Recognizing African swine fever disease and evolution

Introduction

African Swine Fever (ASF) is a devastating highly contagious disease affecting suids. It is caused by a big complex virus, the African swine fever virus (ASFV), that can spread very rapidly within the pig populations by direct or indirect contact. This virus can also become endemic in feral or wild suids, and the transmission cycles existing between these animals and the Ornithodorus spp. ticks, the biological vector, complicate control and eradication programmes.

The domestic pigs (DP) and Eurasian wild boars (WB, Sus scrofa scrofa) are susceptible to the virus infection showing a variety of clinical symptoms. In contrast, a tolerance to virus infection is observed in wild suids in eastern and south sub-Saharan Africa, as while infected they do not show clinical signs or lesions. Acute forms of ASF in susceptible DP and WB are commonly observed at the first stages of the virus introduction into free areas. These animals usually show extensive congestive-haemorrhagic pattern and functional disorders in the digestive and respiratory system, with high mortality rates that may reach up to 95-100%. Other clinical forms of the disease can be also observed after a time of the virus presence in a region, including asymptomatic courses (Arias &Sánchez-Vizcaíno, 2002).

ASF is considered a global animal health priority. Currently, it is present in more than 26 countries in Sub-Saharan Africa, in 18 countries in Europe (importantly in eastern European countries) and 11 countries in Asia. This also includes China with at least 32 provinces/autonomous Regions/municipalities/special administrative regions affected (FAO, February 2020), and Vietnam, with all provinces and municipalities struck by the disease. Its rapid dissemination in Asia since 2018 has resulted in major problems for the swine industry with huge socio-economic consequences. The disease presence in affected countries provokes significant problems at local, regional and global level, and great economic losses by livestock with closure of export markets.

ASF was maintained confined in Africa since the first description in 1921, jumping out the continent three times towards Europe, particularly Portugal twice (1957, 1960) and forty-seven years later Georgia in 2007. When ASF arrived originally to Europe it was spread to Spain (1960-1995) affecting later on to several European countries (in the period 1964-1993), as well as four countries in the Americas (period 1971-1983). Since 1999, the disease was restrained in sub-Saharan Africa and in the island of Sardinia (Italy) until 2007. During this period, several groups of scientists mainly those in Europe, as well as those belonging to the European Union Reference Laboratory (Madrid, Spain) and to the OIE Reference laboratories, in Madrid (Spain), Pirbright (UK) and Onderstepoort (South Africa), kept on working in a wide spectrum of ASF gaps related to disease maintenance, virus transmission, immune mechanisms involved in protection, vaccine development, diagnosis, virus-host interactions, reservoirs, carrier state, and many other epidemiology and control issues. Despite this, more resources in research and education of vets. farmers and producers were needed. However, ASF was not considered important enough, and in spite of the fact of the past cases and its serious consequences, the potential for an ASF re-emerging was underestimating. Therefore, at the time the disease appeared in Georgia, there was a significant unresolved number of gaps, a lack of knowledge about the disease, and a wrong perception about the scale of the problem that ASF and its causative clever virus could cause. What is more, faced with the immediate devastation of peracute and acute ASF, it was not given much thought to the potential dangers of the subacute and chronic infections that were soon to arise. Even this, and because the high virulent virus moving on causing mortality rates near to 100%, at the beginning it was initially considered by some as quite similar to the acute disease caused by other important haemorrhagic disease: Classical swine fever (CSF), which exhibits similar clinical symptoms than ASF. However, nothing could be further from the truth. ASF and CSF are both quite different diseases. ASF is much more complex to combat because of the complex epidemiology, the biological reservoirs, and the lack of neutralizing antibodies and despite the attempts, the lack of a commercial vaccine available. CSF and ASF viruses are completely different: CSFV is an RNA virus of 12,3 kbp. producing around 12 mature proteins, two of them involved in the induction of neutralizing antibodies, which facilitate the availability of a safe effective vaccines (Zhang et al., 2011). By contrast, ASFV is a large complex DNA virus of 170-193 kbp, and, more than 100 polypeptides are induced in the infected cells, with capacity for up 150 (Arias et al., 2017) and despite a high amount of antibodies the virus produces in the infected animals, these are unable to neutralize completely the virus, and this is one of the reason why in spite of the efforts, there is not a vaccine to date.

This review aims to give an updated information about the disease knowledge and recognition, emanating from both the past and the current epidemics, in the different scenarios, pointing out some important issues that matter to veterinarians, producers, hunters and veterinary services. To get success in control and eradication programs is necessary to avoid the solely idea that ASF must give rise to severe lesions and have a mortality rate closely approaching 100%. Based on scientific studies performed in the past epizooties in Europe, and in the Americas, and more recently in the current epidemic in Europe, we give a detailed description of a variety of forms of reduced mortality that can be also present in variable percentage, and therefore arising survivors, recovered and immune animals that play a role in virus transmission, spreading and disease maintenance.

ASF virus (ASFV): a rather unique pathogen

ASFV naturally infects and replicates in macrophages, key actor cell in arising an effective immune response against foreign invaders such as ASFV (Arias et al., 2017). The major components of the viral capsid have been also identified as the most antigenic proteins which are responsible for the production of specific antibodies in the infected animal after a natural infection. However, regardless of the usefulness of these proteins as sero-diagnostic targets, they are not sufficient to develop a protective response based on specific neutralizing antibodies nor developing antibodycell mediated protection against virus infection. Nevertheless, a great amount of specific antibodies against ASFV can be detected from the second week of infection by laboratory diagnostic techniques (Gallardo et al., 2019a). This feature makes ASF specific antibodies good markers to point out the disease presence. ASF specific antibodies are very useful for the detection of survivors and recovered animals, since stay detectable for months and up to years. The specific antibodies are partly responsible for delaying the appearance of clinical signs and reducing viremia levels. However, they are not good markers for the detection of acute infection, which is major in European and Asian affected countries, since a large percentage of infected animals usually died during the first 6-11 days post infection, before ASFV specific antibody appearance.

Due to the fact that not fully neutralizing antibodies are induced during the virus infection, it has not been possible to perform a universal classification of ASF virus isolates based on serotypes. Instead, since the beginning of this century it has been internationally accepted to classify the ASF viruses following genotyping. This is based on sequence analysis of a specific fragment of the gene encoding the major viral protein p-72. Up to 24 genotypes have been described so far, all of them coexisting in Africa (Achenbach et al, 2017; Quembo et al. 2017). Two genotypes (I and II) went outside Africa so far: the genotype I emerged in Portugal in 1957 and 1960, further spreading to Europe and the Americas. Besides the genotype II reached Eastern Europe (Georgia) in 2007, being the causative virus involved in the current epidemic wave by spreading towards west and east territories, getting Asia in 2018. Although belonging to the same genotype II, the molecular analyses of specific variable genome regions of the European and Asian isolates have identified a range of genetic variants as a result of the natural evolution of the virus. However, at the level of the whole genome, the sequence homology among isolates from different regions is greater than 99.9%, which is in the range of the typical genome stability of DNA viruses (Magolokin et al, 2012; Gallardo et al., 2014; Ge et al., 2018; Bao et al., 2019).

It is important to point out that the term “genotype” is a molecular concept, used to establish relationship between viruses and therefore to trace the origin of outbreaks. However, the molecular characterization by genotyping doesn´t correlate or identify with virulence or any specific clinical form of the disease. Instead, the ASF disease presentation is produced by multiple complex factors, including i) the specific virus strain affecting, characteristics, dose and route of infection; ii) the presence of the hosts and transmission cycles (domestic pigs, wild suids and Ornithodoros spp.), the immunological and genetic characteristics of the host, the virus-host interaction, and the role that each actor plays in a specific scenario, in a certain period of time. In addition iii) the environment and human factors, which also play a role in the disease expression, since both elements could influence virus dissemination and maintenance of certain virus strains.

ASFV is highly resistant in the environment and can persist in blood and tissues after death of the infected animal. We should be in mind, the cannibalism of carcasses by WB has been demonstrated and it can contribute to virus transmission in wild suids in the forest. In addition, massive environmental contamination may result by the secretion and excretions of these infected animals, including bloody diarrhea, or if blood is shed during necropsies. These factors, among others make more difficult the control of ASF in wild suids. Importantly, the virus can be maintained alive for long periods of time in frozen and chilled blood or organic infected material. The virus persists as infectious in sera or blood stored at room temperature for 18 months and for up to 15 weeks in putrefied blood (EFSA 2010). It has been also established that virus can remain infectious in contaminated pens for several days and up to several weeks in pig faeces, urine or slurry (EFSA 2010).

In meat products, the virus may persist for several weeks or months in frozen or uncooked meat. Nevertheless, it has been scientifically demonstrated that Spanish cured pig-meat products, such as Serrano hams and Iberian hams and shoulders were free of viable ASFV by day 140 and Iberian loins by day 112 – before the cured period process- (Mebus et al. 1997). In cooked or canned hams, no infectious ASFV has been found when these products were heated to 70°C. Infectivity of ASFV is lost by 110 days in chilled deboned meat, bone-in meat, or ground pork and after 30 days in smoked deboned meat (EFSA, 2010).

On the contrary, ASFV is very sensitive and inactivated by heat treatment at 60ºC for 20 minutes or 1h a 56ºC, and by many lipid solvents and commercial disinfectants (EFSA, 2010).

The Host

The epidemiology of ASF is very complex and varies greatly. Domestic pigs and Eurasian wild suids are susceptible to ASFV infection. In contrast African wild suids (warthogs, bush pigs and Giant Forest hogs) develop asymptomatic infections. The molecular factors involved in this tolerance have not been yet determined. The fact of this asymptomatic wild suids presence in eastern regions of Africa makes eradication very difficult in the region. African wild suids established an ancient virus transmission cycle involving soft ticks. Both act as natural reservoirs in Africa (Detray, 1957; Penrith & Vosloo, 2009). The infection of African wild suids usually results in low virus titers in tissues and low or undetectable viremia. These levels of virus are sufficient for transmission to domestic pigs through tick vectors, but usually not by direct contact between animals. The soft ticks Ornithodorus spp are ASFV biological reservoirs, and transmission vectors. They are present in eastern and southern parts of sub-Saharan Africa, particularly O. moubata, the African tampan. In a similar way, O. erraticus exists in the western regions of Iberian Peninsula and they played a role in the epizooty occurred in 1960-1995 (Arias and Sánchez-Vizcaíno, 2002; Sánchez-Vizcaíno and Arias, 2019). However, it has not been established a potential role of soft ticks in the current ASF epizooty in Europe.

Several Ornithodorus species experimentally tested seem able to transmit ASFV, including, O. porcinus, O. coriaceus. O. turicata and O. savignyi (EFSA, 2010; Groocock, et al.,1980; Hess et al., 1987; Jori et al., 2013; Mellor& Wilkinson, 1985). Other species of soft ticks have been identified in regions of North and South America and should be considered to potentially harbor and transmit ASFV (EFSA 2010; Donaldson et al., 2016). In the absence of viraemic hosts, O. ticks can allow ASFV infection to persist for more than 5 years in a region (Boinas et al., 2011). The geographical distribution of Ornithodorus. spp in many regions of the world has not been yet well elucidated. It should be taken in mind the presence of Ornithodorus spp in the Americas could potentially have a role in the continent, in case of an ASF incursion.

Several studies in Eastern part of Africa have revealed a complex epidemiological situation in which local breeds of domestic pig seem to show greater tolerance to ASFV that can favours endemicity and spread of the disease (Atuhaire et al., 2013; Uttenthal et al., 2013). These “Tolerant" indigenous pigs in Mozambique and in Kenya, and the presence of ASF sero-positive animals in regions of Malawi might reside in the immunogenetics and genetic characteristics of the indigenous pig populations but can also be related to population immunity (Haresnape et al. 1985, 1987; Gallardo, et al., 2012; Okoth et al, 2013). Besides this, virus evolution towards moderate virulent forms in an area could be also contributing to the presence of asymptomatic pigs.

Considering all the three actors, domestic pigs, wild suids and the Ornithodorus spp., as well as the different susceptibilities that domestic and wild suids may show, together with their coexistence in the scenarios and, taking into account their interactions and virus transmission cycles, that will be dependent of many factors (such as the environment -agricultural areas, forest, vast monoculture plantations- as well as the human factors), all these items reflect the great complexity that ASF represents and the difficulties to prevent its spreading.

The Disease

ASF is not associated with pathognomonic lesions, and clinical signs observed in its acute or chronic forms may be similar to other haemorrhagic diseases such as classical swine fever, salmonellosis or erysipelas. The virus is commonly transmitted when unexposed pig populations (domestic or wild) have direct or indirect contact with blood, excretions, secretions, meat or carcasses from infected animals. The incubation period varies from 4 up to 19 days and virus excretion can start already at this stage. Viraemia (virus presence in blood) usually begins 2-8 days post-infection (dpi) and persists for at least 3 to 5 weeks. In the acute phases of infection, the virus titre in tissues and blood is very high. This is due to the absence of fully neutralizing antibodies, and then an intermittent viraemia is usually maintained during convalescence, that can be detected up to 3 months in the recovered animals (Gallardo et al; 2015a). These animals can maintain the virus in tissues for up to 99 days or even more time as demonstrated in past studies what point out the potential for virus transmission by contact or feeding (Hess, 1981; Gallardo et al., 2015a). This is particularly important in the case of death WB in the forest, since it has been already demonstrated a cannibalism behaviour of the WB exists. An investigation carried out in the USA demonstrated virus presence in tissues after 120 days in recovered asymptomatic animals after inoculation with the Brazilian isolate, and healthy animals became infected with this material through feeding (Mebus and Dardiri, 1980).

As mentioned, ASF clinical presentation is influenced by multiple factors such as virulence of the virus strain affecting, the host and breed, the dose and the route of infection (Sánchez-Vizcaíno et al, 2015 Sánchez-Cordón et al., 2017;). This matter has been well studied in the past and again in the ASFV infections caused by the genotype II ASFV strains circulating in Eastern Europe in the wild boar and domestic pig populations since 2007 up to present (Mebus & Dardiri, 1979; Hess, 1981; Mebus et al, 1980,1981, 1983 ; Blome et al., 2013; Vlassova et al., 2015, Gallardo et al., 2018; 2019b).From these studies it is concluded that whatever the genotype is, the disease presentation and the disease evolution in the past and in the current epizooty of Europe and Asia, always exhibits a same pattern. ASF clinical forms vary from peracute, acute, subacute, chronic and subclinical that are produced by highly virulent, moderately virulent or low virulent/attenuated isolates (Mebus, et al., 1980, 1981; 1983; Hess,1981; Pan & Hess, 1984; Gómez-Villamandos et al., 2013; Sánchez-Vizcaino et al., 2015). A good characterization of the circulating viruses will give insights about what should be expected in the field, with a better disease recognition and, therefore, early detection that will influence in the success of the control eradication programmes.

Highly virulent viruses are usually responsible for the peracute and acute forms with high mortality rates that may reach 100% within 4–9 days post-infection. In peracute ASF, affected animals can die suddenly as soon as 1–4 days after the onset of clinical signs with no so evident lesions in organs. Pigs showing the acute forms of the disease display mainly a febrile syndrome moderate anorexia, lethargy and weakness, huddle together and in the final stages, suffer respiratory disorders characterised by rapid laboured breathing, with nasal secretions caused by pulmonary oedema, and deaths are expected from 3-8 days post infection. Exanthemas are very evident (pinkish almost purple skin due to intense hyperaemia), and/or cyanotic foci, which appear as irregular in the skin of the extremities, ears, chest, abdomen and perineum, and also haematomas and necrotic areas, though these lesions usually are more intense in pigs infected with moderately virulent isolates. Internal lesions are mainly characterized by hyperaemic splenomegaly and haemorrhages in organs, particularly in the visceral lymph nodes, petechial haemorrhages in the kidneys, bladder mucosa, pharynx and larynx, pleura and heart, and an excess of natural fluids in body cavities and spaces (Sánchez-Vizcaino et al, 2015). Haemorrhagic discharge from the anus (melena) is sometimes observed. The distribution and frequency of these lesions are variable and most are seen in other swine diseases such as classical swine fever. Acute forms of the disease by virulent viruses have been described in all the epidemiological scenarios were the disease is currently present. (Pan and Hess, 1984; Gómez-Villamandos et al., 2013; Sánchez-Vizcaino et al., 2015).

Moderately virulent viruses lead to the appearance of acute, subacute, chronic and subclinical forms of the disease. The reasons why ASF exhibits this significant variety of clinical signs are not precisely clear, but surely the immune mechanisms of defence developed by the hosts play a role. Pigs may show persistent or fluctuating temperature responses for up to 20 days. Some pigs may stay in good condition, while others show similar but less severe clinical symptoms than those observed in the acute process. Mortality rates range 30–70%, the lowest rate displayed in adults. The death usually occurs after 2-3 weeks post infection. (Pan & Hess, 1984; Mebus & Dardiri, 1979; Gomez-Villamandos et al., 2013; Sanchez-Vizcaıno et al., 2015; Mur et al., 2016). Animals infected with moderately virulent isolates show a variety of clinical symptoms depending on the course of the disease. Some of them can exhibit acute and subacute forms, with clinical signs developing slowly, and deaths between 7 to 20 dpi. In the subacute presentation the vascular lesions, mainly haemorrhage and oedema, are more marked than in acute courses as the lesions develop fully (Gómez-Villamandos et al., 1995, 2013). Pulmonary oedema does not usually occur, though pulmonary haemorrhages does as well as foci of necrotic pneumonia. To further complicate matters, distinction should be made between the chronic disease that is occasionally encountered during the acute and subacute virus infections. Mortality in the chronic animals is low, affecting between 2 and 10% of all the sick animals. Chronic ASF is especially difficult to recognize, because it is extremely variable in its appearance. Clinical signs and lesions are not specific and may persist for several months with no particular signs, other than stunting or emaciation, or it may mimic a variety of illnesses. In addition to stunted growth and emaciation, some clinical signs may include skin ulcers, arthritis, and lameness due to swollen of joints, respiratory symptoms and abortion. Several symptoms are as the result of secondary bacterial infections, the most significant being articular alterations and in reproduction.

It is likely that the incursion of ASF in the Americas were by a moderate virulent virus strain. Although the initial diagnosis were made in outbreaks where mortality was high and the acute disease was prevalent, very shortly thereafter the disease was found to be widespread and considerably reduced in mortality. In all probability, the disease had been in the country for quite some time and had spread extensively. It was diagnosed only after it began to appear in the acute form with mortality high enough to cause alarm. (Hess, 1981)

In areas were moderate virulent virus has been found, the Low virulent or attenuated viruses have been also described, causing chronic and unapparent forms of disease. In vivo experiments with attenuated viruses show both patterns, chronic and subclinical, that may also be observed simultaneously in the same pen.

Virus evolution Disease presentation and recognition

The chronic and subclinical forms of the disease are the result of appearance of moderate and low virulent/attenuated virus strains. This is the natural attenuation and virus evolution of ASFV. Chronic forms have been previously described in the Iberian Peninsula and in the Dominican Republic (Mebus & Dardiri, 1979; Leitao et al., 2001; Sánchez-Vizcaino &Arias, 2019), and it has been recorded in the in vivo experiments with virus strains from Europe in the recent epizooty (Gallardo et al; 2016, Nurmoja et al, 2017). Arthritis and skin ulcers have been associated mainly with the chronic disease that occurred in large numbers of pigs that received modified live virus vaccines in Portugal and Spain during the early 1960s (Sánchez-Vizcaíno, et al., 2015).

Subclinical, unapparent, forms are usually reported in endemic scenarios, in which clinical signs are mild or even absent. These ASF infected animals can be only identified by laboratory diagnosis. The detection of these animals is of major importance since they may act as potential virus carriers, mainly during the recovering time and up to three months after infection. Their presence pointed out new changes in the circulating virus strains and therefore disease evolution. The detection of these animals, survivors, recovered and asymptomatic animals should not be underestimated.

It has been shown that all these clinical forms (acute, subacute, chronic and subclinical) are present in a variable percentage in regions where ASF is present after months or years. Also, it has been well established to be present in the past in the affected countries, as well as in the recent epizooty in Europe. The presence of high-virulent, moderate and low virulent virus isolates belonging to a same genotype moving simultaneously in a specific zone have been demonstrated (Gallardo et al., 2018, 2019 Nurmoja 2017). Therefore, a key point to efficiently combat the disease is to remind that regardless the genotype, and the virulence of the virus in the primary outbreaks, the evolution of the clinical infection after a time (several months or years) shows a common pattern: the presence of per-acute/acute, subacute, chronic and subclinical forms of the disease, in a percentage variable depending on the time and the epidemiological scenario.

Disease recognition is a key point for an efficient early detection of the disease. This recognition may vary due to a number of parameters as explained above. ASF may doesn’t resemble a typical picture. Moreover, ASFV introduction in a farm is not easy to detect at the beginning, since only fever and deaths with some hemorrhagic lymph nodes will be observed in few animals. Several cycles of infection are required to arise a significant virus amount before it began to display the typical characteristics of the acute disease with high mortality of the animals in a pen. Following an initial infection in several pigs, a second wave of infection 12-14 days later should be expected, that will lead to a significant number of deaths, thus increasing the infective pressure. A third wave later and a devastating wave of deaths will take place (Hess, 1981; Arias et al., 2014; Gallardo et al., 2015). In high-risk areas, sudden deaths of few animals should not be merely attributed to common causes but, rather more be treated with the upmost seriousness. Depending on the type of production system, the biosecurity conditions, husbandry, management and organization different evolution patterns of ASFV infection could be expected, that also will influence the movement and spreading of the ASFV infection waves. One key element in prevention ASF introduction is not to subestimate the disease presentation, be alert, and not to minimize the appearance of clinical symptoms, i.e fever, even if they are affecting few animals, as well as to carry out periodical clinical checking.

IPVS - Recognizing African swine fever disease and evolution - Image 1

Published in the proceedings of the International Pig Veterinary Society Congress – IPVS2020. For information on the event, past and future editions, check out https://ipvs2022.com/en.

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Recognizing African swine fever disease and evolution

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