ELISA TESTS:
For antibody detection
o INGEZIM PPA COMPAC K3 (INGENASA)→ blocking ELISA assay which uses a monoclonal antibody (MAb) specific of VP73 ASFV protein o SVANOVIR® ASFV-Ab → indirect ELISA based on a recombinant antigen - the p30 (screening format) o ID Screen® ASF → Indirect Multi-antigen indirect ELISA kit for the detection of antibodies against P32, P62 and P72 ASFV proteins.
For antigen detection
o INGEZIM PPA DAS 1.PPA.K2, commercial kit (Ag-ELISA).
o Tetracore real-time PCR kit based on primers and probes designed by Zsak et al., 2005.
o INGEZIM PPA CROM (INGENASA) based on the technique of Direct Immunochromatography which uses a Monoclonal Antibody (MAb) specific of VP72 of ASFV.
GAPS:
- Field validation data need to be expanded for all tests.
- Penside tests for Antigen detection
- Worldwide distribution of existing commercial tests
- Need for developments of new commercial assays for ASF virus and antibody detection.
· ELISA TESTS:
For ASF antibody detection
- INGEZIM PPA COMPAC K3 (INGENASA)→ blocking ELISA assay which uses a monoclonal antibody (MAb) specific of VP73 ASFV protein
- SVANOVIR® ASFV-Ab → indirect ELISA based on a recombinant antigen - the p30 (screening format)
- ID Screen® ASF → Indirect Multi-antigen indirect ELISA kit for the detection of antibodies against P32, P62 and P72 ASFV proteins.
For ASF antigen detection
- INGEZIM PPA DAS 1.PPA.K2, commercial kit (Ag-ELISA).
· PCR TESTS
- Tetracore real-time PCR kit for ASF; real time PCR based on primers and probes designed by Zsak et al., 2005.
- INgene q PPA real-time PCR kit (INGENASA) based on the use of a Universal Probe Library (UPL) labelled with FAM (Fernández et al., 2013)
· PENSIDE TEST FOR ANTIBODY DETECTION
- INGEZIM PPA CROM (INGENASA) based on the technique of Direct Immunochromatography which uses a Monoclonal Antibody (MAb) specific of VP72 of ASFV.
GAPS:
- Field validation data need to be expanded for all tests.
- Penside tests for Antigen detection
- Need for developments of new commercial assays for ASF virus and antibody detection
Identification of the agent
ASFV genotyping: i) sequencing of the C- terminal end of VP72 gene,which differentiates up to 22 distinct genotypes; ii) full genome sequence of the p54-gene and iii) analysis of the central variable region (CVR) to distinguish between closely related isolates and identify virus subgroups within the 22 p72 genotypes. Described in the OIE Manual of ASF diagnosis (OIE, 2012) and by the EURL (http://asf-referencelab.info/asf/en/).
GAPS:
- Update of the EU and OIE Manual of diagnosis for ASF.
Identification of the agent
o Serological tests
ASFV genotyping: i) sequencing of the C- terminal end of VP72 gene,which differentiates up to 22 distinct genotypes; ii) full genome sequence of the p54-gene and iii) analysis of the central variable region (CVR) to distinguish between closely related isolates and identify virus subgroups within the 22 p72 genotypes. Described in the OIE Manual of ASF diagnosis (OIE, 2012) and by the EURL (http://asf-referencelab.info/asf/en ).
GAPS:
- Update of the EU and OIE Manual of diagnosis for ASF.
High potential to date, due to the current situation.
GAPS:
None
None
None
None
No vaccines available
Needs for authorisation in country to be used in.
Until vaccines developed difficult to assess.
Not at present.
Attempts over many years to develop inactivated or attenuated vaccines to ASF have failed. Inactivated vaccines have conferred no protection. Attempts to attenuate the virus through passage in cell culture have not been successful:
- DNA vaccine strategies have not been successful to date. - Deletion mutant’s strategies have not been successful to date.GAPS:
- Knowledge/molecular basis and understanding immune mechanisms of surviving pigs to resist a challenge with the homologous virus.
- Knowledge of inhibition mechanisms for virus replication.
- New developments based on new , non-conventional, strategies.
- Characterization of the mechanisms/Identification of genes related to virulence of the isolates.
None.
Some studies are on-going. Preliminary results obtained by “in-vitro” experiments using antiviral substances that antivirals might be used as an additional tool in ASF control.
GAP:
- Lack of knowledge on antivirals therapy for ASF.
None at present.
Not applicable.
Low.
None.
Currently there are very good tools in terms of sensitivity and specificity for ASF diagnosis, however some limitations should be considered:
- Few commercial kits are currently available.
- Less sensitivity of current Ab ELISA tests for early detection of the disease compared to that obtained using the confirmatory tests (IPT/IFI).
- Virus isolation and haemadsorption identification is based mainly on the use of primary cell cultures, which difficult the standardisation of the technique at international level.
- A number of PCR tests have been validated for ASFV genome detection in different pig samples. However, virus detection in ticks is carried out by using in-house PCR procedures that are frequently not validated for this target.
- Standardised methods for molecular characterization of ASFV isolates allow the differentiation of at least 22 genotypes and further subtyping analysing some specific genome regions (p72 and p54 coding genes and B602L gene). However, DNA viruses’ variation rate is very low and this approach seems to be not enough to follow deeply an epidemic evolution. A very small number of full genome sequences are available.
- A penside test for antibody detection is commercially available, while the development of a penside test for antigen detection is ongoing.
A different behaviour against the virus of the varied host breeds has been proposed to influence in the disease diagnosis.GAPS:
- Understanding of viral replication at genome and protein level.
- Validated diagnostic tests ready for a fast upgrade to a high-throughput application in case of emergency would be desirable.
- An increased number of commercial kits should be very convenient. Lack of any confirmatory serological test commercially available.
- Improvement of screening ELISA tests for antibody detection.
- Standardisation of virus isolation methods using stablished cell lines cultures.
- Standardisation and international harmonisation of virus detection in ticks by appropriate PCR tests.
- A wide number of full genome sequences of diverse ASFV isolates is required to identify new genetic markers useful for further molecular characterization purposes.
- Penside test and front line diagnostic tests - useful for a very rapid application in case of sanitary emergency.
- Understanding the behaviour of different host breeds to the virus and the kinetics of antibody response.
GAPS: Some gaps that could delay the development of new or improved diagnostics tests:
- More information on host/pathogen characteristics and interaction coming from basic research.
- The availability of field samples from different disease scenarios.
Medium at Laboratory.
GAP:
- Support of programmes for sampling in Africa regions, and European countries through collaboration with the affected countries and international organisations.See section "Requirements for diagnostics development" above.
GAP:
- Determine the potential carrier status of surviving animals infected with different viral isolates and the changes in virulence of the ASFV. See section 18.1 above
- Identify candidate vaccines or components of the genome which should be effective against the mild and acute variants of ASF. Identify candidate vaccines or recombinant DNA based on analysis of the ASFV genome. Production of a molecularly-attenuated ASFV vaccine offers a challenging research project.
- Conventional strategies for a vaccine have not been useful to date. New strategies should be attempted.
GAP:
- Conventional strategies for a vaccine have not been useful to date. New strategies should be attempted.10 years.
High.
- Develop better understanding of the immune response to infection and the humoral and cellular basis for the lifelong immunity post infection. Identification of target proteins or genes
- Understanding of the host-pathogen interactions and whether immunity is humoral or cell mediated.
- Assessment whether antibodies alone can passively protect pigs against ASF virus, has demonstrated not complete protection .There is not protection induced by passively acquired antibodies.
None at present.
Not applicable.
Not applicable.
None at present.
ASF (ASFV) is a complex large icosahedral enveloped DNA virus that exhibits many features common to both Iridovirus and Poxvirus families. This virus is currently classified as the only member of the family Asfarviridae. DNA structure is based on two variable ends and a conserved central region of about 125 kbp.The left and right variable ends of the genome encodes five multigene families (MGF). MGF are involved in the variability of the genome size within isolates, due to deletions and insertions of copies within MGF genes. In addition some MGF are involved in determining virulence and tick host-range.
GAPS:
- Identification and role of viral genes interfering with host gene transcription.
- Persistence mechanisms of the virus in the host.
- The role of the MGF in the generation of antigenic variabilility and evasion of the immune system.
- Role of ASFV proteins and induced proteins for an effective and protective immune mechanism in surviving infected animals, after challenge with homologous isolate.
Suids, wild and domestic, are the natural host of ASFV. Several species of soft ticks have been shown to be ASFV reservoirs and vectors.
ASFV don’t induce neutralising antibodies and this is why non serotype classification can be performed. ASFV genotyping based on the partial nucleotide genome sequence of the B646L gene, encoding the p72 protein allow to differentiate up to 22 distinct genotypes. The complete genome sequence of the E183L gene, encoding the p54 protein might be used also for subtyping. Enhanced discrimination between isolates can be obtained through the analysis of the central variable region (CVR) within B602L gene by characterization of the tandem repeats sequences (TRS). Different isolates belonging the same ASFV genotype vary in their ability to cause disease with some causing severe acute disease and high mortality while others exhibits subacute and chronic forms resulting in seroconversion.
22 different genotypes of ASF virus are already described.GAPS:
- Biological and molecular characterization of current circulating isolates.
- Evolution of circulating viruses in endemic regions
- Genome markers related to virulence
T°: Highly resistant to low temperatures. Heat inactivated by 56°C/70 min; 60°C/30 min
pH: Inactivated by pH <3.9 or >11.5 in serum-free medium. Serum increases the resistance of the virus, e.g. at pH 13.4 - resistance lasts up to 21 hours without serum, and 7 days with serum
Chemicals: Susceptible to ether and chloroform.
Disinfectants: Inactivated by 8/1,000 sodium hydroxide (30 min), hypochlorites - 2.3% chlorine (30 min), 3/1,000 formalin (30 min), 3% ortho-phenylphenol (30 min) and iodine compounds. For animal housing and equipment, soaps and detergents, oxidising agents and alkalis are recommended.
Soaps, detergents and Alkalis are useful to disinfect machinery, clothing, and vehicles, human housing etc and also Vircon is also recommended to be employed in aircraft.
The procedure to be employed in feed, effluents, and manure, include bury or burn, or in the latter also by Acids and Alkalis.
Insecticides (organophosphates and synthetic pyrethroids) for tick eradication,
Survival: Remains viable for long periods in blood, faeces and tissues. Can multiply in vectors.
Swine are the only animal species naturally infected by ASFV. The disease occurs through complex transmission cycles involving domestic pigs, wild boars, warthogs and bush pigs (African wild pigs).
Domestic pigs, wild boars and feral/American pigs are susceptible to ASFV infection showing a range of clinical signs and mortality rates. ASFV usually induces an asymptomatic infection in wild African pigs.GAPS:
- Assessment of the survival pigs in virus transmission, maintenance and dissemination of the disease.
- Geographical distribution of wild boar.
None.
Soft ticks (Ornithodoros spp), including O. moubata and O. erraticus also act as reservoirs and vectors for virus transmission. Transovarial, and sexual transmission can occur. In Africa, ASFV is thought to cycle between newborn warthogs and the soft ticks (Ornithodoros moubata) that live in their burrows. Individual ticks can apparently remain infected for life, and infected soft tick colonies can maintain this virus for years. All the Ornithodoros spp experimentally infected until now were susceptible to ASFV infection.
GAPS:
- Geographical distribution in Europe and worldwide.
- The potential role in the actual ASF European Situation
Wild African pigs, most importantly warthogs (Phacochoerus aethiopicus), bush pigs (Potamochoerus porcus) and giant forest hogs (Hylochoerus meinertzhageni) are also infected but usually do not exhibit clinical signs, acting as reservoir hosts in Africa usually showing an asymptomatic infection. These species of wild pigs act as reservoir hosts of ASFV in Africa. Soft ticks of the Ornithodoros genus have been shown to be both reservoirs and transmission vectors of ASFV (ASFV)
GAPS:
- Host-virus interaction in wild African pigs, determining asymptomatic infection.
Direct and indirect contact between infected and susceptible pigs/wild boar and wild African pigs. See Route of Transmission.
GAPS:
- Studies on Neighbourhood transmission in densely population areas.
- Studies on pig to pig , wildboar-pig, wild and indigenous suids in Africa – domestic pigs .
- Role of carrier state in the transmission of ASFV
Survivors of infection can be virus carriers for life. Around a 10% of survivors of infection are virus carriers.
GAPS:
- Long-term persistence studies in recovered animals
ASF viruses produce a range of syndromes varying from peracute, acute to chronic disease and apparently healthy virus carriers. At present chronic forms is not observed in the actual infection areas.
GAPS:
- Host factors determining the clinical outcome of infection.
- Studies on recovered animals as potential carriers status in affected regions and potential changes in virulence characteristics of the circulating isolates.
Incubation period is 3-15 days.
Depending on the ASFV isolate virulence.
- Acute form (highly virulent virus) -90-100%
- Subacute form (moderately virulent virus) 30-80% varies
- Chronic form –low mortality.
Unknown.
GAP:
- Shedding Kinetic Patterns
ASF is generally spread via oral and nasal routes, but also by tick bite, cutaneous scarification, and intramuscular, subcutaneous, intraperitoneal or intravenous injection. The incubation period varies widely (4-19 days), depending on the ASFV isolate and the route of exposure.
By oral route, monocytes and macrophages of the tonsils and mandibular lymph nodes are the first involved. From these sites, the virus spreads through the blood and/or lymphatic system to the main sites of secondary replication – i.e., lymph nodes, bone marrow, spleen, lung, liver, and kidney. Viremia usually begins 1-8 days post infection depending of ASFV virulence and persists for weeks or months. ASFV is associated with red blood cell membranesGAP:
ASFV has never been shown to infect humans and is not considered to be a zoonotic pathogen.
None.
None.
None.
None.
ASF outbreaks have an impact both due to the severity of the disease and with the introduction of control measures especially movement controls.
GAP:
European species of wild pigs are affected but this is not the case for African wild pigs in which no clinical signed are observed.
Slaughter of infected and in contact pigs.
African swine fever was first recorded in Kenya in 1921 and is present as endemic in most sub-Saharan African countries. It spread to southern Europe in 1957 (genotype I) affecting different countries in Europe and central and south America. In Europe, ASF is still endemic in Sardinia (Italy). Since the introduction of ASF in Georgia in 2007 from East Africa (genotype II) the disease is affecting East Europe. The situation in Russia in wild boar and domestic pigs, with two endemic regions recently described has originated a sporadic spill-over of ASF to the adjacent countries such Ukraine and Belarus. In earlier 2014, ASF cases in wild boar were reported in Lithuania and Poland in bordering regions with Belarus. Since then ASF cases or outbreaks in wild boar and domestic pigs have been detected in the EU countries Estonia, Latvia, Lithuania, and Poland. The events have proved that the threat of ASF spreading to other regions remains and it is potentially devastating to the global pig industry. Source of information: www.oie.int and EU: Animal disease notification system (ADNS)
GAPS:
- Become endemic mainly in developing countries, due to presence of complex transmission cycles which could involve a sylvatic cycle, a domestic cycle and a pig-tick cycle.
- Greatly influenced by presence – and subsequent infection- of wild boar and vectors (wild African pigs and soft ticks acting as reservoirs) GAPS:None.
GAP:
Can be high mainly due to transport and movements of affected animals (domestic and wild board) and products as well as the pig density and farms biosecurity.
GAP:
Potentially high and wide spreading, dependant on the movement of infected pigs/wild boars/ African wild pigs or infected meat or other pig products, and illegal movements.
Direct transmission: contact between sick (domestic and wildboar) and healthy animals; contact with asymptomatic infected African wild reservoirs (carriers) and soft ticks.
Indirect transmission:
Ornithodoros ticks, where present, can act as transmission vector of the ASF virus.
- The virus can survive outside the pig for a long time, so the movement of contaminated vehicles, clothing, footwear and equipment can also spread disease.
- Unspecific clinical symptoms could easily be confused with other diseases such as CSF, Salmonellosis, Erysipela or any other septicaemia condition. It might delay diagnosis.
- Density of wild boar population.
- The use of swill feeding.
- Presence of carrier animals and soft ticks.
- Socio-cultural way of life.
GAP:
- Pigs often die before the development of a humoral response when infected with a virulent strain. Pigs which do not die will mount an antibody response and have significant levels of ASF specific cytotoxic T lymphocytes. Pigs will demonstrate a solid immunity to challenge from homologous strains but not heterologous strains. However there is an absence of neutralizing antibodies against ASFV.
- The ASFV replicates in porcine macrophages.
- The essential effectors immune mechanisms involved in protection against ASF are poorly understood. All attempts to develop an effective vaccine have been unsuccessful to date.GAPS:
Detection of antibodies and evidence of the virus genome or virus antigen. ASFV infection produces a long-term viremia from early stages of infection. Specific IgG antibodies are detectable in blood from the first week and for a long period of time, months even year, in the surviving pigs. The early appearance and subsequent persistence of antibodies is the reason they are so useful in studying subacute and chronic forms of the disease. For the same reason, they play an important role in testing strategies implemented as part of eradication programmes.
- Control of live animals and swine product imports, control of waste food. Movement controls can all be successful. Quarantine. Once established in an area tick control becomes important using acaricides.
- Strict sanitary measures on infected farms, stamping out of animals, cleansing, disinfection, sentinels, serological control of sentinels after a month.
- Identification of carrier pigs. Stamping out.GAP:
- Avoid contact between pigs, wild boar and soft tick vectors, including warthogs in Africa - i.e. prevent pigs from wandering.
- Performing good tracing sources of infection and sources of spreading.Since no vaccine is available, ASF diagnosis by virus detection and serological tests are critical for disease containment.
Virus detection and isolation :
ASF antibody detection:
ASFV genotyping: i) sequencing of the C- terminal end of VP72 gene,which differentiates up to 22 distinct genotypes; ii) full genome sequence of the p54-gene and iii) analysis of the central variable region (CVR) to distinguish between closely related isolates and identify virus subgroups within the 22 p72 genotypes.
GAPS:
None available at present.
GAPS:
No effective treatment at present.
GAP:
Rapid slaughtering of all pigs and proper disposal of cadavers and litter is essential. Thorough cleaning and disinfection. Movement controls.
GAP: Not always fully enforced.
Efficient sterilisation of garbage.
GAP:
Through regular clinical monitoring of animals (wild and domestics), in parallel with appropriate sampling collection and laboratory diagnosis.
Pigs recovered from acute and subacute or chronic infections usually exhibit a viremia for several weeks making the PCR test a very useful tool for the detection of ASFV in pigs infected with low or moderately virulent strains. In addition, Antibody detection techniques are very useful in detecting surviving infected animals.GAPS:Recovered ASFV carrier pigs and persistently infected wild pigs constitute the biggest problems in controlling the disease. Eradication was successful in the Iberian Peninsula and in the Caribbean.
Some common failures have been identified:
- Late detection.
- Low/insufficient resources or political instability.
- Scavenging pig husbandry or free-roaming pigs.
- Uncooked swill feed.
- Presence of wild pigs and ticks as reservoirs.
- Co-circulation of several isolates and or genotypes with different characteristics.
- Failure to identify risk factors.
Invert in prevention is the best measure to avoid the significant socio-economic consequences of this disease.GAPS:
Yes.
http://www.oie.int/fileadmin/Home/eng/Media_Center/docs/pdf/Disease_cards/ASF-EN.pdf
http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_1.15.1.htm
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.08.01_ASF.pdf
None.
None.
Variable depending on the strains involved. With 100% mortality can have a very high cost. This disease produces huge economic and social loses in many African countries, and impairs the development of the porcine industry.
Disease outbreaks have occurred in Europe, South America, and the Caribbean; the cost of eradication has been significant. During outbreaks in Malta and the Dominican Re-public, the swine herds of these countries were completely depopulated. In Spain and Portugal, ASFV became endemic in the 1960s and complete eradication took more than 30 years.
Main indirect impact is on the constraints on livestock production and the trade in pigs and their products. Loss of export markets.
Controls on the movement, of pigs and products from infected countries. Likely ban on imports from affected countries. Quarantine measures.
Movement controls on live pigs and their products from infected areas.
Restrictions and controls on internal movements form the protection and surveillance zones.
- Lack of vaccines, carrier recovered pigs, reservoirs in ticks and wild pigs in Africa.
- Improvement of sanitary infrastructures at animal holdings and Biosecurity in some countries. .
- Individual identification of every animal.
- Census update, in some countries.
GAPS:
o improvement of sanitary infrastructures at animal holdings and Biosecurity in some countries. .
o Individual identification of every animal. Census up date in some countries.
o epidemiological surveys.
o promoting associations
- Ability to identify carriers, control of ticks.
- Improvement of sanitary infrastructures at animal holdings and Biosecurity in some countries. .
- Individual identification of every animal.
- Census up date, in some countries.
GAP:- appropriate vaccinesFew information available.
GAP:
Not known.
GAP:
No.
Not known.
GAP:
Changes in production practices and increased globalization have in-creased the risk of African swine fever being introduced into free areas.
The main risk of ASF introduction into Europe is via infected pig meat or pig meat products, for example illegally imported pig meat or bush meat from infected countries or legally imported meat from areas with undetected infectionGAPS:
ASF has the potential to enter free countries if sanitary and border controls are ineffective. The development of effective vaccines for contingency use in Europe and for routine use in endemic countries would be advantageous.
Names included are included where permission has been given
José Manuel Sánchez-Vizcaíno - Director del Laboratorio de Referencia de la OIE, Universidad Complutense de Madrid (Leader)
Marisa Arias/ INIA-CISA, Madrid, SpainYolanda Revilla/ CSIC, Spain
Carmina Gallardo/ INIA-CISA, Madrid, Spain
Cristina Jurado/ Universidad Complutense de Madrid, Spain
Project Management Board.
9th of April 2015.
Defra
http://www.defra.gov.uk/animalh/diseases/vetsurveillance/az_index.htm
OIE
http://www.oie.int/eng/normes/MMANUAL/A_index.htm
http://www.oie.int/eng/maladies/fiches/a_a080.htm
http://www.oie.int/eng/ressources/en_diseasecards.htm
http://www.oie.int/eng/maladies/en_alpha.htm?e1d7
Centre for Food Safety and Public Health Iowa State University
http://www.cfsph.iastate.edu/DiseaseInfo/animaldiseaseindex.htm
USAHA The Seventh Edition Foreign animal diseases- Grey book.
http://www.aphis.usda.gov/emergency_response/downloads/nahems/fad.pdf
ASF experts at INIA-CISA, European Union Reference Laboratory for ASF.
http://asf-referencelab.info/asf/en/
OIE Reference Laboratory for ASF: http://www.sanidadanimal.info
EFSA: http://www.efsa.europa.eu/en/scdocs/scdoc/1556.htm