1. 1 Kristīne Lamberga Food and Veterinary Service, Latvia Workshop on Equine identification and prophylaxis Legislative background of horse infectious disease surveillance principles in EU – with particular reference to Equine infectious anaemia Baku,13 - 14 December 2010

2. 1 Content 1.EU legislation related to EIA 2.Map - EIA situation in EU /November, 2010, ADNS/ 3.Equine infectious anaemia (EIA) 4.Transmission of the EIA virus 5.Incubation period 6.Course of infection 7.Clinical signs 8.Differential diagnosis 9.Testing 10.Information from the First Workshop for EIA, by EU Reference Laboratory 11.Action if EIA is confirmed 12.Criteria for eradication programs 13.Vaccination

3. 1 1. EU legislation related to EIA (1) -Council Directive 2009/156/EC on animal health conditions governing the movement and importation from third countries of equidae -Council Directive 82/894/EEC on the notification of animal diseases within the Community -Commission Decision 2010/346/EU on protective measures with regard to EIA in Romania -Commission Regulation (EC) No 180/2008 concerning the Community reference laboratory for equine diseases -Council Directive 92/65/EEC laying down animal health requirements governing trade in and imports into the EU of animals, semen, ova and embryos not subject to animal health requirements laid down in specific Community rules referred to in Annex A (I) to Directive 90/425/EEC -Regulation (EC) No 1774/2002 laying down health rules concerning animal by- products not intended for human consumption

4. 1 Council Directive 2009/156/EC on animal health conditions governing the movement and importation from third countries of equidae Defines rules for the movement of equidae between MS Sets common standard form for health attestation for registered equidae and form of health certificate for movement of equidae for breeding, production and slaughter Defines rules for importation of equidae in EU from third countries (only from countries included in the list of countries and territories from which equidae may be imported (Council Decision 79/542/EEC)) Provides for restrictions concerning the movement of equidae from holdings where the presence of EIA has been confirmed until, following the slaughter of the infected animals, the remaining animals have undergone two Coggins tests with negative results: Article 4 point 5 Equidae must not come from a holding which has been subject of prohibitions: (a) prohibition period concerning holding of origin must be at least: (iii) in case of EIA until the date on which the infected animals have been slaughtered, the remaining animals have shown a negative reaction to 2 Coggins tests carried out 3 months apart 1. EU legislation related to EIA (2)

5. 1 EIA is a compulsorily notifiable disease in accordance with Annex A to Council Directive 90/426/EEC In addition, Council Directive 82/894/EEC on the notification of animal diseases within the Community provides that outbreaks of EIA are to be notified to the Commission and other Member States through the Animal Disease Notification System (ADNS) 1. EU legislation related to EIA (3)

6. 1 The disease situation (endemic disease) in Romania presents an animal health risk for equidae in the EU. Therefore protective measures are adopted laying down a specific regime for the movement of and trade in equidae and equine semen, ova and embryos and certain equine blood products from Romania (Commission Decision 2010/346/EU) Regulation No 180/2008 concerning the Community reference laboratory for equine diseases lays down the functions, tasks and procedures of the reference laboratory in the EU for equine diseases Council Directive 92/65/EEC laying down animal health requirements governing trade in and imports into the EU of animals, semen, ova and embryos includes test requirements for EIA Regulation (EC) No 1774/2002 laying down health rules concerning animal by- products sets out the requirements for serum of equidae. 1. EU legislation related to EIA (4)

7. 1 2. Map - EIA situation in EU /November, 2010, ADNS/ (1) 2010: UK, Belgium, France, Greece, Italy, Hungary, Germany, Romania, (Croatia)

8. 1 2. Map - EIA situation /November, 2010 WAHID OIE/ (2)

9. 1 2. Map - EIA situation in EUROPE /November, 2010, WAHID OIE/ (3)

10. 1 3. Equine infectious anaemia (EIA) - EIA is an exotic viral disease that affects horses, mules and donkeys (donkeys are naturally protected against disease and are asymptomatic carriers) - EIA caused by Genus: lentivirus, Family: Retroviridae - Disease is notifiable - Affected animals remains a lifelong carrier of the disease, it is infectious to other animals - Therefore infected animals should be humanely destroyed

11. 1 4.Transmission of the EIA virus - biting insects - large biting flies (tabanid species) or stable flies (stomoxys calcitrans) Adult flies are transmitting the virus. Flies overwinter as larvae and the larvae cannot transmit the disease - when travelling to territories where disease is endemic - from the use of biological products infected with EIA virus - contaminated equipment (by blood, milk or placental fluid), veterinarian instruments - saliva, nasal secretions, feces, semen, ova and embryos (acute infection) - pregnant mares may pass the disease to their foals via placenta or rarely via virus contaminated colostrum (transmission through semen uncommon)

12. 1 - Generally 1 - 3 weeks (days till few months) - Antibodies usually develop 7-14 days after infection and last for life - Horses have been known to take several months before clinical signs or antibodies are apparent 5. Incubation period

13. 1 6. Course of infection -Acute -Subacute -Chronic ( If disease become chronical, animals are predisposed to secondary infectious) -Sub-clinical (latent)

14. 1 7. Clinical signs -Recurring fever, anaemia, oedema, death -Signs may be very mild and only serological testing then will show the infection -Acute form - fever, depression, increased heart and respiratory rate, haemorrhaging, bloody diarrhoea, loss of coordination, ataxia, rapid weight loss, skin swelling (carry high level of virus in the blood) -Chronic form – recurring fever, depression, anaemia, weakness and weight loss

15. 1 8. Differential diagnosis African horse sickness, Anthrax, Dourine, Equine viral arteritis, Japanese encephalitis, Equine influenza, Equine herpes virus, Babesiosis, Leptospirosis, Fascioliasis, other causes of oedema, abscess, fever and anaemia etc. Laboratory diagnosis (blood testing) is essential

16. 1 9. Testing (1) Blood sample to test for the presence of antibodies against EIAV proteins Antibodies at detectable level may be present in the blood 7-14 days after infection, but horses in the first 2-3 weeks after infection will usually give negative serological reactions (in rare cases – 60 days) Virus isolation is usually not necessary to make diagnosis Virus titer is higher in horses with clinical signs

17. 1 9. Testing (2) Methods - Coggins test – agar gel immuno-diffusion (AGID) – currently officially recognized for international movement or trade According to relevant Chapter of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the OIE the agar gel immunodiffusion (AGID) is used for the detection of EIA in horses (It is an accurate and reliable test except in certain circumstances specified in the Manual therefore two AGID tests for EIA with negative results are used to compensate the limitations of that test) - ELISA also used as screening test in some MS (ELISA detects antibody earlier and at lower concentrations, but positive samples should be confirmed by Coggins due to false positives) For confirmation also: - PCR (complementary test if serology unclear, confirmation of infection before antibodies develop, confirmation of the status of a foal from an infected mare, ensuring freedom from disease if horse is used for production of antiserum, vaccine or used as blood donor) - IB (immunoblot technique)

18. 1 10. Information from the First Workshop for EIA, by EU Reference Laboratory for Equine Diseases Paris, October, 2010 1.Harmonization of the serology tests for EIA in Europe (regarding 2010 proficiency testing results): training session are planned for NRLs in November 2011 2. Some ELISA kits might be used for screening: - ELISA positive samples should be confirms by AGID or IB - ELISA negative samples are AGID negative 3. Implementing PCR reaction for EIA diagnosis (project under development with other NRL) 4.Molecular characterization of EIA viruses circulating in Europe. The EU-RL has started a collaborative project with NRL from Belgium, Italy, Germany and Romania. By the end of the year, the EU-RL should receive biological samples (tissue, blood) and then will start the molecular characterization of those isolates The aim of this study is to better understand and describe the different populations of EIA viruses circulating in Europe. This phylogenetic analysis will help to develop a molecular diagnostic tool (PCR) allowing detection of EIA viruses in biological samples Recommendations:

19. 1 - Positive animal should be humanely euthanized or slaughtered - Each animal traced as contact with infected animals should be kept under restrictions and tested - Restriction period concerning infected holding must be at least until the date on which the infected animals have been slaughtered, the remaining animals have shown a negative reaction to 2 Coggins tests carried out 3 months apart - Detection of infected animals and their prompt destruction, prevention of all modes of transmission are basic control measures 11. Action if EIA is confirmed (1)

20. 1 - stop all movement of horses - isolate the horse - implement vector control - appropriate treatment of equipment (including veterinary equipment) - disinfect equipment, stable, vehicles - implement good hygiene - quarantine in-contact horse for 90 days post exposure, blood test - Depending EIA situation in country – surveillance and/or eradication program should be implemented 11. Action if EIA is confirmed (2)

21. 1 12. Criteria for eradication programs (Council Decision 90/638/EEC) (1) 1.Description on epidemiological situation 2.Designation of competent authority for supervision and coordination of program implementation 3.Objective and likely duration of the program 4.Estimated costs 5.Description of area in which the program will be implemented

22. 1 6. Notification system of suspected and confirmed cases or outbreaks 7. Control procedures (movement restrictions, on farm inspections, etc.) 8. Registration of holdings (animals) involved in the program 9. System of traceability of animals 10. Different statuses applicable to holdings or areas, conditions of movement of animals between holdings (areas) of different statuses 11. Consequences of loosing the status 12. Sampling, testing principles 12. Criteria for eradication programs (Council Decision 90/638/EEC) (2)

23. 1 13. Measures to be taken if positive results are found: - slaughter of animals - restrictions on meat - destruction/ treatment of products which could transmit the disease to avoid possible contamination - disinfection procedures of infected holdings, equipment - procedures for re-stocking with healthy animals 14. Compensation to farmers (if possible) 12. Criteria for eradication programs (Council Decision 90/638/EEC) (3)

24. 1 13. Vaccination (1) Although live attenuated vaccines are available internationally, these are not considered to be sufficiently effective or safe!

25. 1 WHAT DO WE EXPECT FROM A SAFE AND EFFICIENT LENTIVIRAL VACCINE?* A vaccine strain should: – protect from disease and/or infection – elicit protection from a large spectrum of infecting viruses (homologous and heterologous strains) – induce and maintain broad, specific and mature humoral and cellular immune responses – be genetically stable over years and not display genetic reversion to virulence – not have an increased pathogenicity in individuals with minor or major immune disorders – keep its protective effect in immunosuppressed horses In 2010, the best candidates are 2 live attenuated EIAV vaccines: American and Chinese 13. Vaccination (2) * Information from the First Workshop for EIA, by EU Reference Laboratory for Equine Diseases, Paris, October, 2010

26. 1 -Only live-attenuated vaccines are able to protect horses -Both the Chinese and the American vaccines elicit good levels of protection from disease and infection -They elicit broad and mature immune responses -The Chinese vaccine protects against heterologous strains, the American vaccine protects only against homologous strains -Protective vaccine immunity is maintain even after dexamethasonemediated immunosuppression -The Chinese live-attenuated vaccine developed 30 years ago is probably the first successful lentivirus vaccine -EIAV vaccination seems reachable … but they are live attenuated vaccines and may potentially revert and give rise to new pathogenic EIAVs 13. Vaccination (3)

27. 1 oie Recommendations for the importation of equines: - Veterinary authorities of importing countries should require the presentation of an international veterinary certificate attesting that: the animals showed no clinical sign of equine infectious anaemia (EIA) on the day of shipment and during the 48 hours prior to shipment; and no case of EIA has been associated with any premises where the animals were kept during the 3 months prior to shipment; and if imported on a permanent basis, the animals were subjected to a diagnostic test for EIA with negative results on blood samples collected during the 30 days prior to shipment; or if imported on a temporary basis, the animals were subjected to a diagnostic test for EIA with negative results on blood samples collected during the 90 days prior to shipment

28. 1 Thank you for attention!