibit PCR and/or give a false positive result in haemadsorption (HAd) identification. Blood is the target for virus detection by PCR and virus isolation. The plasma separated during the centrifugation process can be used to detect antibodies using an indirect immunoperoxidase test (IPT) or an indirect fluorescent antibody test (nMFA).

Dry blood spot microvolume testing (DBS) on a filter paper card is a convenient way to collect and store blood for further DNA and/or antibody detection. These cards are very useful in remote areas or when a cold chain is not available, such as hunting or rural areas in the tropics. However, ASSF or antibody genome detection tests are less sensitive with DBS than with whole blood or serum. DBS samples are the collection of a few drops of blood using a lancet or a sterile needle from a syringe from a vein or skin onto a specially made absorbent filter paper (card). The blood thoroughly soaks the paper and dries within a few hours. Samples are stored in low gas permeability plastic bags with a desiccant added to reduce moisture. They can be stored at room temperature even in tropical climates.

b. Serum

Collect whole blood from the jugular vein, inferior vena cava, or ear vein, or at the time of autopsy using sterile vacutainers without anticoagulant (red stopper). When sent to the laboratory to obtain serum, the blood must be incubated for 14-18 hours at 4 ± 3 °C to separate the clot. The clot is discarded and, after centrifugation for 10-15 minutes, a supernatant (serum) is obtained. If the serum is red, it means that the sample is hemolyzed, and this can lead to a false positive reaction in the ELISA test. Hemolysis usually occurs when an animal, such as a wild boar, is already dead. Serum can be immediately tested with antibody and virus detection methods or stored at a temperature<-70 °С до дальнейшего использования. Для обнаружения антител температура хранения может быть -20 °С, но для обнаружения вируса это не оптимально.

in. Tissue and organ samples

Although all organs and tissues of the pig can be used to test for the presence of ASFV (mainly in acute and subacute forms of the disease), the preferred organs are the spleen, lymph nodes, liver, tonsils, heart, lungs and kidneys. Of these, the spleen and lymph nodes are considered the most important, as they usually contain large amounts of virus. Bone marrow is also useful in the case of dead wild animals, as it may be the only tissue that is relatively well preserved if the animal has been dead for some time. Intra-articular tissues of the joints can be examined to check for the presence of low virulence isolates. It is recommended that samples be stored at 4°C and delivered to the laboratory as soon as possible (within 48 hours). If this is not possible for technical reasons, samples can be stored either in a freezer or in liquid nitrogen. For histopathological examination, samples in 10% buffered formalin can be used in parallel. While they cannot be used for further virus isolation testing, they can be used for PCR and immunohistochemistry.

For virus detection by PCR, virus and/or antigen isolation by ECBA, a 10%(w/v) homogenized tissue suspension in phosphate buffered saline should be prepared. After centrifugation, it is recommended to filter the supernatant and treat it with 0.1% antibiotic for 1 hour at 4±3°C. The homogenized tissue suspension can be used immediately for ASFV and genome detection, or stored at< -70 °С для дальнейшего использования. Для ПЦР рекомендуется обработать разведенный 1/10 супернатант параллельно с неразведенным материалом. Экссудаты тканей, полученных, главным образом, из селезенки, печени и легких, очень полезны для проверки на наличие антител с использованием ИПТ и нМФА (Гайардо, 2015 г.).

d. Soft mite specimens

Ornithodoros soft ticks can be tested for ASFV and genome. Ticks can be found in African boar burrows, crevices/holes in pigsties, and occasionally in rodent burrows inside pigsties. Different types of ticks have different preferred habitats and habitats. There are three methods for collecting mites: manual collection, carbon dioxide capture, and vacuum aspiration. After collection, the ticks should remain alive or stored in liquid nitrogen to ensure optimal virus retention within the ticks and avoid DNA degradation.

Packaging and transport of samples

To obtain a correct diagnosis, it is important to select the right samples, carefully pack them, label them, and, with proper temperature control, quickly send them to the laboratory. The diagnosis of ASF is urgent and specimens should be sent to the nearest appropriate laboratory by the shortest route. Samples must be accompanied by an accompanying document indicating the number and type of samples, animal species, sampling location (address, county, oblast, district, country of origin). It should also list the tests required, the name of the person submitting the samples, the clinical signs observed, significant lesions, morbidity, mortality, number of affected animals, history, and what types of animals are affected. In the case of pets, the owner, farm name and type of holding, and a list of differential diagnoses should be provided. Care must be taken that each sample can be associated with the animal from which it was taken.

However, the minimum required information may vary from laboratory to laboratory. It is prudent to call the laboratory prior to sampling to follow the correct procedure for sending samples and to ensure that the prescribed number of samples can be analyzed or that the samples are kept for the required time.

Samples should arrive at the laboratory as soon as possible to avoid deterioration in quality and ensure the best results. They must be shipped under safe conditions to avoid contaminating other animals or people during transport, and to avoid contaminating the samples themselves. Shipped samples must be delivered with sufficient cooling materials, such as ice packs, to prevent degradation. Remember that an accurate diagnosis can only be made when the specimens are in good condition.

Ground transport

When transporting specimens to the nearest laboratory, national rules and regulations must be followed, even if specimens are transported by veterinarians. For Europe, the main document is the European Agreement concerning the International Carriage of Dangerous Goods by Road (ADR). In other regions, national codes and regulations must be observed.

If not available, the UN Model Regulations set out in the OIE Manual for Diagnostic Tests and Vaccines for Terrestrial Animals (2016; chapters 1.1.2 and 1.1.3) should be followed.

Triple packing should be used even in the case of road transport. A detailed example of the characteristics of a triple pack is shown in Figure 27.

Air transportation

Samples must be transported in accordance with regulations3‚ using a “triple packing” system. In particular, if samples are transported by air, the sender must comply with the International Air Transport Association (IATA) International Dangerous Goods Regulations (DGR) and packaging must comply with Packing Instruction 650 in the DGR.

African swine fever diagnostic specimens are considered hazardous and must be properly packaged and labeled to prevent the spread of the virus. Therefore, it is necessary to use materials that meet the technical requirements (i.e. the relevant IATA requirements for the transport of diagnostic samples, such as 95 kPa pressure test, drop test). To find suppliers for such containers and packages, search the Internet with keywords such as "95 kPa" and "UN3373" and "vial", "tube" or "bag", and in this way you can get the information you need.

• primary containers. Specimens should be stored in an airtight, watertight, sterile container (called the “primary container”) as shown in Figure 27. Each primary container should not contain more than 1 liter. The lid of each container must be sealed with adhesive tape or parafilm. These primary sealed containers must be separately packed in cushioning and absorbent material which, if leaked from containers or vials, can absorb liquid and protect against shock. It is important to label each container with waterproof ink so that the animal from which the sample was taken can be identified.
• secondary packaging. All of these primary containers should be placed in secondary leak-proof, hermetically sealed, watertight containers made of plastic or metal. The secondary packaging must, without leaking, withstand an internal pressure of 95 kPa (0.95 bar) over a temperature range of -40 °C to 55 °C. The absorbent material must also be placed inside the second container. If several fragile primary vessels are placed in one secondary container, each should be wrapped or separated from the others to avoid contact.

WARNING 1) dry ice must not be placed inside the primary or secondary vessel due to the risk of explosion. 2) The primary container must be capable of withstanding, without leakage, an internal pressure of 95 kPa (0.95 bar) over a temperature range of 740 °C to 55 °C.

• Rigid outer packaging. The secondary container shall be packed in an outer packaging using suitable liner material. It must successfully pass the 1.2 m drop test and be specifically marked UN3373. The outer packaging shall not contain more than 4 liters of liquid or more than 4 kg of solids. The quantities indicated do not include ice, dry ice, or liquid nitrogen, which are used to keep samples cold.

Samples shipped at 4°C, usually for short shipments (1-2 days)
Such samples, packaged as above, must be shipped with refrigerants (sufficient to maintain the desired temperature) in insulated and secure packaging, in accordance with IAEA Packing Instruction (IAEA) No. 650, if transported by air.

Samples shipped frozen (-20°C or -70°C)
For shipments longer than three days, specimens must also be packaged as specified, with sufficient dry ice added in an insulated bag to maintain temperature. It is important to ensure that the secondary packaging is in the center of the box, because as the dry ice "melts" the secondary container may leak. The carbon dioxide (CO2) released as a result of the "melting" of dry ice lowers the pH and inactivates the virus; therefore, all primary and secondary containers must be hermetically sealed. When dry ice is used to keep specimens cold during transport, the outer packaging must be vented (i.e. not hermetically sealed) to prevent pressure build-up that could rupture the container. Never freeze whole blood or serum containing a coagulant.

1. Danger labeling and labeling

The outer part of the box (rigid outer packaging) shall bear the following marking:

1. sign “Biological substance Category B” (Figure 28) and the proper shipping name next to it: “Biological substance, Category B” (“Biological substance, Category B”);
2. full name, address and telephone number of the sender;
3. the full name, address and telephone number of the recipient;
4. full name and telephone number of the responsible person who knows about the shipment, for example: responsible person: first name, last name ‚+ 123 4567 890;
5. sticker that reads: "store at 4 degrees Celsius" or "store at -70 degrees Celsius".
   When using dry ice:
6. sign "dry ice" (Figure 29);
7. UN number and proper shipping name for dry ice with the words "HOW TO COOL". The net weight of dry ice in kilograms must be clearly written alongside (Figure 29), for example: UN 1845, DRY ICE, AS COOLANT, NET. ## KG.

2. documentation

Samples sent to the laboratory must be accompanied by an accompanying document, the form of which was previously submitted by that laboratory or, if not available, a cover letter. This letter should include information about the owner of the animal, name of the farm and area, type of animal husbandry system, details of the affected animal/animals, history, clinical signs, and autopsy data. It is also necessary to specify the required tests. Transport documentation: if the consignment crosses national borders, sometimes an import permit or an export permit is required, as well as a copy of the permission from the receiving laboratory that they can accept the infectious substance for diagnostic purposes, etc. Such requirements vary from country to country. It is advisable to ask the recipient's laboratory in advance what documents are required for importing diagnostic specimens.

3. Transportation

Before sending samples, contact the receiving laboratory as early as possible and inform them about the planned shipment, provide details, approximate date and time of arrival. It is best to use a door-to-door courier service that will deliver directly to the laboratory. Once the samples have been shipped, the courier service will be required to provide the receiving laboratory with their company name and postal identifier, waybill and/or air waybill number, if any. If samples are transported by air, prior arrangements must be made with the receiving laboratory to collect the shipment upon arrival at the airport (some international laboratories have this system, but not all). The receiving laboratory should be provided with the airline name, flight number and air waybill number as soon as possible. People are prohibited from carrying infectious substances with them as checked or carry-on baggage, or on themselves.

Transport of isolated/cultured ASF virus

Isolated/cultured ASFV must be transported as infectious substance category A. UN number UN2900, proper shipping name Infectious substances affecting animals (African swine fever virus) . Packaging in accordance with packing instruction 620 must be used. Danger labels and markings on the outside of the box also differ.

The Dangerous Goods Regulations require that all employees involved in transport receive appropriate training. This is particularly important in the transport of Category A infectious substances, where personnel must be trained in accordance with the requirements, including attending special courses, passing examinations and obtaining a certificate (for a period of two years). For more information, please refer to the WHO Guidelines for the Transport of Infectious Substances.

Laboratory diagnosis of ASF

Since there is no vaccine, early and early detection of the disease is essential to implement strict sanitary and biosecurity measures to prevent the spread of the disease. The diagnosis of ASF means the identification of animals that are or have previously been infected with ASF. In order to obtain the appropriate information for the implementation of control and eradication programs, it is necessary to make a diagnosis, which includes the detection and identification of ASFV-specific antigens or DNA and antibodies. When choosing a diagnostic test (Figure 30), it is important to consider the course of the disease. As animals may be at different stages of the disease, both virus detection and antibody detection tests should be performed during outbreaks and in disease control/eradication programs.

The incubation period for natural infection varies from 4 to 19 days. Within two days before the onset of clinical signs, ASF-infected animals begin to shed large amounts of the virus. Virus shedding may vary depending on the virulence of a particular strain of ASFV. Serological conversion occurs around the seventh to ninth day after infection, and antibodies can be detected throughout the rest of the animal's life (Fig. 30).

A positive test for the presence of the virus (i.e. antigen) indicates that the animals tested were already infected at the time of sampling. On the other hand, a positive ASF antibody test indicates a current or past infection when the animal has recovered (and may remain seropositive for life).

Since the end of 2015, epidemiological serological data in Eastern Europe have shown a significant increase in the incidence of seropositive animals, which is especially noticeable in the wild boar population in disadvantaged EU countries. These results indicate that some animals survive for more than a month and can recover from ASF, and in some cases even remain subclinically infected, as previously observed in the Iberian Peninsula, the Americas and Africa. Therefore, antibody detection methods are necessary to obtain complete information for the implementation of disease control and eradication programs.

ASF virus detection

Detection of the ASFV genome by polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is used to detect the ASFV genome in samples taken from pigs (blood, organs, etc.) and ticks. Small fragments of viral DNA are amplified by PCR to detectable amounts. All validated PCR tests can detect viral DNA even before the onset of clinical signs. PCR makes it possible to diagnose ASF within hours of specimens arriving at the laboratory. In the detection of ASFV, PCR is a sensitive, specific, and rapid alternative to virus isolation. PCR has higher sensitivity and specificity than alternative antigen detection methods such as enzyme-linked immunosorbent assay (ELISA) or direct fluorescent antibody (MFA) assay. However, too high a PCR sensitivity poses a risk of cross-contamination, so proper precautions must be taken to minimize this risk.

The conventional and real-time PCR recommended in the OIE Guidelines for Diagnostic Tests and Vaccines for Terrestrial Animals (2016) are fully validated and are good tools for the routine diagnosis of this disease. Other real-time PCR tests are more sensitive than those recommended by the OIE Guidelines and can be used to detect the ASFV genome in recovered animals. The different sets of primers and probes used in these molecular methods are designed to amplify the locus in the VP72 coding region, a well-studied and highly conserved region of the ASFV genome. A wide range of isolates belonging to all 22 known p72 viral genotypes can be detected using these PCR methods even in inactivated or degraded samples.

PCR should be chosen in case of hyperacute, acute or subacute ASF infection. In addition, since PCR detects the viral genome, the reaction can be positive even when no virus is found during virus isolation, making PCR a very useful tool for detecting ASFV DNA in pigs infected with low or moderate virulent strains. Although it is not possible to determine the infectivity of the virus using PCR, this method provides information on its quantity.

ASF virus isolation
Virus isolation is based on sample inoculation into susceptible primary cell cultures of porcine origin, monocytes and macrophages. If ASFV is present in the sample, it will replicate in susceptible cells, inducing a cytopathic effect (CPE) in infected cells. Cell pisis and CPE usually occur after 4872 hours of haemadsorption. The importance of this finding lies in its specificity, because none of the other porcine viruses is capable of haemadsorption in leukocyte cultures. When the virus replicates in these cultures, most ASFV strains induce a hemadsorption reaction (HAd) by adsorbing porcine red blood cells onto ASFV-infected leukocytes to form so-called “rosettes” (Fig. 31).

However, it is important to note that CPE, in the absence of haemadsorption, may be caused by inoculum cytotoxicity, the presence of other viruses such as ADV, or a non-haemadsorbing VASF isolate. In these cases, the presence of ASFV in the cell sediment must be confirmed by other virological tests, such as MFA, or using PCR. If no change is observed, or if MFA and PCR are negative, the supernatant should be sub-inoculated into fresh cultures up to 375 passages before ASFV can be ruled out.

Virus isolation and identification by GAd are recommended as reference tests to confirm the positive results of a preliminary positive antigen test (ELISA, PCR or MFA). These tests are also recommended when ASF has already been confirmed by other methods, especially in the case of the first outbreak of ASF in the area. In addition, virus isolation is mandatory if your goal is to obtain viral material for subsequent characterization by molecular and biological methods.

Detection of ASF antigen using the direct fluorescent antibody method (MFA)
MFA can be used to detect the ASFV antigen in swine tissues. The test consists in the microscopic detection of viral antigens on smears-imprints or thin cryosections of organ tissue. Intracellular antigens are detected using specific antibodies conjugated with fluorescein isothiocyanate (FITC). MFA can also be used to detect ASFV antigen in leukocyte cultures that do not show AHAD, and thus non-hemadsorbing ASFV strains can be identified. MFA can also distinguish between CPE caused by ASFV and CPE induced by other viruses or inoculum cytotoxicity. Positive and negative controls are used to ensure correct interpretation of slides. It is a highly sensitive test for cases of hyperacute and acute ASF and can be performed fairly quickly. This is a reliable test, but in most cases it is being replaced by PCR and reagents are not always available. However, it is important to note that in the subacute and chronic form of the disease, the sensitivity of MFA is much less (40%).

Detection of ASF antigen by antigen-ELISA
Viral antigens can also be detected using enzyme-linked immunosorbent assay (ELISA), which is cheaper than PCR and allows large-scale testing of samples in a short time without special laboratory equipment.

However, as in the case of MFA, in the subacute and chronic form of the disease, the sensitivity of the antigen-ELISA is significantly reduced. In addition, field samples are often in poor condition and this can also reduce the sensitivity of the test. Therefore, it is recommended to use the antigen-ELISA (or any other ELISA test) only as a "group" test, together with other virological and serological tests.

ASF antibody detection

Serological assays are the most commonly used diagnostic tests due to their simplicity, relatively low cost, and the fact that they do not require a large amount of specialized equipment or laboratory. Since there is no vaccine against ASF, the presence of antibodies to ASF always indicates a current or past infection. In addition, ASFV antibodies appear soon after infection and persist for several years. However, in hyperacute and acute infections, pigs often die before antibody levels reach detectable levels. Therefore, it is recommended to collect samples and to detect viral DNA already in the early stages of an outbreak.

The following tests are recommended for the detection of antibodies to ASF: ELISA for antibody screening and, as confirmatory, immunoblotting (IB) or indirect fluorescent antibody (nMFA). The indirect immunoperoxidase test (IPT) can be used as an alternative confirmatory test to detect ASF antibodies in serum and tissue exudate. It can be used with a large number of samples, does not require expensive fluorescence microscope equipment, and provides sufficient sensitivity.

Detection of ASF antibodies by ELISA test
ELISA is a very useful technique and is widely used in large scale serological studies of many animal diseases. Some of the most outstanding characteristics of this method are high sensitivity and specificity, speed of execution, low cost, and easy interpretation of the results. Large populations can be screened quickly with automated equipment.

In order to detect antibodies to ASF in serum samples, ELISA uses labeling of antibodies with certain enzymes. When the antigen and antibody bind to each other, the enzyme causes a reaction that causes a color change, thereby identifying the presence of ASF. A variety of commercial and in-laboratory methods, such as indirect or blocking ELISA, are currently used to detect ASF antibodies.

Incorrectly processed or poorly preserved serum (due to inadequate storage or transport) and hemolyzed specimens can result in up to 20% false positives. Thus, all positive and questionable samples after the ELISA test should be tested by alternative serological confirmatory methods.

Immunoblotting (IB) is a fast and sensitive assay for the detection and characterization of proteins. It uses specific deterministic antigen-antibody recognition. This test uses antigen strips that carry viral antigens. The test includes solubilization, electrophoretic separation and transfer of proteins to membranes (usually nitrocellulose is used). The membrane is overlaid with primary antibodies to a specific target and then labeled secondary antibodies to visualize a positive reaction.

The first viral proteins that induce ASF-specific antibodies in pigs invariably react to IB in all infected animals. In surviving animals, reactions become positive with sera obtained from animals 7-9 days after infection and up to several months after infection. Sera from animals vaccinated against other viruses may give false positive reactions. In such cases, alternative confirmatory methods such as IPT or MFA should be used.

Detection of ASF antibodies using indirect fluorescent antibodies (nMFA)
The test is based on the detection of ASF antibodies bound to a monolayer of African green monkey kidney cells infected with adapted ASFV. The antigen-antibody reaction is detected using a conjugate labeled with fluorescein. Positive samples show specific fluorescence in the cytoplasm of infected cells. nMFA is a rapid method for the detection of ASF antibodies in serum, plasma or tissue exudate, with high sensitivity and specificity.

Detection of ASF antibodies using an indirect immunoperoxidase test (IPT)
IPT is a fixed cell immunocytochemical method for determining the formation of an antigen-antibody complex under the influence of peroxidase. In this method, green monkey kidney cells are infected with an ASFV isolate adapted to these cell cultures. Infected cells are fixed and used as antigens to determine the presence of specific anti-ASF antibodies in samples. Like MFA, IPT is a rapid, highly sensitive, and highly specific method for detecting ASF antibodies in serum, plasma, or tissue exudates. Interpretation of results is easier than in MFA due to the enzymatic imaging system used.

Summing up, we can say that modern diagnostic tests make it possible to confidently diagnose ASF by combining methods for detecting both the virus and antibodies. Real-time PCR is the most widely used virological diagnostic method for the sensitive, specific and rapid detection of ASFV DNA. Because of the possibility of cross-contamination, one positive PCR result from a single animal from a natural habitat (e.g. wild boar) or one positive PCR result from one group of animals must be confirmed by additional virological tests in combination with serological, pathological and epidemiological results. Because PCR detects the presence of viral DNA and not live virus, it is strongly recommended that virus isolation be performed from infected specimens before an outbreak is confirmed if a new region is affected.

Given the limitations of different methods, validated ECBA tests are the best method for detecting ASF antibodies, especially for screening serum samples. Confirmatory tests such as IB, nMFA, or IPT are key to identifying false positive results from the ECB. In addition, nMFA and IPT are the recommended methods for the analysis of tissue exudates and plasma samples, providing a complete epidemiological picture and allowing time of infection to be determined.

An accurate diagnosis of ASF should be based on virological and serological results, as well as clinical, pathological and epidemiological data. Table 5 shows the characteristics of the main laboratory methods for diagnosing ASF.

Prevention and control

African swine fever differs from most other transboundary animal diseases in that there is no vaccine or cure available to prevent or treat the disease. Therefore, it is especially important that regions free from this disease remain so in the future. Preventing the introduction of ASFV into domestic and feral pig populations and controlling and eradicating the disease as soon as it is detected are the best ways to minimize the impact of this disease. There are, however, also successful examples of ASF eradication, for example in Brazil, Portugal, Spain or Côte d'Ivoire.

Prevention begins with the introduction of tough measures at the border and raising awareness among all stakeholders. Early detection, early diagnosis, early response and good communication are critical to minimizing the spread of the disease after an introduction. In order to understand which measures will be most effective, it is important to keep in mind how ASF is transmitted: i.e. first of all, when moving infected pork and products from it (infection occurs after eating); by direct contact with live animals, including wild pigs; and through the bites of Ornithodoros ticks.

Measures can be taken at the institutional or individual (eg farmer) level, most of these measures involve improving biosecurity. Prevention and control actions can be carried out through private or public initiatives, but reaching the optimum level usually requires a combination of both. Farmers play a key role, but they may need technical and financial support.

For more information, please refer to the two FAO guidelines: Good Emergency Management Practice (GEMP): Fundamentals (FAO, 2011), and Good Biosecurity Practice in the Swine Sector (FAO, 2010).

Awareness
Raising awareness as well as providing information/technical assistance and training to all stakeholders has a direct positive impact on the implementation of all disease prevention, control and surveillance activities. Therefore, raising awareness is considered the most cost-effective measure. Awareness helps pig producers make quick, effective decisions when implementing prevention and control measures.

Persons in contact with pigs should be aware of how to prevent and respond to ASF. These include veterinarians and farmers, as well as all those involved in the market chain, i.e. persons involved in the transportation, sale, slaughter and butchering of pigs; service providers (eg private veterinarians, feed distributors, etc.); and in some cases, the general public. In the case of wild boars, hunters, foresters and logging workers are also the target audience.

It is very important to establish regular contacts between the veterinary service (professionals or para-professionals) and livestock keepers/traders. These should be not only routine visits, but also "home visits" to investigate and provide assistance in connection with the disease. In this way, farmers will gain confidence to seek official veterinary help when faced with unusual and potentially devastating diseases like ASF. This bottom-up approach will also allow farmers' input to be taken into account when developing prevention, management and strategy tools. For those countries where the private sector is the provider of official veterinary services, additional interaction between them and the veterinary authorities is needed (GEMP, 2011).

All stakeholders should be aware of the potential severity of ASF, how to detect and prevent it (i.e. the clinical presentation), and the need to immediately report any suspicion of ASF to the veterinary service (i.e. passive surveillance). The latter is particularly important as farmers may perceive large numbers of pigs to die as “normal”. Measures to reduce the likelihood of infection should also be communicated. The dangers of feeding on food waste and other violations of biosecurity need to be emphasized, especially for smallholders and the private sector. If ASF is introduced into the country, the issue should be well publicized in the press, emphasizing the importance of strengthening biosecurity at all levels, regularly checking pigs, and promptly reporting suspicious lesions and deaths to the authorities. Even information on control policies, such as slaughter, compensation and restocking, will help farmers understand their role in this process and strengthen their willingness to cooperate.

Livestock traders, traders and dealers are often overlooked, despite the fact that this is an important target group that needs to be informed. The movement of animals carried out by traders is often a key factor in the spread of epizootic diseases such as ASF. Building trust between veterinary authorities and those involved in the animal trade is just as important as with farmers. The main themes should be general, although emphasis should be placed on the importance of acquiring animals from disease-free regions, so that they do not buy or sell sick pigs or pigs from groups where there have been cases of the disease, and that they comply with the rules of quarantine, vaccination , testing, identification of animals and their accounting. However, the potential impact of ASF on domestic and international trade should be highlighted (GEMP, 2011).

The development and dissemination of information and training is carried out mainly by government agencies (and sometimes NGOs) through agricultural extension and advocacy services rather than by the private sector. There are many ways to communicate information, such as flyers, booklets, posters, TV and radio messages, meetings organized by religious leaders or village elders, etc. The format depends on the target group. In some cases, however, more thorough preparation is required. When it comes to awareness materials, there are several formats, from online courses to traditional face-to-face training. When there is a need to provide information to a large number of people, the train-the-trainer model may be the best approach. This approach is also referred to as "cascading training" because these programs are designed to train people who in turn will train others.

Prevention
The risk of introducing ASFV (or any other pathogen) is reduced if good biosecurity practices are applied not only on the farm, but at every stage of the supply chain, such as live animal markets, slaughterhouses, animal transport, etc. Particular attention should be paid to small commercial operations, such as backyards, which have low biosecurity standards, markets where animals flock from many sources. They are key to the spread of ASF and although the same biosecurity concepts apply, specific measures and instructions have been developed specifically for them.

Biosecurity measures should be used to avoid the entry of pathogens into the herd or farm (external biosecurity) and to prevent or slow the spread of disease in non-infected animals in the herd or farm after infection (internal biosecurity) and stop the infection other indoors or wild pigs. With government-mandated biosecurity regulations on farms, needs and expectations vary by farming system and local geographic and socio-economic conditions (from large-scale, indoor farms to small village grazing pig farms). Global biosecurity issues are relevant to all production systems, but they are especially problematic for small households in developing countries and countries with economies in transition. However, a wide range of options for improving biosecurity, for example sometimes as simple as improving record keeping, means that all farms can improve their disease prevention and control practices.

The ability of farmers to implement on-farm biosecurity measures depends on the characteristics of their production system, their technical knowledge and financial resources. Those responsible for improving biosecurity programs need to have a thorough knowledge of the various systems and understand the people involved in pig production, such as why they keep the animals and what resources they have. By taking these factors into account, they will be able to develop sustainable biosecurity strategies on farms and along production and value chains.

There are differences between on-farm biosecurity measures before an outbreak (biological containment) and after an outbreak has occurred (biocontainment), although these good prevention and management measures are closely related. In order to distinguish methods of ASF prevention from general disease prevention, it is necessary to take into account the ways of transmission of ASF. Some of the most important biosecurity measures are listed below. More information on biosecurity can be found in the FAO Guidelines for Good Biosecurity Practices in the Swine Sector.

Feeding food scraps
Feed is an important control point for the spread of both ASF and other diseases. By its nature, food waste is a convenient, affordable, but very dangerous way to feed. Feeding offal poses a very high risk of potentially infecting a healthy pig population with a range of diseases. An effective ban on feeding offal would be the ideal solution, but it is unlikely to be enforced at the household level as it goes against the main motive for keeping pigs, i.e. minimum feeding costs due to food waste or pasture. In any case, pigs should not be given food waste containing pork, but should be boiled for 30 minutes, stirring occasionally, and fed chilled to the pigs.

Restriction on the movement of pigs
The construction of pigsties that allow for hygienic conditions should be encouraged. Also, a fenced perimeter will prevent direct contact and spread of potential disease from domestic pigs to wild boars (and feral pigs) and vice versa from wild African pigs to domestic pigs. A fenced perimeter can also limit feral and domestic pigs' access to litter, offal, or animal carcasses that may be contaminated. The fence not only keeps the domestic pigs inside the building and the wild ones outside, but it must also go underground to a depth of at least half a meter, as the pigs can dig the ground under the fence. In general, the authorities should prevent the establishment of grazing pig farms, as they provide pigs with access to potentially infected offal or animal remains, allow contact with infected wild boars, other free-range pigs or feral pigs.

However, like waste feeding, traditional ways of keeping pigs are not easy to change, as many farms may decide that it does not make sense to keep (and feed) pigs under such conditions. A significant part of the pig sector operates on the basis that pigs can be freely grazing. Thus, any move towards a more closed system, with the subsequent increase in feed costs, is likely to provoke resistance from many small farmers.

It is difficult to implement an effective biosecurity system if the pigs spend most of the day freely rummaging through the garbage. However, some simple precautions at a minimum cost of money and time can still be recommended. It is possible to apply perimeter fencing around the entire village because the pigs of the same village are assumed to have the same health status. However, this solution is not always practical. It is useful to note the benefits of insulation in preventing theft, traffic accidents and predators. In general, when maintaining biosecurity in open-air farms, more attention should be paid to the control of feed, water and pastures, as well as wildlife and visitors.

Cleaning and disinfection
On the farm, equipment and facilities must be cleaned and disinfected frequently. Pigsties, equipment, vehicles, etc. must be cleaned of organic contamination before disinfection. Employees and vehicles (shoes, equipment, etc.) must be disinfected at the entrance / entrance to the farm and exit / exit from the farm. Disinfectants that have been proven effective include detergents, hypochlorites, and glutaraldehyde. VASF is sensitive to ether and chloroform. The virus is inactivated with 8/1000 sodium hydroxide (30 minutes), hypochlorites - 2.3% chlorine (30 minutes), 3/1000 formalin (30 minutes), 3% orthophenylphenol (30 minutes) and iodine compounds (OIE, 2013 .). Effective commercial products are also available. Consideration should be given to the environmental impact of these agents. Equipment that is not easily disinfected should be exposed to sunlight.

Other biosecurity measures

• The number of visitors should be kept to a minimum and should only be allowed in after shoes have been cleaned and disinfected or clothing and shoes have been changed, especially in the case of high-risk visitors such as livestock owners and veterinary staff. People working with pigs should avoid contact with other pig populations.
• Vehicles must not enter the farm and the loading and unloading of pigs in particular must take place outside the perimeter of the fence. Trucks carrying pigs must be cleaned and disinfected after unloading.
• Equipment should not be exchanged between farms/villages without proper cleaning and disinfection.
• Workers should be provided with work clothing and footwear allocated for this purpose only.
• Where practicable, farms should operate as closed herds, with a limited supply of new animals.
• Newly acquired animals must come from reliable sources and be quarantined (i.e. kept in isolation for the purpose of observation) for at least 14 days.
• Farms should be located at an appropriate distance from each other.
• In raising pigs, age segregation should be observed (according to the “empty-busy” system).
• Dead pigs, sewage and carcass residues left after slaughter must be disposed of properly, out of the reach of wild pigs and domestic pigs on the range.
• Pigs that have been in the live market should not be returned to the farm. However, if brought back while she is, they must be kept in quarantine for 14 days before being introduced into the herd.
• Personnel must be trained in good sanitation and hygiene practices and disease recognition.
• Keep wild birds, pests, and other animals away from pigsties, animal feed, and water systems.

Risk analysis and import-export procedures
The concept of biosecurity can also be applied at the national level. Just like on a farm, the only way to prevent the entry of ASF into countries free of this infection is through a strict policy for the safe importation of pigs and high-risk products, i.e. pork and pork products, pig semen, skins, etc. Such preventive measures help to reduce the incidence of the disease and its consequences. Detailed guidelines can be found in the OIE International Terrestrial Animal Health Code (2016). GEMP (2011) provides the following:

• Adequate awareness should be maintained to provide early warning of changes in distribution and epidemiology in affected countries and trading partners. Information should be collected on points of entry into the country of pigs and pork supply chains, distribution of holdings according to their production cycle, feral pigs, animal sales, slaughterhouses, etc. This data will assist in risk analysis of all potential entry and distribution routes. This should be carried out on a regular basis and depending on the risk assessment. The measures taken must be dynamic and appropriate to the degree of risk.
• Prevent the introduction of the pathogen as part of legitimate imports through additional targeted restrictions in accordance with recognized international standards. Restrictions on imports will reduce the risks existing in trade and ensure the maximum effectiveness of the “quarantine barrier”.
• Customs, regulators and quarantine authorities must effectively “intercept” illegal/unregulated food and other hazardous materials at international airports, seaports and border crossings. Confiscated materials should be destroyed or safely disposed of and not thrown within reach of people or animals. Recent events indicate that special attention should be paid to the proper disposal of food waste from aircraft, ships or vehicles arriving from disadvantaged countries, preferably by incineration or, if possible, by processing of non-food animal raw materials.
• Consider testing products for certain diseases of concern before and after import, depending on the level of risk.
• Create and expand cross-border information exchange with neighboring governments.

Control
When an outbreak is suspected, it is important to take appropriate immediate action. Veterinarians, as well as farm owners, workers and other stakeholders, must do everything possible to contain and prevent the further spread of this disease. Because ASF-infected animals begin to shed the virus 48 hours before clinical signs appear, the elimination of feed, bedding and animals (both live and slaughtered) from infected premises is critical.

After the disease has been detected and confirmed, it is necessary:

1. resort to a contingency plan;
2. evaluate the initial outbreak (eg size, geographic distribution, epidemiology) and determine what control measures may be needed;
3. implement control measures promptly and fully;
4. monitor progress and adjust policies;
5. continue to exchange information and data with neighboring administrations;
6. communicate with the public and all interested parties, including the OIE (GEMP, 2011).

The measures taken to control and eradicate the disease will depend greatly, at least initially, on how widespread the disease is and how severe the incursion was before it was discovered. The wider the spread of the disease and the more farms it affects, the less likely it is that slaughter will be effective as a means of eradication. Slaughter is the most effective measure when it can be carried out within the first few days. To do this, you need to quickly identify the disease, and slaughter the affected animals immediately after detection, for which compensation is paid. If this is not possible, animal movement controls and other actions may need to be introduced. Therefore, it is extremely important to establish the geographic distribution and number of affected farms at the start of the outbreak (i.e. epidemiological surveillance). Usually the so-called "index case" (first case found) is not actually the first one (GEMP, 2011).

Equally important are actions at the final stage, when the clinical manifestations of the disease have ceased. If foci of infection go unnoticed, the results of the campaign to eradicate the disease can be nullified. One should not lose vigilance or abandon surveillance and control efforts when the clinical manifestations of the disease seem to have disappeared and there is no longer any socio-economic losses. If surveillance is terminated prematurely, ASF may re-emerge.

Contingency Planning (GEMP, 2011)

Preparing for an emergency is the key to effective emergency management. However, preparation should be carried out at the warning stage, that is, in "peacetime". It is important to agree in advance and have a clear understanding of who is responsible for what, and create a single chain of command and lines of communication. In peacetime, the distribution of responsibility often occurs differently. A key benefit of planning is that it predetermines the people who will be involved in the process and forces them to think carefully about the problems that may arise. This allows you to prevent possible errors or shortcomings even before the outbreak.

Farmer participation can make a significant contribution to emergency planning. Rural communities are more likely to cooperate in emergencies if they see that quick and decisive action is being taken and that it will ultimately benefit them. They should also be aware that they have contributed to the planning and that their input has been taken into account.

These plans and instructions are "living" documents that should be reviewed and updated regularly (at least every five years) to reflect any changes that have taken place since then.

Participants should be regularly trained in disease detection, reporting and response procedures, outbreak investigation and analysis, etc. Regular simulation and field trainings with the participation of all stakeholders help to implement emergency plans and operational instructions in practice. Regular training and practice is a key aspect in maintaining real control capacity as well as filling gaps in the existing system.

Legal Framework (GEMP 2011)

Appropriate legal authority is needed to take swift action to control the disease. These include the right to enter the farm (for surveillance, prevention and control purposes), slaughter and destroy infected and contacted animals, establish quarantine and movement controls, identify infected and restricted areas, provide compensation, etc.

Giving legal powers takes time, so they must be established in "peacetime". Since it is not possible to develop a set of rules for every disease, there should be a common set of legal powers and regulations that apply to the diseases listed for notification and control.

Sometimes it becomes necessary to enlist the support of the police and law enforcement agencies, for example, when restricting the movement of livestock, establishing quarantines and protecting personnel.

In countries with a federal system, uniform and consistent legislation should apply throughout the country. The same should be observed between countries in duty-free (i.e. unrestricted foreign trade) regions of animals and animal products, such as the Economic Community of West African States (ECOWAS), the South African Development Community (SADC), the Common Market for Eastern and South African States (SOMEBA), the East African Community (EAC), the Eurasian Economic Union (EEC) or the European Union (EU).

Financing (GEMP, 2011)

Experience has shown that the delay in obtaining funding is one of the main barriers to rapid response to unexpected outbreaks. Immediate application of even modest amounts will help to avoid significant expenses in the future. Therefore, advanced financial planning is an important component of preparedness. The financial plan should cover both current costs (eg supervision, risk analysis) and costs that may arise during an emergency (eg control). Such costs should be included in the Contingency Plan.

Funding may cover the cost of the entire campaign. As a rule, they cover only the initial stages, spending further funds occurs after a review of the campaign and the funds needed to complete the eradication of the disease. In some countries, it would be better if funds for emergency programs against certain diseases were provided not only by the government, but also by the private sector (cost-sharing).

Communication
An important aspect of disease control is communication with stakeholders at all levels, from farmers to the general public. It is best to agree on who will be interviewing and limit communication to only insiders and trained individuals.

Movement control
The spread of ASF is mainly due to human activities, and not due to the movement of wild boars or other vectors of infection. The spread of the disease due to the movement of live animals and animal products can be controlled by restricting their movement, which should be supported by legislation. It is best if the owners of animals or animal products themselves understand that compliance with the requirement is in their interest.

Unfortunately, quite often, when a disease outbreak is suspected, pig farmers rush to sell animals for slaughter. Selling contaminated meat from sick animals is a serious risk. Sick pigs, even in the incubation period of the disease, can spread ASF, especially if the animal is sold alive.

Following an outbreak or suspected case on a farm, a strict quarantine should be introduced as soon as possible, i.e. no pigs, pork meat or potentially contaminated materials should leave the farm. No one should leave the farm without changing clothes or disinfecting clothes and shoes. Free range pigs should be driven indoors and locked up.

In the outbreak area (restriction zone), authorities must prevent any illegal trade in dead or sick animals and their products. The exact boundaries of these restricted areas do not need to be circular, but should be taken into account and natural barriers and administrative boundaries and any relevant information should be used. The boundaries of these zones must be clearly marked by road signs.

Various zones and periods of animal movement restriction can be created to prevent the spread of the disease. Such restrictions will be most effective if they have minimal impact on pet owners. It is recommended that:

1. all pig farms were registered and registration of all animals was carried out;
2. all susceptible animals on these holdings were subject to regular veterinary examinations;
3. susceptible animals (or products of their processing) were not taken out of the farm;
4. the exception is forced slaughter under official supervision.

Inspection of animals and the establishment of checkpoints are an important part of the process of implementing traffic control. However, checkpoints on major roads can cause unacceptable traffic disruptions or be prohibitively expensive. In addition, pigs may be smuggled out of the restricted area by hiding them in vehicles or along unguarded secondary roads (GEMP, 2011).

Stamping out and disposal
Infected and actively shedding animals are the largest source of ASF. Such animals can also lead to indirect contamination by contaminating objects (fomites), including vehicles, clothing and, in particular, footwear. ASF replication stops when the animal dies. However, animal carcasses can remain contaminated for a long period after death, hence the need for prompt and efficient disposal (GEMP, 2011).

Stamping out includes the slaughter of infected animals, plus usually all other susceptible animals on the farm and sometimes in neighboring holdings or in contact, i.e. those who came into contact due to the movement of animals, people or vehicles. It is very rare to produce large-scale slaughter, in particular, annular, solely on the basis of geographical location. Animal slaughter must be carried out locally and humanely, using gentle methods. Production capacity at such a mass slaughter can be overloaded, so careful planning of resources, equipment and personnel is necessary. This is especially true when it comes to stamping out large commercial swine herds.

After stamping out, carcasses should be disposed of locally, if possible, in a safe manner, i.e. they must be incinerated, composted, recycled or buried to prevent access by wild pigs, wild boars and other scavengers (including humans). Disposal of a large number of pigs in a short time is a big problem both from the point of view of logistics and from the point of view of ecology.

The only major problem with stamping out is that pig owners object to the slaughter of animals in the absence of timely and adequate compensation. Without appropriate compensation mechanisms, it is likely that farmers will not always report disease outbreaks and the disease will spread through the illegal movement of infected animals and products. Therefore, no stamping out campaigns can be applied in the absence of a proper compensation program.

Cleaning and disinfection
Destruction of carcasses must be accompanied by thorough cleaning and disinfection of all premises, vehicles and equipment. Although disinfection with appropriate substances helps to eliminate the virus, ASF can survive in a protein-rich environment for long periods of time and under a wide variety of conditions.

Organic matter must be removed from pigsties, equipment, vehicles and all surfaces that have been in contact with contaminated material. Cars (especially underbody, bedding if live pigs were transported, body) and employees (shoes, equipment, etc.) must be cleaned and then disinfected at the entrance/entrance and exit/exit from the farms.

Proven effective disinfectants include detergents, hypochlorites, and glutaraldehydes. VASF is sensitive to ether and chloroform. The virus is inactivated with a solution of 8/1000 sodium hydroxide (30 minutes), hypochlorites - 2.3% chlorine (30 minutes), 3/1000 formalin (30 minutes), 3% orthophenylphenol (30 minutes) and iodine compounds (OIE, 2013). ). Effective commercial products are also available. Consideration should be given to the environmental impact of these agents. Equipment that is difficult to disinfect should be exposed to sunlight.

Compensation (GEMP, 2011)

The compensation policy is the cornerstone of any disease control policy that requires the slaughter of animals or the destruction of property. Compensation is key to ensuring that farmers notify authorities in a timely manner of an outbreak. While compensation may be seen as costly by some, the incentive it creates for early and rapid warning will reduce the overall cost of dealing with an outbreak. All in all, this is a very likely opportunity to save money.

Compensation can take many forms, which have been and are being discussed extensively. In order to implement an accurate compensation strategy, all aspects must be carefully analyzed, taking into account the local context and with the participation of all stakeholders. Compensation can be in cash or goods, such as replacement animals. But regardless of the type of compensation - cash or animals, farmers should be consulted, if possible, before an outbreak occurs. The advantage of cash is that it allows breeders to choose the type and number of animals they want to buy and, last but not least, the timing. However, paying cash can lead to corruption and theft.

Compensation should be paid for any animals slaughtered as part of mandatory slaughter, whether they were infected or slaughtered for possible exposure to infection, or for animal welfare, as sometimes happens. In reality, the government buys the animals and then kills them. Compensation must also be paid for goods and property destroyed during the mandatory stamping out campaign. Given that compensation is primarily intended to encourage farmers to report outbreaks in a timely manner, it should not be paid for animals that died or were slaughtered by the producer before the outbreak was confirmed.

Compensation is only effective when it is paid shortly after the losses incurred. Therefore, it is necessary to plan in advance how compensation will be paid to those who are entitled to it.

Compensation amounts should be based on the fair market value of the animals at the time of slaughter and, where possible, their full market value. However, some experts recommend that compensation be just below market value, arguing that farmers should also contribute, for example, 10 percent. Inadequate or overly generous compensation mechanisms may encourage behaviors that are detrimental to the control system.

Lack of adequate and timely compensation for animal slaughter can lead to:

1. that the outbreak will not be reported;
2. slaughter of animals by farmers for their own consumption or sale;
3. hiding animals or moving them to other premises;
4. improper disposal of the carcass of an animal in places accessible to domestic or wild pigs.

Too generous compensation can encourage dishonest farmers who rely on the fact that if the animals become infected, they will receive compensation.

Producers suffer the most serious losses due to production losses during the outbreak, not due to dead animals or restrictions on movement (for example, because they are unable to sell animals). However, these losses are not predictable because they depend on the overall duration and severity of the outbreak. Thus, other support mechanisms (eg financial and social, apart from compensation) are needed and should be included in the plan to assist the affected farmers.

Livestock replenishment

Once the disease has been eradicated, the next step in the ASF management system is to restore production on the farm or region. After a massive outbreak, some owners are unwilling to restock or continue to raise farm animals. But most farmers still want to return to the traditional way of life and replenish the number of pigs.

Before starting this process, you should make sure that the pathogen on the farm is destroyed. This can be achieved by cleaning and disinfection, which should be carried out twice. In addition, it is desirable to improve the biosecurity system on the farm before restocking. After cleaning and disinfection of empty rooms, at least 40 days should elapse, but this period always depends on the situation and can only be established after a risk analysis. If indicator pigs (sentinels) are introduced, which is highly recommended, then the condition of the animals should be observed (clinically and serologically) in order to identify possible reinfections. If no signs of infection are observed after 40 days, these sentinel pigs can be used as part of a restocking program.

Pigs for restocking, if possible, should be purchased from the same area or nearby. Such animals are adapted to local conditions and farmers are usually very familiar with their needs. Buying from multiple sources means buying animals that have different health and immune status. Mixing different animals creates a stressful situation and can lead to cross-infection.

Mite Control

Eradication of Ornithodoros mites from infested piggeries is a difficult task, especially in older buildings, due to the longevity of the mites, their hardiness and their ability to hide in crevices that acaricides cannot penetrate. Destruction of the tick's habitat (for example, treating cracks where ticks hide or building new structures with materials that do not have cracks) helps to reduce their numbers and the possibility of transmission. Infested premises should not be used as pigsties. They must be isolated in such a way that the pigs cannot enter them, or destroyed and rebuilt in some other place. If farmers are able to rebuild previously contaminated premises, then this should be done. This is also an opportune moment to consider improving biosecurity.

Acaricides and other pesticides can be applied to disinfect bedding or, depending on the product, directly on the skin of pigs.

Because blood-sucking insects can mechanically spread ASFV in a herd, it is recommended that insect control programs be carried out in infested premises.

Wildlife Management

There are no realistic measures that can be taken to prevent the transmission of ASF in feral pig and Ornithodoros tick populations. The only option is to implement preventive measures to protect domestic pigs from infection. In parts of southern and eastern Africa where the forest infection cycle occurs, the construction of appropriate enclosures or permanent housing for domestic pigs has successfully demonstrated complete protection for over a century. Fences and walls should go at least 0.5 m deep into the ground to prevent access by African boars burrowing the ground. The recommended height of the fence is 1.8 meters. In addition, in South Africa, in areas where the forest cycle of infection occurs, control of Ornithodoros ticks in African wild boars and in burrows is carried out along the perimeter of farms.

If ASF affects a wild boar or feral pig population, effective control becomes much more difficult. The strategy is to minimize contact between wild boars and domestic pigs by fencing pigsties, limiting the number of free range or feral pigs, and ensuring proper disposal of kitchen waste and carcasses. There are different views on how best to control ASF in wild boar populations. The removal of wild boar carcasses during an epidemic and the subsequent decontamination of these areas, although expensive, have been widely and successfully used in Eastern Europe. Intense hunting can be counterproductive as it can push wild boars to move to other areas. Feeding can keep wild boar within a known, well-defined area, thus limiting the dispersal of wild boars and the spread of the virus. However, feeding will also promote close contact between animals, thereby facilitating disease transmission. Fencing open areas to avoid the movement of wildlife is difficult and expensive, not only in terms of construction, but also maintenance. This interferes with movement and migration in the wild, and its effectiveness is questionable as feral pigs will be able to find their way under or over the fence. The use of deterrents is also problematic. Hunters and hunting clubs, as well as forestry services, are important partners in the surveillance and control of ASF in wild boar populations.

Zoning and compartmentalization

When a disease is present in only one part of a country, zoning becomes an important strategy for the gradual eradication and eradication of the virus without hindering trade from disease-free zones. In order to apply zoning, national authorities need to define infected zones and disease-free zones, and enforce tight controls on the movement of pigs and products between them. Compartmentalization is another approach based on creating a sub-population with its own supply chain under a common biosecurity management system. These sub-populations are clearly defined and separated from other sub-populations, with a different or potentially different status. Compartmentalization is very suitable for commercial pig farms and allows business activity to continue even in the infected area. Costs and responsibility for compartments are the responsibility of the manufacturer and his suppliers, but monitoring and authorization remain the responsibility of the competent veterinary authorities.

African swine fever is also called Montgomery's disease. It was first recorded at the beginning of the 20th century in South Africa. After that, in a fairly short period of time, she "moved" to Spain, Portugal, America, Central and Eastern Europe, Asia, and cases of swine diseases in Russia and Ukraine became more frequent. Initially, only wild boars were sick with it, but over time, it began to threaten ordinary domestic pigs as well.

What is African swine fever?

African swine fever (ASF) is an infectious disease that causes a number of very serious life-threatening symptoms in pigs. When examining the internal organs of diseased animals, many foci of hemorrhage are found, some organs are greatly enlarged, others swell.

The causative agent of the disease is the Asfivirus virus and this is what distinguishes the disease from simple swine fever, which is caused by the Pestivirus virus. At the moment, several genotypes and seroimmunotypes of the virus are known, each of which has, in fact, minor differences.

The genome of the African plague is very strong, it can survive at very low and high temperatures, drying, high acidity, rotting, freezing. And with all this, it remains active.

In pork meat, this virus can live up to several months, and is transmitted if it is not thoroughly cooked. But experts and doctors assure that ASF is harmless to humans if the meat is well fried or boiled at a temperature of 70 degrees Celsius and above before eating.

African swine fever is not dangerous to humans.

How is the virus transmitted?

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African swine fever is transmitted through the skin, oral cavity by direct contact with an infected individual. It is for this reason that the disease always reaches a large scale. Almost all individuals in the stall die if they live together and among them there is at least one infected pig.

Also, the virus can enter the body of a pig through the bites of insects that carry it (lice, ticks, zoophilous flies). The disease is also carried by rodents, birds, and even people who have contact with infected pigs. So healthy individuals in the stall do not provide 100% certainty that the disease will never appear.

The disease can “come” to the farm with poor-quality feed. African swine fever lives quietly in spoiled food waste, which is usually fed to pigs. Walking pigs in places where the influence of the virus was previously noticed is not recommended, since it can also live in the ground.

The lesion can occur regardless of the sex, breed or age of the pig. So all the animals that live together are at risk.

The main symptoms of the disease

The incubation period of the virus is 5-15 days. But in real life, it can be delayed for 1-2 weeks. It all depends not only on the virus itself, but also on how, where the pig was infected, its immune system and the amount of virions that entered its body. There are hyperacute, acute, subacute and chronic forms of African swine fever.

• Hyperacute illness develops instantly, and death occurs suddenly. In this case, the breeder may not be aware of the disease, and only then find out about the causes of death of the animal. This form has no symptoms.
• The acute form develops up to a week. It proceeds with high temperature (40.5-45 degrees), weakness, shortness of breath, lethargy, paresis of the limbs, purulent discharge from the nose, eyes, vomiting, diarrhea with blood. Bruises appear on the skin in the lower part of the neck, perineum, abdomen, ears. Perhaps the development of pneumonia, pregnant females lose babies. A few hours before death, the temperature drops dramatically, then the pig falls into a coma and dies.
• Subacute form lasts 15-20 days. There may be fever, lethargy. Death usually occurs as a result of heart failure.
• The chronic form is accompanied by secondary infections. Symptoms are periodic bouts of shortness of breath, fever. Wounds appear on the skin that do not heal even with enhanced treatment. The pig lags behind in development, looks very lethargic, does not eat up. Develops tendovaginitis, arthritis.

How to diagnose African plague?

As mentioned above, not all forms of this disease generally have symptoms, but in most cases the disease can be recognized. The first characteristic feature is cyanotic spots on the body of the animal. Immediately after their appearance, you need to contact the veterinary service and isolate the sick individual from any contact with other animals.

Veterinarians usually do tests (without them it is impossible to reliably identify the virus), conduct studies of the general herd and the sick individual, monitor their changes, and then make a diagnosis. In case of detection of ASF, the establishment of the causes of its occurrence and further development begins. African swine fever is distinguished from simple swine fever by differential diagnosis.

African swine fever treatment

There is currently no vaccine for African swine fever. Treating the disease is useless and even prohibited, given the rapid spread of the virus. This can only lead to new cases of infection and lead to a real epidemic.

It is worth noting that earlier the mortality rate from African swine fever was 100% and it usually proceeded in severe forms. But now cases of the chronic course of the disease have become more frequent.

The measures that are taken when a disease is detected can be called abruptly cardinal, but only this can stop the spread of the virus. The first thing to do is to destroy the entire herd of pigs that are on the farm, even those individuals that seem healthy. They are slaughtered in a bloodless way. After that, all pigs are burned along with items for their care, food, bedding in the barn. Ideally, the barn should also be burned, but this is not always possible.

The resulting ash is mixed with a large amount of lime and buried in the ground to a considerable depth. Pig farms and all nearby areas, buildings, are treated with a 3% hot solution of sodium hydroxide and 2% formaldehyde solution. For a whole year, the owners of the farm where the disease was discovered are prohibited from having animals.

All pets within 10 km of the disease outbreak are slaughtered and processed into canned food, and the region is quarantined. This is the only way African swine fever is currently contained.

What preventive measures are in place?

To protect the herd from African swine fever, breeders must take preventive measures.

African swine fever (ASF) is a very dangerous and incurable infectious disease. The fatal outcome is almost one hundred percent, all animals are affected, regardless of age and the method of penetration of the virus into the body. It is important to know how dangerous African swine fever is to humans and its symptoms in order to prevent the massive spread of the disease.

The first information about the virus appeared relatively recently, in the first decade of the last century. Then the famous researcher R. Montgomery was in East Africa, where he registered a dangerous virus with a fatal outcome, so the disease is sometimes called by his name. Over time, the disease spread throughout the African continent, was introduced to Europe, then to America, and appeared later on the territory of the Russian Federation.

Carriers of the virus can be both animals that have been ill and recently ill (the pathogen can live in their body for about two years), excretion occurs with saliva, during urination, with blood or feces.

To understand how dangerous ASF is, you need to talk about possible ways of infection. There are several of them:

The symptoms and manifestations make African swine fever almost indistinguishable from classical swine fever. An incubation period of at least two days, but not more than two weeks, depends on a number of symptoms. This significantly complicates the correct diagnosis. The disease can be acute, subacute, hyperacute, chronic and asymptomatic. If ASF is acute, the animal dies about seven days after infection, hyperacute - a day or three, subacute - after two to three weeks. If during this time the death did not occur, most likely a chronic form develops, and the animal will die after complete exhaustion of the body.
It is important to know that African plague can affect not only domestic but also wild adult pigs or piglets, regardless of age, sex and breed. The disease manifests itself in different periods of the year. Long-term studies allow us to conclude that on the European continent most outbreaks of infection appear in winter and spring periods.
The final diagnosis is obtained after complex laboratory studies are completed.

Blood samples are obtained from infected animals, parts of internal organs (spleen) are obtained from dead pigs.

Blood is taken from animals that have been ill for a long time, as well as from those that have been in direct contact with sick animals of different ages.
In many cases, African plague is acute. During this time you can see:

The virus can mutate, the symptoms change, so not all, but only some of the alleged symptoms can manifest themselves in a particular territory.

Symptoms of African plague in humans

There are no vaccines or drugs that can be used to treat animals. Almost all sick pigs die.
If we talk about the danger of African swine fever for people, then it is absent. Meat products can be used and will be completely suitable for consumption, there is a long-term full and high-quality heat treatment (boiling, frying). It is necessary to take into account the fact that after smoking the virus will not be destroyed. When a person eats the meat of such a pig, nothing will threaten his life, because the disease is not transmitted to people from sick animals. But the veterinary service, in any case, after the establishment of the African plague virus, will introduce quarantine in a 20-kilometer zone, and will deal with the destruction of the entire pig population in this territory in order to prevent the spread of ASF. A person can also become a distributor of a dangerous disease. Let's take one simple example. The owner slaughtered one of the pigs he kept without even knowing it was infected. When this meat is eaten, the virus can spread to other animals. It is known that the unused residue of the pig breeder is put in a separate container, then thrown somewhere, most often as a feed for the remaining animals. So the disease will spread, and a person after eating meat products will become a distributor of the virus, without knowing it.

African swine fever (ASF, Montgomery's disease) is a contagious disease that occurs acutely, subacutely, chronically, asymptomatically and is characterized by fever, hemorrhagic diathesis, inflammatory and necrodystrophic changes in parenchymal organs. The disease has been reported in Africa, Spain, Portugal, France, Brazil and Cuba. Pigs of all ages and breeds get sick at any time of the year. The virus was described by Montgomery in 1921 and placed in a separate family.

Clinical signs and pathological changes. They are similar to those in CSF. ASF manifested itself as intense hemorrhagic septicemia, a highly contagious, rapidly progressing disease that caused the death of all contaminated animals. Under natural conditions, the incubation period lasts 5-7 days; in the experiment, its period varied depending on the strain and dose of the virus. There are hyperacute, acute, subacute, chronic and latent course of the disease. Superacute and acute course is more often observed.

At super island In the course of the body temperature in a sick animal rises to 40.5-42 ° C, depression and shortness of breath are strongly expressed. The animal lies more, and after 24-72 hours it dies. At Ostrom(the most characteristic) course of the disease, the temperature rises to 40.5-42 ° C and decreases one day before the death of the animal. Simultaneously with an increase in temperature, the first symptoms of the disease appear: a depressed state, paresis of the hind limbs. Red-violet spots appear on the skin of the ears, snout, belly, perineum and lower neck. In parallel, signs of pneumonia appear: breathing becomes short, frequent, intermittent, sometimes accompanied by a cough. Symptoms of indigestion are mild: prolonged constipation is usually observed, feces are hard, covered with mucus. In some cases, there is diarrhea with blood. In the atonal stage of the disease, the animals are in a coma that lasts 24-48 hours, the body temperature drops below normal, and after 4-10 days from the moment the temperature rises, the animal dies.

Subacute The course of symptomatology is similar to acute, but the signs of the disease develop less intensively. The disease lasts 15-20 days, the pigs usually die. In single surviving individuals, a chronic course of the disease develops, which is characterized by intermittent fever, exhaustion, stunting, mild painless edema in the joints of the wrist, metatarsus, phalanges, subcutaneous tissues of the muzzle and lower jaw, skin necrosis, keratitis. Animals get sick for 2-15 months, death, as a rule, occurs after involvement in the infectious process of the lungs. Clinically, most of the recovered animals turn into healthy carriers of the pathogen, i.e. they develop a latent course of ASF. The pathogenesis of the chronic course of ASF has some similarities with such diseases as INAN, Aleutian mink disease, etc. This similarity is expressed in the persistence of the virus, weak, if not completely absent, virus-neutralizing activity of sera, and hypergammaglobulinemia. The latter, apparently, is due to constant antigenic stimulation by a persistent virus, since it is released from the organs of most chronically infected animals, and its titer correlates with an increase in the level of gamma globulins and AT.

In the last 20 years in Portugal, Spain, Angola and other countries, there has been a change in the form of manifestation of ASF - mortality has significantly decreased, the number of cases of inapparent infection and latent carriage has increased.

latent flow It is typical for natural carriers of the virus - warthogs, forest and bush pigs in Africa and domestic pigs in Spain and Portugal. Clinically, this form is not expressed and is manifested only by intermittent viremia. When stressed, they secrete the virus and infect healthy pigs. At least 3 feral pig species found in Africa may carry the ASF virus without visible clinical signs of the disease. However, if this virus is introduced into domestic pigs, it will cause a highly contagious hyperacute febrile illness with a fatal outcome. Individual individuals that survive this form of the disease are usually resistant to a massive dose of a highly pathogenic homologous strain. Although high titers of specific (CS, PA) antibodies can be detected in the sera of such convalescent pigs, their immunological significance remains unclear. Such animals are almost always chronically infected, carrying both AT and virus in their blood.

In pigs that have died from an acute or subacute form of the disease, fatness is preserved, rigor mortis is pronounced, the skin of the dewlap, the ventral part of the abdominal walls, the inner surface of the thighs, the scrotum is reddened or purple-violet. The nasal cavity and trachea are filled with a pinkish frothy fluid. The lymph nodes of the carcass and internal organs are enlarged, the incision surfaces are marble. Often they are dark red, almost black in color and resemble a blood clot. The spleen is enlarged, cherry or dark red in color, soft in consistency, its edges are rounded, the pulp is juicy, easily scraped off the incision surface. The lungs are plethoric, enlarged, grayish-red in color. The interlobular connective tissue is heavily impregnated with serous-fibrinous exudate and appears in the form of wide strands that clearly limit the pulmonary lobules and lobes. Often, small-focus hemorrhages under the pleura and foci of catarrhal pneumonia are found. The kidneys are often enlarged, dark red in color, with spotted hemorrhages. The renal pelvis is edematous, dotted with spotted hemorrhages. Sometimes hemorrhages are found against the background of anemia of the kidneys. The liver is enlarged, plethoric, unevenly painted in a grayish-clay color. The mucous membrane of the gallbladder is swollen, riddled with petechial hemorrhages, the latter are also localized in the serous membrane. The mucous membrane of the gastrointestinal tract is reddened, swollen, in places (especially along the folds) with hemorrhages. In some cases, hemorrhages are localized in the serous membrane of the large intestine. The vessels of the brain are filled with blood, the medulla is edematous, with hemorrhages.

In the chronic course of the disease, pathomorphological changes are manifested by a sharp increase in bronchial lymph nodes and bilateral lung damage. The asymptomatic course is characterized by marble coloration of the portal or bronchial lymph nodes and focal lung lesions. histological changes. In acute and subacute course of the disease, hemodynamics in the lymph nodes and spleen are sharply upset as a result of mucoid swelling and fibrinoid necrosis of the walls of blood vessels; devastation of lymphoid tissue and cell breakdown by the type of karyorrhexis. In the central nervous system and in parenchymal organs, inflammatory-dystrophic changes of varying severity are noted. IF virus and its AG are found in macrophages, reticular cells, lymphocytes and Kupffer cells, in megakaryocytes and hemocytoblasts of smears-prints of the spleen, lymph nodes, bone marrow, liver and lungs of sick animals. Perinuclear inclusions are visible.

In a chronic course, the pathological process is localized mainly in the bronchial lymph nodes and lungs. At the same time, changes inherent in serous-hemorrhagic lymphadenitis and croupous-necrotic pneumonia are recorded. The transition of inflammation to the heart shirt and myocardium is possible. The asymptomatic course of the disease of a limited nature is manifested by uneven hyperemia of the bronchial or portal lymph nodes, focal serous-cata-ral or serous-fibrinous pneumonia. In sick pigs, the virus initially causes hyperplasia of lymphoid cells. In the process of its reproduction and accumulation, the bulk of them (70-80%) die according to the type of karyopyknosis and karyorrhexis. In a culture of bone marrow cells and porcine blood leukocytes, erythrocytes are adsorbed on the surface of cells infected with the ASF virus when the virus titer reaches 103.5-4.0 HAEzo/ml. In the perinuclear zone of infected cells, inclusions appear, located at the sites of virus synthesis. Later, infected cells round out, lose contact with each other, and flake off the wall.

Pathogenesis. AT Under natural conditions, the virus enters the body of pigs through the respiratory, digestive, damaged skin and mucous membranes. The nucleic acid of the virus induces a restructuring of cellular metabolism and activates hydrolytic enzymes, resulting in increased proliferation of lymphoid tissue cells. Proliferating cells provide a favorable environment for the reproduction of the virus. In the body, the virus quickly spreads through the blood and lymphatic vessels, affects the lymphoid tissue, bone marrow and the walls of blood vessels. Its action is exacerbated by the development of allergic reactions, manifested by an increase in the number of mast cells, eosinophils, as well as the development of mucoid swelling and fibrinoid necrosis of the vascular walls.

The ASF virus multiplies in the cells of the lymphoid and reticuloendothelial tissues. In the acute course of the disease, it depresses the immune system, destroying or changing the functions of lymphoid cells, in chronic or latent cases, it disrupts the ratio of leukocyte subpopulations, the function of macrophages, the synthesis and activity of mediators of cellular immunity. Pathological processes that develop in the late stage of the acute course of ASF (a sharp deterioration in the general condition, an increase in vascular permeability, multiple hemorrhages), as well as during a long course of the disease (croupous necrotic pneumonia, tissue infiltration with lymphoid cells, skin necrosis, arthritis, hypergammaglobulinemia) are caused by hyper-ergic, allergic and autoimmune processes. Allergic and autoallergic processes play a significant role in the pathogenesis of ASF. In the acute course of the disease, blood properties change dramatically (leukopenia, increased adhesion of leukocytes, activation of enzymes in the blood and organs), severe degenerative changes in RES cells, multiple hemorrhages as a result of impaired vascular wall permeability, activation of phosphatases and the disappearance of glycogen in the liver.

In the chronic course of ASF, a systemic manifestation of an allergic reaction is detected, turning into an autoimmune disease with damage to target organs. Deposition of antigen-antibody complexes with complement fixation was found in the lesions. During the period of recurrence of the disease, cyclic changes in the picture of white blood, autoimmune damage to neutrophils and inhibition of phagocytic activity are detected. In subacute and chronic course of ASF, extensive local inflammatory processes, called tumor-like formations, often develop at the site of re-introduction of the virus. They are extensive swelling in the submandibular space and neck with a diameter of up to 30-40 cm. At the same time, pain and an increase in local temperature are not expressed. However, within 12-14 days these formations increase, which is accompanied by an increase in temperature and a deterioration in the general condition of the animals. At slaughter and autopsy of such pigs, formations that are not clearly limited from normal tissues with severe edema along the periphery and necrosis in the central part are established. In the tissues, the accumulation of the virus in a non-hemadsorbing form up to 107.5 TCC50/ml and specific AG detected in CSC and IF was established. Histoexamination revealed changes characteristic of hyperergic inflammation: infiltration of tissues with lymphoid-histiocytic elements with an admixture of eosinophils, neutrophils and plasmocytes.

Inflammatory-allergic reactions at the site of re-introduction of the virus or its AG contribute to the localization of the pathological process. Allergic sensitization in ASF can be detected by intradermal allergy testing. Allergens are concentrated virus-containing materials, inactivated U- Rays that are injected intradermally. At the injection site of the allergen in animals infected with the ASF virus, after 24-48 hours, an inflammatory reaction develops, accompanied by infiltration of the connective tissue layer of the skin with mononuclear cells, which is manifested by hyperemia and swelling from 10 to 40 mm in diameter. An allergic reaction is detected from 3 to 150 days after infection in 68.7% of animals. The above information suggests that allergic or autoallergic reactions play a significant role in the pathogenesis and immunogenesis of ASF.

Morphology and chemical composition. Virions are rounded particles with a diameter of 175-215 nm, consisting of a dense nucleoid, a two-layer icosahedral capsid and an outer shell. The nucleoid contains DNA and protein and is surrounded by an electron-transparent layer. The bilayer capsid consists of 1892-2172 capsomeres. The outer lipoprotein envelope of virions has a typical structure and is not necessary for the manifestation of the infectious properties of the virus. There is an electron-transparent layer between the outer shell and the capsid. The floating density in CsCl is 1.19-1.24 g/cm3, the sedimentation coefficient is 1800-8000S. The infectivity of the virus persists at 5 ° C for 5-7 years, at room temperature - 18 months, at 37 °С - 10-30 days. The virus is stable at pH 3-10, sensitive to fat solvents and inactivated at 56°C for 30 minutes.

The ends of the DNA are covalently linked and contain inverted repeats, similar to those in the DNA of poxviruses. DNA is not infectious. 54 polypeptides were found in ASF virus virions. Virions are associated with several enzymes necessary for the synthesis of early mRNAs.

The ASF virus reproduces in the cytoplasm of cells, but the function of the nucleus is also necessary for its reproduction. In infected cells, 106 virus-specific proteins were found, of which 35 are synthesized before viral DNA replication (early proteins) and 71 after DNA replication (late proteins). Virions mature in the cytoplasm and acquire an outer envelope when they bud through the cytoplasmic membrane. The virus multiplies in the body of pigs and ticks of the genus Ornithodoros. In pigs, the virus replicates in monocytes, macrophages, and reticuloendothelial cells. In female ticks, the virus persists for more than 100 days, is transmitted transovarially and transphasically.

It is known that the penetration of viruses into the body is accompanied by the formation of VNA. The exception is primarily the ASF virus. Infection with this virus does not induce the synthesis of VNA in animals, although KSA, PA, and type-specific inhibitory GA ATs are detected in the blood serum. The absence of VNA results in the body's inability to bind and eliminate the virus, which in turn leads to exceptionally high mortality in infected animals. On the other hand, the noted paradoxical phenomenon nullifies attempts to create an effective vaccine, since attenuated strains of the virus cause a chronic course of the disease in pigs and prolonged virus carriage, which is very dangerous from an epizootological point of view.

The ASF virus has distinct characteristics of irido and poxviruses. It is the only representative of a unique family. DNA encodes over 100 polypeptides, of which more than 30 were found in purified virus preparations. A number of enzymatic activities are associated with virions, including DNK-dependent RNA polymerase, phosphatohydrolase activity, as well as protein kinase and acid phosphatase. DNA-dependent RNA polymerase is located on the periphery of the capsid, and ATP hydrolase is located between the capsid and the nucleoid. The capsid is formed mainly by polypeptides with a mol. m. 73 and 37 kD. A DNA-dependent RNA polymerase, which is involved in the initial stages of virus reproduction, is also associated with the capsid. DNA is a two-stranded structure. m. 100-106 D, consisting of 170 thousand p. 58 nm long with covalent end-links in the form of inverted repeats of 2.7 thousand bp.

The ASF virus has a 20-sided shape, its size is 175-215 nm, it is covered with a two-layer lipoprotein shell, which has antigenic affinity with the host tissues. Next is a three-layer capsid of periodically placed capsomeres, inside there is a nucleoprotein of dense fibrils containing DNA. The surface membrane and capsid contain a large amount of lipids. ASF virus DNA pcs. BA71V has a length of 170101 bp. and 151 open reading frames. DNA sequencing showed that the ASF virus occupies an intermediate position between poxviruses and iridoviruses and belongs to an independent family of viruses. Under the action of restrictase ECo-R-l, 28 DNA fragments (value 0.3-21.9 kD), which is 96% of the entire molecule, were detected, and 11-50 fragments (0.3-76.6 kD) were detected by other restrictases. The expression of 16 DNA fragments in E. coli was obtained, the location of 80 sites was determined by molecular hybridization, and a map of the location of the fragments was compiled. Differences between individual isolates and variants of the virus, as well as the mechanism and sequence of synthesis of virus-specific proteins, their role in the pathogenesis of the disease were revealed.

According to other data, 28-37 virus-specific proteins were found in the composition of virions and infected cells, according to other data, 100 structural and 162 non-structural virus-specific proteins with a mass of 11.5-245 kD were registered. Major polypeptides (172, 73, 46, 36, 15, 12 kD), early and late proteins, glycoproteins (54, 34, 24, 5, 15 kD) were identified, a relationship with AT 25 proteins was established. It is believed that early proteins are synthesized from the terminal sections of DNA, and late ones from its central part. Virus-specific proteins in infected cells are located as follows: in membrane proteins - 220, 150, 24, 14, 2 kD, in viroplasts - 220, 150, 87, 80, 72, 60 kD, in the cell nucleus - 220, 150, 27 kD. A certain order of location of individual proteins in the virion (starting from the surface) has been established - 24, 14, 12, 72, 17, 37 and 150 kD. Physical maps of the DNA of the virulent strain of the ASF virus K-73 (serotype 2) and the avirulent variant KK-262 isolated from it, adapted to the culture of pig kidney cells (PPK-666), were constructed. Each strain has its own, different physical map of DNA, with a certain similarity. Proteins 32 and 35 kD are strain-specific. The virion contained DNA polymerase, protein kinase and other enzymes that are necessary for the early synthesis of virus-specific structures.

The ASF virus is heterogeneous. It is a heterogeneous population consisting of clones that differ in terms of hemadsorption, virulence, infectivity, plaque formation and antigenic properties. The biological properties of the virus used for experimental infection of pigs differ from virus isolates isolated later from the same pigs. In 1991, a report was published on current data on the architecture of morphogenesis and the distribution of structural polypeptides in the ASF virion. Based on the general plan of the structure of the ASF virus, the localization of viroplasts in infected cells, the virus was assigned to the group of iridoviruses. R. M. Chumak hypothesized about the hybrid origin of the ASF virus, the ancestors of which were viruses of the smallpox group and one of the insect iridoviruses. In the author's opinion, this virus should be allocated to a separate family, where other viruses will be assigned later.

A. D. Sereda and V. V. Makarov identified an isolate-specific glycopeptide of the ASF virus. Three glycosylated polypeptides with a mol. m. 51, 56, 89 kD and three radio-labeled monochrome shell components with a mol. m. 9, 95, 230 kD, the biochemical nature of which has not been elucidated. Five virus-induced glycosylated polypeptides with a mol. m. 13, 33, 34, 38, 220 kD were identified in ASF virus-infected Vera cells. The polypeptide (110–140 kD) seems to be directly related to GAD AG, the existence of which was previously judged only by the GAD phenomenon. The authors showed that oligosaccharide proteins make up about 50% of the mass of the glycosylated polypeptide (110–140 kD). The lipid composition of the ASF virus depends on the cell culture system.

Restriction analysis and cross-hybridization of restriction fragments showed that the genome of the CAM/82 ASF virus isolate does not change during passaging on pigs (for 20 passages) and in cultured pig bone marrow cells (for 17 passages). The ASF virus genome is quite stable during transmission of the virus in natural and experimental conditions. Comparison of physical mapping data and biological properties of ASF virus strains made it possible to assume that the left terminal region contains DNA regions that are directly related to such manifestations of the virus phenotype as virulence and immunogenicity. This assumption is based on the fact that in avirulent strains there is a loss of a large portion of DNA in this region, while in natural isolates the length of the left terminal region is much more significant. On the basis of the obtained results, physical maps of the genomes of the reference strains of ASFV of all 4 serotypes were constructed and the certification of vaccine strains was carried out, which makes it possible to further control possible changes in the genome. Using primers complementary to the nucleotide sequences of the structural gene of the VP2 protein of VASHF, a test system for the identification of VASHF by PCR was developed. The B438L open reading frame, located on the EcoRI-L fragment of the African swine fever virus (VALS) genome, encodes a protein of 438 residues with a mol. m. 49.3 kDa, having an RGD cell attachment motif and not homologous to proteins from databases. The B438L gene is transcribed only at the late stage of infection with VALS. The protein was expressed in Escherichia coli, purified and used to obtain a rabbit antiserum that recognizes a protein with a mol. m. 49 kD in VALS-infected cells. This protein is synthesized at the late stage of infection by all studied VALV strains, is located in cytoplasmic viral factories, and is a structural component of purified VASF virions.

The genomes of African swine fever virus isolates isolated in Cameroon in 1982-1985 are indistinguishable by restriction analysis. Isolate CAM/87 differs slightly from the isolates of 1982-1985. However, significant differences were found in the DNA of the CAM/86 isolate using 4-restriction enzymes in 2-fragments (within the right terminal region and in the central region).

Sustainability. The ASF virus is exceptionally stable over a wide range of temperatures and pH environments, including drying, freezing and decay. It can remain viable for a long time in faeces, blood, soil and on various surfaces - wood, metal, brick. In the corpses of pigs, it is inactivated not earlier than after 2 months, in feces - within 16 days, in soil - within 190 days, and in a refrigerator at -30-60 ° C - from 6 to 10 years. The sun's rays, regardless of the infected objects (concrete, iron, wood), completely inactivate the ASF virus (st. Dolizi-74) after 12 hours, and pcs. Mfuti-84 - in 40-45 minutes. Under the conditions of a pigsty at 24 ° C, natural inactivation of the virus (pcs. Dolizi-74) occurred in 120 days, and pcs. Mfuti-84 - in 4 days. 0.5% formalin solution turned out to be optimal for disinfection of infected premises. Freezing does not affect the biological activity of the virus, but is the initial stage of genome damage. The virus with percol is resistant to DNase after freezing at -20 °C and -70 °C and is damaged at -50 °C. Drying the virus without a stabilizer causes the loss of its infectivity. months.

The long-term stability of the pathogen in the blood, excreta and corpses is taken into account when planning veterinary and sanitary measures. Since the virus remains viable in infected pigsties for 3 months, this period corresponds to the exposure, after which the importation of a new batch of pigs is allowed. The stability of the virus is affected by the composition and pH of the medium in which it is suspended, the content of protein and mineral salts, the degree of hydration, and the nature of the virus-containing material under study. At 5 °C, it remains active for 5-7 years, when stored at room temperature - up to 18 months, at 37 °C - 10-30 days. At 37 °C, its infectivity decreased by 50% within 24 H In a medium with 25% serum and for 8 hours in a medium without serum. At 56 °C, a small amount of virus remained infective for more than 1 hour, so a 30-minute serum inactivation at 56 °C used in practice is not enough to destroy the pathogen. At 60 °C, it was inactivated within 20 minutes. and in an alkaline environment. Most disinfectants (creolin, lysol, 1.5% NaOH solution) do not inactivate it. The greatest virucidal effect on it is exerted by chlorine-active preparations (5% chloramine solution, sodium and calcium hypochlorites with 1-2 % active chlorine, bleach) with a 4-hour exposure. Sodium hydroxide in the form of a 3% solution is recommended for disinfection only in hot form (at a temperature of 80-85 ° C). When disinfecting, special attention is paid to thorough mechanical cleaning and rinsing hot water, as manure organic matter can reduce the effectiveness of disinfection.

AG structure. It's complex with the virus. The causative agent contains group KS-, pre-cipitating and typical GAD antigens. DNA-binding proteins have been found, including major and minor ones with a mol. m. from 12 to 130kD. Their total number reaches 15, of which 7 are structural. Proteins P14 and P24 are located on the periphery of the virion, and P12, P17, P37 and P73 - in the intermediate layer; protein P150 was discovered - a major viral protein, which is located in the nucleoid or in one of the vertices (corners) of the virion. All eukaryotic cells have a special protein, consisting of substances of amino acid residues and covalently linked to various cellular proteins (for example, histone). This connection is provided by the ubiquitin-configuring enzyme UBS. One of the proteins encoded by the ASF virus is able to activate ubiquitin.

Questions about the nature of infectious hypertension that induces the formation of VNA are still open. The situation is different with AGs that induce the formation of ATs that delay hemadsorption. Serums with anti-HAD properties are widely used by all researchers studying the problem of ASF. Polypeptides with a mol. m. 120, 78, 69, 56, 45, 39, 28, 26, 24, 16 and 14 kD are most intensively detected on electrophoregrams and immunoblotograms of purified ASF virus preparations. A mixture of proteases and pancreatic lipase in low concentrations removes polypeptides from these preparations with a mol. m 120 and 78 kD, in medium concentrations - polypeptides with a mol. m. 69, 56, 45, 39, 28 and 14 kD, in high concentrations - a polypeptide with a mol. m. 26 kD. Polypeptide with mol. m. 21 kDa, which did not react in the immunoblot with specific antiviral serum, was resistant to the combined action of proteases and lipase. Treatment of the virus with Triton X-100 and ether led to an increase in the activity of virus-associated DNA-dependent RNA polymerase, and treatment with ether and subsequent reprecipitation led to a significant decrease in activity in the precipitated preparation. Treatment of the virus with ether did not affect its activity. On the basis of the obtained results and literature data, a scheme for the arrangement of viral polypeptides and enzymes in the virion structure was proposed.

AG variability and relatedness. Based on the hemadsorption delay, two AG A- and B-groups (types) and one subgroup C of the ASF virus were identified. Within the A-, B-groups and C-subgroup, many serotypes of this pathogen have been identified. Two genetic groups (CAM/88 and CAM/86) of African swine fever virus isolated in Cameroon cause similar clinical signs and lesions in domestic pigs. 3-6 days after infection, fever, loss of appetite, lethargy, loss of coordination, trembling, diarrhea and shortness of breath develop. There is hyperemia of the lungs and the appearance of hemorrhage in the kidneys and visceral lymph nodes. Virus titers in pigs infected with isolates of different groups did not differ statistically.

With the help of immunoassays and RZGA, 7 reference strains of each group were established: L-57; L-60; Hinde-2; Rhodesia; Dakar; 2743; Mozambique. Reference strains include - pcs. Hinde; No. 2447; 262; Magadi; Spencer; L-60 and Rhodesia. MAB immunoblotting revealed 6 groups, and restriction analysis revealed 4 groups and 3 subgroups. This is a reference piece. Uganda, Spencer, Tengani, Angola, L-60, E-75. There are reports of high variability of the ASF virus in terms of antigenicity, virulence and other properties, as well as the existence of mixed populations of it, which are difficult to attenuate. For example, pcs. Kerovara-12, isolated from a warthog in Tanzania, shows the typical heterogeneity of the ASF population. Features of the virus are interrelated with pathological and immunological processes in the body of infected pigs. Most of the isolates isolated during the epizootic from domestic pigs in Africa had various GA AGs. Isolates passaged in vivo in porcine macrophages change faster and more profoundly than when passaged in Vero cells. In African isolates, P150, P27, P14 and P12 turned out to be the most variable proteins, in non-African isolates - P150 and P14, the P12 protein does not change, and P72 - the main AG - was stable when diagnosed with EL1SA. AH differences between strains of the ASF virus cannot be determined using solid-phase ELISA, RDP and IEOP, since these methods reveal only common AG for all strains of the ASF virus. This can only be done by depleting the cultured ASFV antigen with heterotypic serum. As can be seen from the above facts, the serological and immunological plurality of the ASF virus is one of its main properties.

Virus localization. The virus is found in all organs and tissues of sick animals. It appears in the blood during the initial rise in temperature and is found there until the death of the animal in titers from 103 to 108 GAd5o / ml - In the chronic course of the disease, the titer of the virus in the blood decreases rapidly, viremia is intermittent. In the absence of viremia, it can persist for a long time (up to 480 days) in the spleen and lymph nodes. The exact localization of the virus in the latent course of the disease has not been established. In the initially infected organs (lymphoid tissue in the pharynx), the virus remained in a titer of about 107 HAD50L until the death of the animal. Its highest titers (10s) were observed in tissues containing a large amount of reticuloendothelial elements: spleen, bone marrow, liver, which is consistent with the detection of significant lesions in these tissues. The primary site of localization of the virus is the tonsils. Its presence in leukocytes from the 1st day of infection indicates that the pathogen is introduced into other tissues by leukocytes. The appearance of the virus in the spleen and bone marrow after 2 days and the rapid increase in the titer of the virus in these tissues suggests that they are the site of secondary reproduction of the pathogen.

From the body of infected animals, the virus is shed in the blood, nasal excretions, faeces, urine, saliva and, probably, through the lungs with exhaled air. In most surviving animals, the virus carrier is almost lifelong. Periodically, the virus can be isolated from the blood, lymph nodes, lungs, spleen. It is difficult to isolate it from other tissues. Virus shedding occurs 2-4 days after the onset of fever. Stress factors contribute to the exacerbation of infection and the release of the virus into the external environment. At the same time, the seasonality of virus excretion is associated with farrowing. In Ornithodoas ticks, the ASF virus multiplies in the intestines and then spreads to the salivary glands and reproductive organs. Ticks can remain persistently infected and transmit the virus for up to 3 years; along with warthogs, they create a permanent reservoir of the virus for domestic pigs. Ticks are able to transmit it transovarially and transpha-zovo. The concentration of the virus in ticks is higher than in virus-carrying pigs.

AG activity. In the sera of convalescents, precipitating SCs and retaining GAd AT appear, which do not affect the CPP of the virus. PA and KSA are not type-specific, they are common for all individuals, while ATs that inhibit GAd are strictly type-specific and are used for ASF virus typing. KSA and PA are not associated with the formation of immunity. BHAs are not formed, but an AT-mediated mechanism operates in defense. These antibodies are active in two systems: A) In vitroantibody-dependent cellular cytotoxicity; b) complement-dependent lysis. The sera of convalescent animals specifically retain GAD in cultures infected with the homologous ASF virus. The titer of such AT reaches its maximum 35-42 days after the clinical recovery of the animals. The ASF virus does not cause the formation of VNA and the humoral components of the immune response are of little importance. The inability to produce VNA against the ASF virus is probably due to the properties of the pathogen itself.

Interaction of the virus with AT. One of the reasons for the lack of knowledge of the immunology of ASF is the lack of neutralization of the AT virus, the main property of other viruses that have been the traditional basis for studying their immunogenicity since the discovery of serological reactions. In this regard, there is only one zoopathogenic analogue - parvovirus of the Aleutian disease of minks, but the low ability to neutralize typical representatives of iridoviruses is also known. Many attempts have been made to study this unique phenomenon, but no satisfactory explanation has yet been offered; There are many versions - from the absence of virion glycoproteins to antigenic mimicry and heterogeneity. In an effort to clarify this issue, the authors studied step by step the results of the interaction of the virus with AT, the virus with susceptible cells in culture, and the virus + AT complex with sensitive cells. It has been shown that the immune complex (AG+AT) freely penetrates into sensitive cells, and the virus retains its original reproductive activity. In ASF, neutralization of the virus in vitro is accompanied by the opposite effect - increased viral reproduction and extensive pathology due to the spread of infected macrophage monocytes.

The question of the interaction of the ASF virus with AT needs further experimental study. In seropositive native animals, specific CSA and PA are found in the blood in titers up to 1:128 and 1:64, respectively. Specific antibodies in the blood of piglets appear only after taking colostrum from seropositive sows. The level of AT in colostrum was equal to or exceeded their concentration in the blood.

experimental infection. Cats, dogs, mice, rats, rabbits, chickens, pigeons, sheep, goats, cattle and horses are immune to experimental infection. In experimentally infected argasid ticks Ornithodoros turicata, the virus was detected by bioassay during the year. In the intestines of the tick, the earliest and longest presence of the virus was established. Its rapid distribution and replication in other tissues occurs through the hemolymph. As early as 24 hours after infection. AH was detected using MFA. After 2-3 weeks, the virus was found in hemocytes, and by the 6-7th week - in most tissues.

Cultivation. For the cultivation of the ASF virus, gilts of 3-4 months of age can be used, which are infected by any method. More often, they are infected intramuscularly at a dose of 104-106 HAd50. With the development of clinical symptoms of the disease on the 4-6th day after infection, animals are killed and blood and spleen are used as virus-containing material, in which the virus accumulates in a titer of 106-8 HAd50. Attempts to cultivate the virus ASF has not been successful in other animal species.

Cultures of blood leukocytes and bone marrow macrophages of pigs were sensitive to the virus. Cells are usually infected on the 3-4th day of growth at a dose of 103 HAD of the virus per 1 ml of nutrient medium. After 48-72 hours, it accumulates in cell cultures in the titer JO6-7 5 HAD 50/ml - ASF virus infected most macrophages (monocytes), if not all, then only about 4 % Polymorphonuclear leukocytes in peripheral blood. B - and T-lymphocytes, which are at rest or stimulated with PHA, liposaccharide or mitogen from American phytolacca, are not susceptible to the virus. The latter replicates exclusively in macrophages, and is found in the highest titers in porcine erythrocytes. It enters the cell mainly in a receptor-independent way, its replication occurs in the cytoplasm, but for synthetic processes, the participation of the nucleus is necessary. Infection with more than one particle of the virus is possible, which implies the presence of several of its subpopulations in one cell and their interaction. The number of cells containing AG on the surface reaches its maximum level after 13-14 hours. A large amount of unused virus-specific material, having a membrane, cylindrical or eccentric structure, remains in the infected cells. It is assumed that their shells contain GAD AG.

The virus multiplies in cultures of leukocytes and bone marrow of pigs with the development of GAD and CPP without adaptation. At the optimal dose of infection, GAd manifests itself after 18-24 hours, CPP - after 48-72 hours and is characterized by the formation of cytoplasmic inclusions, followed by leakage of the cytoplasm and the appearance of multinucleated giant cells (shadow cells). It enters CV-1 or Vero cells by adsorptive endocytosis or receptor-mediated endocytosis. "Undressing" of virions occurs in endosomes or other acidic intracellular vesicular organelles. When ASFV is incubated with porcine peripheral blood mononuclear cells, it inhibits the proliferative response of lymphocytes to phytohemagglutinin and other lectins. This inhibition is believed to be induced by soluble fractions that are released by peripheral mononuclear cells after co-incubation with the virus. The HAD of the virus in infected cultures is so specific that it is used as the main test in the diagnosis of the disease. In other types of cell cultures, the virus does not multiply without prior adaptation. It is adapted to a number of homo- and heterologous cultures: continuous cell lines of pig kidney (PP and RK), green monkey kidney (MS, CV), macaque kidney Vero cells, etc. In the literature, little attention is paid to the effect of carbohydrate components, which can from 50 to 90% of the mass of glycoproteins, on the immunogenicity of the virus: one of the reasons for the weak immunogenicity of the enveloped glycoprotein (gp 120) of the immunodeficiency virus (HIV) is that 50 % Its mass is due to the “atmosphere” of sugars, which can play a negative role, for example, preventing AT from accessing the fixation site on the HIV envelope, i.e. vital areas of HIV are “chemically” protected from the action of the immune system. It is possible that the presence of highly glycosylated proteins on the surface of virions may be the reason for the non-neutralization of the ASF virus. The coexistence of glycosylated components of unknown nature in the envelope of ASF virions was reported by Mudel Wahl et al. in 1986.

The presence of such components on cell membranes can also contribute to the escape from other effector mechanisms of the host immune system and increase its pathogenicity. The study of subcellular localization and activity of ASF virus transprenyltransferase in infected cells showed that the enzyme is an integral membrane protein and exhibits geranylgeranyldiphosphate synthase phenyltransferase activity in membrane fractions, a 25-fold increase in the formation of geranylgeranyldiphosphate in infected cells. Thus, the membrane-bound protein synthesizes predominantly trans-GGDP synthetase. reproduction features. The methods of in situ hybridization, autoradiography and electron microscopy have been used to study the ultrastructural organization of replication DNA ASF virus in infected Vera cells. At an early stage of viral DNA synthesis, it forms dense foci in the nucleus near the nuclear membrane, and at a later stage it is exclusively in the cytoplasm. Sedimentation in an alkaline sucrose concentration gradient showed that at an early stage, small DNA fragments (=6-12 S) are in the nucleus, and at a later stage, longer fragments (=37-46 S) are labeled in the cytoplasm. Pulse labeling showed that these fragments are precursors of mature cross-linked viral DNA.

Head-to-head forms were found at the intermediate and late stages. These data suggest that ASF virus DNA replication follows a de novo start mechanism with the synthesis of short DNA fragments, which then turn into long fragments. Ligation or elongation of these molecules gives two-unit structures with dimeric ends, which can generate genomic DNA As a result of the formation of a site-specific single-strand break, rearrangements and ligation. Biochemical methods for analyzing ASF virus capsid assembly, assembly, and envelope formation have been used to study cellular processes that are important for enveloping the virus in membrane cisterns. Assembly of the ASFV capsid on endoplasmic reticulum (ER) membranes and ER cisterna envelopment is inhibited when ATP or calcium is depleted as a result of incubation with A23187 and EDTA or with taxiharpine, an ER calcium ATPase inhibitor. The EM method has shown that Ca-depleted cells cannot assemble icosahedral VASF particles. Instead, the assembly sites contain comb-like or bulbous structures, in rare cases, empty closed 5-gonal structures. Recruitment of the VALS capsid protein from the cytosol to ER membranes does not require ATP or Ca2+ reserves. However, the subsequent stages of capsid assembly and shell formation depend on ATP and are regulated by the Ca2+ gradient in the membrane cisternae of the ER.

GA and GAd properties. The virus does not have GA properties. When multiplying it in vitro in cultures of leukocytes or cells of the bone marrow of pigs, the phenomenon of adsorption of erythrocytes on the surface of the affected cells is observed. Erythrocytes attach to the wall of the leukocyte, forming a characteristic corolla around it and sometimes closing the cell from all sides, as a result of which the affected leukocytes look like a mulberry. The time of appearance of HAD depends on the inoculated dose of the virus and can appear already after 4 hours, but in most cases - after 18-48 hours, and at low virus titers - after 72 hours. With an increase in incubation time, the number of affected cells increases, then they begin to clear up , and the CPD of the virus is manifested. The sensitivity of RGAD depends on the properties of the virus and the degree of its accumulation in the infected cell culture. It is detected under a light microscope when the infectivity titer in the culture is not lower than 104 LD50/ml - According to some authors, the time of onset of HAD depends on the virus titer in the material sample under study. A decrease in the titer of the ASF virus entails a decrease in the sensitivity of RAd. In this regard, in some cases, it becomes necessary to conduct up to three consecutive serial "blind" passages of the virus in the culture of leukocytes or bone marrow in order to confirm its presence in the test material in the case of the HAD variant.

Sometimes non-hemadsorbing strains of the virus are isolated, which have only pronounced cytopathogenic properties. When passing them in cell culture at least 50 times and infecting pigs, hemadsorption was not restored. In South Africa, a non-hemadsorbing strain was isolated from domestic pigs during a natural epizootic. Later, a non-hemadsorbing variant was isolated there from suspensions of O. moubata mites collected in foci of infection.

Since specific GAD characterizes the virulence of ASF strains, isolation of less virulent non-haemadsorbing viruses from pigs with chronic pneumonia is of great interest. However, individual non-hemadsorbing isolates or clones may be highly virulent. The mechanism of the GAD reaction, as well as the localization of the AGs responsible for GAD, have not been established. The role of the outer membranes of virions in their binding to erythrocytes is important, since virions that do not have membranes are not adsorbed on erythrocytes. The antigens involved in GAD are localized in the envelopes of virions originating from the cytoplasmic membranes of host cells.

ASF at the initial manifestation usually proceeds acutely and subacutely with the death of up to 97% of the pig population. In isolated farms in tropical conditions, the cause of secondary foci is ill pigs - latent carriers of the pathogen. Thus, the ASF virus in the Congo circulates among local animals in the form of a hard-to-detect non-hemadsorbing population, without causing any visible symptoms of the disease and creating a positive immune background in local pigs. An epidemiological survey of the local pig population indicates that, under certain conditions, native domestic pigs, as a reservoir of the virus in nature, play a significant role in the epizootology of ASF. In seropositive native animals, specific KSA and PA are found in the blood in titers up to 1:128 and 1:64, respectively.

In order to study the formation of passive immunity, experiments were carried out with piglets of different ages obtained from seropositive animals. In the blood of unborn fetuses, as well as colostrumless piglets, specific AT were absent. Also, the virus was not isolated from these animals. Specific AT in the blood of piglets appeared only after taking colostrum from seropositive sows. The dynamics of specific AT in the blood of 82 piglets from seropositive sows was traced over a 5-month period. In the control infection of 2-5 month old piglets, in the blood of which CSA and PA were found in titers of 1:16-1:32 and 1:2-1:4, respectively, all animals died with clinical signs of ASF. Seropositive piglets of the same age in contact with them were resistant to infection.

The ASF virus can persist both in susceptible pigs and in vitro in cell culture. In African settings, domestic pigs can become infected through contact with wild warthogs (Phaco choerus) and bush pigs (Patomochoerus), in which it causes a latent infection. Argas mites O. moubata porcinus are a natural reservoir and carrier of the ASF virus. Ornithodorina ticks (carriers of the ASF virus) can live for 9 years, the ASF virus persists in their population for a long time. O. turicata is found in North America in Uta, Colorado, Kansas, Oklahoma, Texas, New Mexico, Arizona, California, and Florida. Ticks can migrate up to 8 km from their habitat. In addition to O. turicata, the ASF virus can also be carried by rattling tick species: O. puertoriceusis, O. tolaje, O. dugersi.

The stability of the virus in dead ticks, as well as its reproduction and persistence in 70-75% of ticks for 13-15 months, have been established. Arthropods get the virus by sucking sick animals during the period of viremia. The virus multiplies in arthropods, which have a long period of persistence, and finally, mites transmit it to healthy pigs during feeding. ASFV has been isolated from coxal fluid, saliva, excreta, Malpighian vessels and genital exudate from naturally and experimentally infected ticks, as well as from eggs and nymphs of the first stage of infected females. Thus, transovarial and transspermal transmission of the virus is possible in this tick species. This contributes to the maintenance and circulation of the virus in the population, even in the absence of regular contact of carriers with infected animals. It is enough to bring the agent into the tick population once, and its circulation occurs, regardless of the contact of this population with sensitive animals in the future. Due to the long lifespan of ticks (10-12 years), the focus of the disease, if it occurs, can exist for an indefinitely long time. In areas where this has occurred, the possibility of eradicating ASF appears doubtful.

Thus, the main route for the rapid spread of the pathogen and the emergence of new outbreaks of the disease is probably alimentary. The respiratory route contributes to its spread within the epizootic focus, and the transmissible route contributes to the creation of persistent natural foci. Due to the close biological relationship between the virus and argasid mites, a natural focus can exist without re-introduction of the virus for an indefinite period. Although the Malawi Lil20P (MAL) strain of AHS virus has been isolated from ticks Ornithodorus sp., attempts to experimentally infect these ticks by feeding the MAL strain have been unsuccessful. 10 populations of O. porcinus porcinus ticks and one population of O. porcinus domesticus ticks were fed VALS MAL. At 10 days post infection, less than 25% of ticks contained ASFV. In more than 90% of mites, VALS was not detected 5 weeks after inoculation. Upon oral inoculation of VAS MAL to O. porcinus porcinus mites, the VALS titer decreased 1000-fold after 4-6 weeks and became below the detection limit. However, after inoculation of the VALS isolate Pretoriuskop/90/4/l (Pr4), the VALS titer increased 10-fold after 10 days and 50-fold after 14 days. In the midgut of ticks inoculated with ASFV, expression of early but not late viral genes was found and no synthesis of ASFV DNA was observed.

Progeny virions are rarely present in ticks after oral inoculation with VALS. If they are present, they are associated with a strong cytopathology of phagocytic midgut epithelial cells (MECs). With parenteral administration of VALS MAL, a persistent infection is established in the hemocoel, but delayed generalization of MAL is observed, and its titer in most tissues is 10-1000 times lower than after infection with VALS Pr4. Ultrastructural analysis showed that MAL VALS replicates in many cell types, but not in EXSCs, and Rg4 VALS can replicate in EXSCs. Thus, MAL VALS replication is limited in ESC ticks.

ASF in Madagascar was confirmed by PCR and nucleotide sequencing after virus isolation. After inoculation of leukocytes, no haemadsorption or CPE was observed, but viral replication in the cells was confirmed by PCR. The determination of the ASF viral genome was carried out by amplification of a highly converted region encoding the p72 protein. 99.2% identity was found between the Maladasi strains and the virus isolated in 1994 during an outbreak in Mazambika. Serological studies were carried out on 449 sera samples, as a result, it was found that only 3-5% of sera isolated from pigs between 1996 and 1999. were positive.

Under natural conditions, domestic and wild pigs are sick with African plague. In some wild African pigs, the disease is subclinical. Such animals pose a great danger to pigs of cultural breeds. In nature, there is a vicious circle of circulation of this virus between wild pigs carrying the virus and ticks (genus Ornithodorus). The ASF virus is a heterogeneous population consisting of clones with different biological characteristics in terms of GAD, virulence, infectivity, plaque size, and antigen properties. The virulence of an isolate is determined by the virulence of the dominant clone in the population, and not by the amount of virus introduced. Passaging of ASF virus isolates in pigs and in Vero cell culture can lead to a change in the ratio of different clones in the viral population and a change in all its characteristics. The cultural and virulent properties of the ASF pathogen are modified during the natural course of the epizootic and during experimental selection. The cultural and virulence properties of the ASF virus are extremely labile: it can lose its HA ability, reduce its virulence, up to its complete loss, during the natural evolution of an epizootic and in an experiment when passaged in tissue cultures.

Immunity and specific prophylaxis. Allergic or autoallergic reactions play a significant role in the pathogenesis and immunogenesis of ASF. Under the action of attenuated virus strains on lymphoid cells, defective ATs are synthesized, unable to neutralize the virus. Antigen-antibody complexes are formed, which are concentrated in the tissues of target organs, leading to a violation of their functions and the development of allergic and autoimmune processes; they observe the stimulation of cellular immunity - the lysis of infected cells by sensitized lymphocytes, the release of mediators of cellular immunity: lymphotoxin, the factor of inhibition of blast transformation migration, etc. The development of these processes depends on the biological properties of the strains used and the individual characteristics of the organism (state of the immune system).

A certain role in the pathogenesis of the disease is played by the interaction of the virus with erythrocytes and a violation of the mechanism of blood coagulation. The effect of the virus on the cells of the lymphoid system and erythrocytes is characterized by their destruction or change in function, as well as the development of allergic and autoimmune processes.

Animals that have been ill or vaccinated (with inactivated material or attenuated virus) have a certain degree of resistance to the homologous isolate of the virus (delay in the death of pigs), a change in the severity of clinical signs of the disease, recovery and a complete lack of response to control infection). The lack of specific protection against isolates isolated in other areas indicates their AH and immunological differences.

S. Anderson observed long-term carriage of the virus and its engraftment upon re-infection in recovered and vaccinated animals. Passive and colostral immunity is weakly expressed. AT does not sufficiently neutralize the virus. The reasons for the weak immunity tension, as well as the neutralizing activity of AT, are associated with the features of the antigen structure of the virus (blocking of antigen by lipids, competition or masking of protective antigen by the species antigen of the virus or host), as well as with a change in the function of lymphoid cells - a violation of the interaction of the virus and antigen with macrophages and cooperation of the latter with T - and B-lymphocytes. The first assumption is supported by a weak or altered response to inactivated antigen drugs in both susceptible and other animal species. Under conditions of low AT activity, cellular immune responses are enhanced, which are essential in blocking infection, and also cause the development of delayed-type hypersensitivity, allergic and autoimmune complications.

The process of protection in ASF is presented as a dynamic balance between etiological factors (virus) and immune defense mechanisms. It can be predominant in both directions, it depends on the properties of the applied strains and the state of the animal's immune system. There are no reliable prophylactic drugs against ASF. No one has succeeded in obtaining inactivated vaccines against ASF by classical methods using modern techniques. Most of the vaccinated animals died during the control infection, and only an insignificant part of them survived after a long illness. The results of testing an inactivated vaccine suggest that the structure of AG and their interaction with each other, and not the state of the immune system of the macroorganism, is of primary importance in the anomaly of immunity in ASF.

Preparations from live attenuated virus were more effective, causing a weak post-vaccination reaction, they protected 50-90% of vaccinated animals from infection with a homologous virus. However, the most significant disadvantages of live vaccines are prolonged virus carrying after vaccination, the development of complications in some immune animals, engraftment of a virulent virus in vaccinated animals without clinical signs of the disease, which is also dangerous in practical conditions. Given these shortcomings, the question of the use of live attenuated vaccines to eliminate foci of the disease in combination with other veterinary and sanitary measures has been called into question.

The multiplicity of immunological types of the pathogen and the existence of mixed or modified virus populations significantly limit the use of such drugs. However, there is information about the selection of effective agents for the treatment of sick pigs and removal of virus carriers, which can be used in combination with attenuated strains of the virus. Proceedings of the European Economic Community ASF Expert Meeting (1978-1987) and other reports outline the development of scientific research aimed at creating component, chemical and genetically engineered vaccines. For this purpose, the fine arterial structure of the ASF pathogen and infected cells, the structure and functions of the genetic material are studied, and protective antigens are searched using modern methods of molecular biology, genetics, mAbs. These directions may lead to the development of new approaches to the development of effective and harmless vaccines against ASF. The 9GL gene of the ASF virus is homologous to the yeast ERV1 gene involved in oxidative phosphorylation and cell growth, and to the ALV gene with the hepatotrophic fraction.

The 9GL gene encodes a protein of 119 residues (I) and is highly conserved in all ASF field isolates studied. I has been shown to be a late VASV protein. A mutant of the MAL strain with a deletion of the 9GL gene (A9GL) multiplies 100 times worse in macrophages and forms small plaques compared to the MAL parent. I affects the normal maturation of virions: 90-99% of virions in macrophages infected with the A9GL mutant have acentric nucleoid structures. The mortality of pigs is 100% when infected with the MAL strain, and when infected with the A9GL mutant, all pigs survive, and they have a temporary fever. All pigs infected with the A9GL mutant remain clinically normal, and their viremia titer is reduced by 100-10,000 times. All pigs previously challenged with the A9GL mutant survived subsequent challenge with a lethal dose of ASFV MAL. Thus, the A9GL mutant can be used as a live attenuated VALS vaccine.