African swine fever (ASF) is an infectious disease causing high mortality of pigs, and is notifiable to the World Organisation of Animal Health (OIE). The aetiological agent, African swine fever virus (ASFV), is classified as the sole member of the
ASFV resistance and stability have attracted the interest of numerous investigators over the years (5, 7, 10, 23, 26, 27, 28, 41, 42, 47). It has been proved that ASFV shows high resistance to environmental conditions and remains infectious over a long storage time either below 0°C or at 4°C. The curing process of infected meat (a process like that which Parma, Iberian, or Serrano ham undergoes) facilitated survival of ASFV in ham for over a year (28). ASFV can survive many freeze–thaw cycles, and furthermore it is stable at pH levels between 4 and 13 and can survive a temperature of 56°C for over an hour (42).
Due to its high stability, ASFV is able to persist for a long time in contaminated fomites or meat; therefore they could play a role as vehicles for the transboundary or even transcontinental spread of the pathogen. Such a mode of dissemination is one of the most frequent routes of ASFV introduction into territories previously free of it. For example, in 2007 an ASF outbreak in Georgia was caused by improper disposal of contaminated pork meat from a ship at Poti docks. Similar events occurred in history to cause other ASFV introductions, namely to Portugal (1957), Cuba (1971), Brazil (1978), and Belgium (1985) (10, 31). Besides through the negligence by which humans spread the disease transnationally, ASFV is present in an environment because of its long persistence in wild boar carcasses, which can in effect be a virus reservoir. Therefore eradication of the disease is extremely difficult, due to the necessity of actively searching for decaying boar cadavers to make possible the proper disposal of infected carcasses (3).
Over a span of many years, numerous experiments were dedicated to ASFV stability. As far back as 1921, Montgomery (30) demonstrated that ASFV is extremely resistant to high temperatures, putrefaction and desiccation. Much later, Coggins (5) evidenced high ASFV resistance to selected chemical (trypsin and EDTA) and physical treatments (freezing/thawing and ultrasonic waves). In the same study he successfully collected viable virus after 1 h incubation at 56°C and one week at 37°C. Plowright and Parker (42) in 1967 showed that storing at 4°C preserves infectivity of viraemic blood for at least 75 weeks and of virus-spiked medium without Ca2+ or Mg2+ for 61 weeks. At 37°C, medium containing ASFV remained infectious for 11–22 days, but at 60°C it was only for 30 min. It was also shown that the virus is stable over a wide pH range (from pH 3.9 to 13.4) for seven days (in serum-supplemented media) (42). Another study concerning ASFV resistance to temperature was conducted more recently, and partially confirmed previous results. It was shown that the virus was stable at 4, 22, and 40°C and lost only less than 101 50% haemadsorption doses (HAD50)/mL during 24 h incubation in EMEM. At 50°C, only a small fraction of virus remained infectious, and at 60°C no infectious virions could be detected after only 15 min (47). In summary, ASFV in tissues can survive deep freezing (−70°C) for many years without significant loss of titre, but at −20°C it systematically loses its titre, nevertheless remaining viable for at least 105 weeks (2 years) (42). At 4°C it is also very stable when contained in medium; it remains infectious for at least 61 weeks (1 year and 2 months). In higher temperatures ASFV is inactivated relatively quickly. At 37°C traces of viable virus could be found after 22 days, at 56°C after 1 h, but at 60°C no longer than after 15 min.
Direct contact between sick and susceptible animals has repeatedly been proved to be an effective transmission route for ASFV (2, 14, 17, 21, 38, 41, 48). Recent experiments conducted with current ASFV European strains showed that viral DNA and/or infective virus might be detected in blood (first detection at 3.75 ± 1.4 dpi) (2, 14, 17, 21, 33, 38, 41, 48), nasal, rectal, and oral fluids (21, 33, 37, 41, 48), and faeces and urine (7, 21, 37, 38) of infected animals. The highest viral loads in blood are recorded between 5 and 27 dpi by intramuscular or intranasal inoculation or 9 and 29 dpi when pig-to-pig contact is investigated, and maximum ASFV titres in blood range from 106 to 109 HAD50/mL in the acute phase (Table 1).
Current Eurasian ASFV strain levels of maximum viraemia in blood and shedding potential in other body fluids after various modes of inoculation
Maximum viraemia |
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Inoculation mode | ASFV strain | Dose HAD50/(mL) | Titre (log10 HAD50/mL) | Dpi | Virus detection in other body secretions/excretions | Reference |
Georgia 2007/1 | 102 | 6–8 | 5 | Nasal fluid: VI+ (102–104), 5 dpi Rectal fluid: (10–102), 6 dpi Urine: Excretions: PCR+*, PCR+*, VI+ VI+ Oral fluid: PCR+*, VI− *1st day of fever | Guinat |
|
Intramuscular | Lithuania LT14/1490 | 101 | 6.4–8.7 | 6 | n/a | Gallardo |
5 × 103 | 6.5–7 | 7 | Nasal fluid: PCR+ | |||
Russia K/08/13 | 50 | 6.5–7 | 9 | Rectal fluid: PCR+ | Vlasova |
|
Georgia 2007/1 | 103 | 7–8 | 7 | n/a | O’Donnell |
|
Odintsovo 02/14 | 5 × 103 | 7.56 | 11 | n/a | Elsukova |
|
Caucasian | 2 × 106 | Cq: 22–39 | 5 | Oropharyngeal Faecal fluid: PCR+, fluid: 5 PCR+, dpi 6 dpi | Blome |
|
5 × 103 | 7.5 | 7 | ||||
Kashino 04/13 | 50 | 6.5–7.5 | 7 | Nasal fluid: PCR+ | ||
5 × 103 | 6.5–7.5 | 9 | Rectal fluid: PCR+ | Vlasova |
||
Boguchary 06/13 | 50 | 6.5–7 | 5 | |||
Oral/nasal | Pol/15/Lindholm | 2 × 104 | ~9 | 6 | Nasal fluid: PCR+*, VI+ (101.6–4.8) Rectal fluid: PCR+*, VI+ (102.8–3) Oral fluid: PCR +*, VI− *1st day of fever Viral isolation at day of euthanasia | Olesen |
Odintsovo 02/14 | 103 | 7.45 | 11 | n/a | Elsukova |
|
50 | 7.45 | 27 | ||||
Caucasian | n/a | Cq: 20–29 | 11 | n/a | Blome |
|
Georgia 2007/1 | n/a | 6–8 | 10 | Nasal rectal fluid: fluid: VI+ VI+ ((1010––101022) ) from from 7 12 dpi, dpi | Guinat |
|
Kashino 04/13 | n/a | 6.5–7 | 15 | n/a | ||
Boguchary 06/13 | n/a | 7 | 9 | n/a | Vlasova |
|
Contact | Lithuania LT14/1490 | n/a | 6.4–8.7 | 14 | n/a | Gallardo |
Odintsovo 02/14 | n/a | 7.45–7.66 | 29 | n/a | Elsukova |
|
Pol/15/Lindholm | n/a | ~9 | 12 | Nasal fluid: PCR+, VI+ (101.8–2.8) Rectal fluid: PCR+, VI+ (101.6–8) Oral fluid: VI− PCR + in many prior to the PCR+ from blood | Olesen |
n/a – not applicable, dpi – day post infection, HAD50/mL – haemadsorbing doses per millilitre, PCR+(−) – viral DNA detected (not detected) by real-time PCR, VI+(−) – infectious virus detected (not detected) by virus isolation in cell culture
As regards excretions and secretions, it has been demonstrated that they might contain viable virus (1.6–4.8 log10HAD50/mL) (Table 1) on the day of euthanasia, but in the case of contact animals, the virus was molecularly detected in nasal fluid prior to being evident in blood (38). ASFV survivability in these contaminated excretions depends mainly on temperature; however, in favourable conditions it may retain its viability for long time, increasing the risk of the disease spreading, particularly under low-biosecurity conditions (13).
It has been clearly and repeatedly shown, that solely air contact (without direct physical contact between sick and healthy animals) is sufficient to develop a clinical course of the disease in susceptible pigs (9, 38, 50). Air sampled during experimental infection was consistently molecularly and virologically positive during the first 25–30 days after infection, with virus titres up to 103.2 TCID50 eq./m3 (9).
As the viable virus has been identified in excretions, it raises the question of how long the virus can survive in them. Montgomery (30) showed as long ago as 1921 that when faeces are stored in the dark at room temperature they remain infective for at least 11 days, but other later studies showed that this time might be extended up to 160 days (13). The most recent investigations indicated that ASFV stability in faeces is much lower than previously thought and depends largely on the temperature. Faeces collected from experimentally inoculated pigs remained infectious for 8 days at 4°C, and for 3–4 days at 37°C. When it comes to urine, it might contain viable virus for up to 15 days at 4°C, 5 days at 21°C, and 2–3 days at 37°C (7).
The study by Olesen
Since infectious ASFV is secreted and excreted, it therefore easily contaminates the environment, which subsequently may act as a virus source. Numerous epidemiological studies have proved that ASFV can be easily transmitted, either by direct contact or indirectly,
In 2018, ASF unexpectedly emerged in eastern Asia where dozens of outbreaks in pigs were reported in China and Mongolia, and in 2019 ASF struck also in Vietnam (35). Molecular characterisation of the intergenic region (IGR) between the I73R and I329L genes revealed a high level of DNA sequence similarity between recent Chinese and Eastern European (IGR II) ASFV isolates, but not between Chinese and Siberian ones (of the Irkutsk 2017 strain) (IGR I) (19, 22, 25). The exact origin of the disease in eastern Asia still remains unknown and needs further investigation; nevertheless a recent phylogenetic analysis indicated that ASFV in China might have had at least two independent introductions due to some level of divergence in nucleotide sequences obtained from cases which occurred far from each other (49). Most of the outbreaks which recently emerged in China were separated by thousands of kilometres, suggesting that the spread of the disease might be associated with contaminated feed. This hypothesis seems to be probable, in particular having regard to the fact that ASFV DNA has been detected in pig feed and feed ingredients like dried pig blood (24, 44, 49). In Europe, besides on backyard farms, the disease has been reported on numerous high-biosecurity operations (12). In Romania, ASFV was introduced into a high biosecurity breeding farm containing up to 140,000 pigs; however, the exact source of the disease has not been determined. Hypothetically it may have originated from contaminated water in the nearby Danube River (3).
Several experimental studies have demonstrated that transmission
Accordingly to these experimental data and recent epidemiological findings in Europe and Asia, long-distance (transboundary and transcontinental) movement of ASFV with contaminated feed and feed ingredients should be considered a possible mode of virus spread, especially within ASF-free areas.
Historically, ASFV introduction into distant disease-free territories has been attributed to the consumption of contaminated pork or pork products (31, 45). Moreover, although prohibited in the EU and in contravention of biosecurity measures, swill feeding is still a common practice all over the world, especially in free-ranging and backyard farms (1). Therefore, contaminated pork presents a possible mode of transmission for ASFV. Heated, cooked, and canned meat products are generally considered safe as regards any viable pathogen presence, which has been experimentally demonstrated (5, 42, 46). Several experiments provided data concerning ASFV stability in raw and processed meat and other pork products (13, 26). Frozen raw meat and organs provide ASFV viability for periods lasting from 103 to 118 days, but according to Adkin
The matter of ASFV survival in products which cannot be heat-treated but are preserved through salting and drying is more complicated than in raw pork (12). The studies regarding ASFV survival in dry-cured processed meat are limited to ham, Spanish and Italian shoulder, loin, smoked pepperoni and salami, pork belly, and corned meat (26, 27, 28 40, 46). Salami and pepperoni might remain infectious up to 30 days (26). Pork belly and loin were demonstrated to still contain viable ASFV after 60 and 83 days, which is longer than the duration of their commercial curing processes (14–21 and 60 days, respectively) but still within the shelf-life of the products. These pork products pose a low potential short-term risk if in swill fed to pigs (40). Corned meat stored at 4–6°C remained infectious for at least 60 days (the study duration), nevertheless the time reduced to 16 days as the temperature increased to room temperature (42). Ripening hams, like Iberian loin (112 days), shoulders (140 days), and Serrano and Parma (respectively 180 and 300–399 days) hams might remain infectious relatively long, but still cease to be within the duration of the curing process, which lasts much longer (13, 27, 28). Therefore the curing time is sufficient to inactivate ASFV and these products should be considered safe.
ASFV is a tick-borne virus; therefore ticks as well as pigs may host the virus. Nevertheless, so far only soft ticks of
Nevertheless, as several cases of ASFV outbreaks have been reported on high-biosecurity farms in Eastern Europe and the Baltic States where the density of infected wild boars was high, the question arises whether arthropods may play a mechanical vector role between wild and domestic pigs. Hard ticks were investigated to determine their competence as an ASFV vector. ASFV was not detected either in the field-collected ticks or ticks fed infectious blood which transmitted the virus to susceptible animals in laboratory conditions (8). ASF virus does not replicate within the tick organisms; however, viral DNA could be detected from six to eight weeks after feeding with infected blood. Therefore, hard ticks may represent only a potential mechanical but not a biological vector in transmission between wild boars and pigs (8).
The stable fly,
Adducing evidence from fieldwork, viral DNA was detected in Poland in stable flies collected from farms during disease outbreaks in pigs as well as in hard ticks
Along with increasing globalisation, the introduction of human and animal diseases is going to pose a continuous threat to public and livestock health, trade of animals and their products, and food security. Worldwide, the pig-farming industry is constantly growing in reply to rising demand for pork meat. Nevertheless, this branch of the economy is particularly vulnerable to production decimation because of transmission of various transboundary infectious diseases, amongst which ASF is currently causing the greatest concern. During recent years, ASFV has been spreading towards new areas; however, the most dramatic turn occurred in 2018, when the disease emerged in China, the top pig-farming nation providing half of global pig production. Due to the lack of a safe effective vaccine and the common presence of infected wild boars in particular areas, the only method to control the disease is strict biosecurity measures allied to international cooperation on this matter. Knowledge and epidemiological understanding of how the virus may be introduced into susceptible populations of pigs is crucial to provide awareness to prevent the outbreaks and detect and control them immediately and appropriately when they do occur. Therefore, identification of potential sources and pathways of transmission in regards to ASFV is exceptionally important to prevent further disease spread.
ASFV stability in different environmental conditions was the subject of numerous investigations, but most of them were conducted in the previous century. The virus has been identified as extremely resistant to physical treatment such as high temperatures, putrefaction and desiccation, freezing/thawing, ultrasonic waves, or extreme pH values. Low temperatures, such as the −20°C usually required to preserve pork meat, facilitate virus survival for years. Raw meat and other pork products can provide long ASFV survivability, but the temperature conditions are the main factor directly influencing virus stability. Ripening hams and dry-cured meat products may contain viable virus, nevertheless it depends greatly on the preparation and conservation techniques, which differ widely between regions and countries.
In reference to the disease’s transmission, it was proved that ASFV infectivity without a susceptible animal having direct contact with infected blood is rather moderate; nevertheless, transmission only
As regards ASFV survivability and its indirect transmission, there are still data that are missing,