1. Bluetongue virus serotype 8 in Northern Europe Talk by Dr Ruth Watkins to Pedigree Beef Society Group on 24 10 2007 at the Emergency Bluetongue Meeting held at Stoneleigh National Agricultural Centre First found near Maastricht in the Netherlands in July 2006 Documented in >35,000 holdings in 2007 Netherlands, Belgium, Luxemburg, Germany, France, England, Denmark Could spread over the whole palaearctic Infection of ruminants by infected female midge; insect animal cycle

2. Bluetongue infection of a ruminant Inoculation of virus into blood from saliva of biting midge (or needle) Viraemia within a few days Infectious day ~3 to day ~21, occ. up to 60 or even 70+ days Cells of the immune system infected, RBCs, platelets and endothelial cells lining small blood vessels Vasculitis, leukopaenia, D.I.C.

3. Test for the presence of virus RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) This test detects the RNA genome of the virus Blood sample required for lab.

4. Immune response to infection Within first week T-cell specific immune response Then virus specific antibody formed from B-cells within 14 days, tested for by ELISA Antibody is serotype specific – BTV-8 Neutralizes virus Protects against reinfection Immunity for life

5. Meaning of test results Antibody –ve, PCR –ve Antibody –ve, PCR +ve Never been infected = susceptible, or within first few days of infection after inoculation Infected within last 2 to 14 days, infectious to female midges

6. Meaning of test results Antibody +ve, PCR +ve Antibody +ve, PCR -ve Infected within last 14 to 180 days – may or may not be infectious to midges (red blood cell half life is 180 days; virus sequestered in RBCs until end of their life and not infectious to midges) Infected more than 6 months ago, or vaccinated

7. Disease There may be no disease inapparent infection cattle > sheep 1ary viral disease, high temp, no appetite, nasal discharge, swollen face & conjunctivitis, excoriation of mucosa, frothy saliva, bloody nasal discharge and pneumonitis, laminitis & coronitis, muscle stiffness, ulceration of udder, diarrhoea, loss of fleece, long illness, abortion and sterility 2ary bacterial infection

8. Treatment of ill animals Get animal in shade Supportive treatment, fluid and electrolyte replacement, soft food (cut grass) Anti-inflammatory – flunixin ? Dexamethasone Antibiotics for secondary infection which follows 1ary viral infection

9. Morbidity and mortality No European breed exempt from illness, figures below approximate Cattle 10 – 15% ill <1 – 3% die Sheep Up to 50% ill 10 - 40% die

10. The virus Orbivirus family Capsid encloses double stranded RNA genome 10 segments encode at least 10 proteins At least 25 serotypes, each serotype only gives protective immunity against itself Only serotype in N Europe is BTV-8 Btv-8 Originates south of the Sahara, virus from Nigeria best RNA match Introduction unknown – infected animal, midge (lab escapee? Too far for Nigerian midge)

11. The midge The female midge is the vector – nuliparous (immature), parous (mature egg laying) The vector midge must take several blood meals, one to infect her, subsequent meals to infect susceptible ruminants Implicated species are Culicoides obsoletus complex and Culicoides dewulfi C obsoletus @woodland, eggs in leaf litter, damp hay C dewulfi @ farms, eggs in dung, horse & cow Females must secure blood meal, will fly 2 km Attracted to ruminants by CO2 Prefer to feed on bovines- cattle bitten more than sheep Follow animals into housing Bite most at dusk and at night but also during daylight Midge is tiny, prefers wet warm climate, faunal home

12. Cycle of virus infection between midge and ruminant Parous infected female midge bites a susceptible ruminant Period of infectivity of ruminant for midge about 21 to 60 days after 3 day gap (incubation) Uninfected female midges (1000s) bite an infected ruminant She ingests a blood meal (lays some eggs) and virus infects her midgut, haemocoel and then the salivary glands It takes 7 or more days depending on the ambient temperature for the newly infected female midge to shed virus in the saliva (called the extrinsic incubation period EIP). She sheds virus for life. About 1 in a 1000 parous females are infected. She lives for several weeks and takes several more blood meals injecting virus with her saliva She dies Her eggs become larvae then nuliparous females (or males) but are not infected (no vertical infection).

13. The winter - midge free or not? Cold kills midges = midge free season = no transmission of BTV between ruminants 2006 in N Europe new cases in December, restart in May 2007 infection rate highest July through October BTV-8 overwintered in N Europe 2006/2007 *Importance of housed animals and associated midges* A few animals infectious for 4 months? Infected female parous midges slow down, life is prolonged until spring? A few nuliparous females of C obsoletus were trapped throughout winter, Low level of continuous breeding? Or Immune reservoir of gamma delta cells in infected animal activated next year or vertical infection of midges (these last 2 possibilities not proven)

14. The summer of 2007 in Europe Spread of BTV-8 despite movement restrictions by the midge flying, ~2km a day Spread by plume of midges on suitable winds – some distance within Europe and across the sea to England; midge tsunami Prevalence of infection in 2007 very high in areas infected in 2006. Average 80% (30 – 96%)? No premises uninfected. Infill of areas that escaped last year No re-infections proven

15. Prevention of infection Cannot eliminate midges Repel or control midges? Ivermectin or Doramectin in blood or dung (killing) or affecting attractants on sheep wool Deltamethrin or OP or CP sheep dip or shower (repellent) *No studies on N European midges *Probably made no difference in experience of EU farmers Vaccination Used for a century in S Africa Prevents disease and infection against serotypes in vaccine Either MLV (modified live virus) using attenuated virus Or inactivated whole virus vaccine Could vaccination eliminate BTV-8 infection?

16. Inactivated vaccine for BTV-8 CVO must order Technology proven and available (conditional license) 1ary course 2 doses, annual booster Safe for very young and pregnant animals One circulating serotype = monovalent vaccine Experience of using it in Europe, BTV-2, -4 and -16 Merial vaccine BTV-2 in Balearic Islands led to elimination of BTV-2. Could BTV-8 be eliminated? Animals could be screened before vaccination for previous infection in hyperendemic areas Infection of vaccinated animals could be detected by testing for RNA (infection within last 6 months)

17. Live attenuated vaccine (MLV) Not available as a monovalent vaccine for BTV-8 from S. Africa, would take long period to attenuate and prepare Advantage Life long immunity Disadvantages Spread to susceptible animals via infection of vector May recombine with other BTV if co-circulating Vertical infection of ruminants, infection of newborn Can go off if not stored or made up correctly Therefore not an option for Europe