About BluetongueDr. Ruth Watkins' talk at the meeting held in Scotland on 22nd February 2008
There is general agreement that vaccination against bluetongue virus serotype 8 [BTV-8] of susceptible ruminants is the only way that bluetongue infection can be prevented where there is threat of exposure.
We have not had any serotype of bluetongue virus infecting our ruminant livestock of Northern Europe before.
It was believed that we would become vulnerable as global warming proceeded because the African midge called Culicoides imicola (I think of thirsty Africans drinking coca cola) has been creeping northwards in the last decade to reach the Northern shores of the Mediterranean- Portugal, Spain, S France, Italy, Greece and the islands- C imicola midge ‘plumes’ have been carried on the wind from North Africa or across from Turkey and Israel. Several different BT viruses have been brought this way in the last 10 years, BTV serotypes 1, 2, 4, 9, and 16.
Why should all these outbreaks remain relatively smaller and localized in contrast to BTV-8 that has spread so quickly to such a large area of N Europe? We know that restriction of animal movement will not have any effect on the movement of the midge- either the 2 km per day that she can fly to secure a blood meal or longer journeys on favourable winds of up to 150 km. These small outbreaks have involved the midge species C imicola acting as the main vector. In the Balkans it was found that BTV 9 was transferred from C imicola via infected ruminants to native midge species and this has been called ‘the baton effect’ (like a relay baton). This phenomenon was described by entomologists at Pirbright. [Prof Rudi Meiswinkel an entomologist expert on midges and bluetongue originally from South Africa has pointed out that the midge C imicola likes coastal flats and deltas in Italy for instance and its range does not overlap that of C obsoletus which is the midge predominant inland blanketing the rest of Italy.]
What none of the scientists foresaw was that bluetongue could jump to midge species not previously recorded as vectors - most likely by an infected ruminant was inadvertently imported into Belgium in the summer of 2006 whilst infectious for BTV-8. C imicola midges wrapped up with imported flowers has also been suggested as a source (the baton effect) by Pirbright. The details of exactly what happened will never be known. However detective work such as sequencing of the North European BTV-8 2006 strain and looking for the closest match has revealed that this was a BTV-8 circulating in sub-saharan West Africa. The virus arrived from Sub-Saharan Africa by global trade- it is much too far for infected midges to be carried on the wind in a plume.
The midges C obsoletus and C dewulfi are known to be the most important vectors of BTV-8 in Northern Europe – there are no C imicola midges in Northern Europe. Unluckily for us the strain of BTV-8 introduced to N Europe must possess a property that allows it to infect these 2 novel midge vector species. [As well as C obsoletus and C dewulfi, BTV-8 may infect other midges such as C scoticus and C pulicaris but these have not been shown to be significant in Northern Europe.] As the summer temperature rises the midge is rendered more susceptible to infection. The fact that BTV-8 can infect native North European midges, that these are prevalent over our farmed and wooded temperate lands, that pastoral farming in the temperate conditions of Northern Europe has populated the landscape with 10s of millions of domestic ruminants, as well as millions of wild ruminants (deer), has resulted in an explosive outbreak of BTV-8 ever expanding and dwarfing all previous outbreaks of any BTV serotype in Southern Europe.
How far could the BTV-8 outbreak spread? The midge species C obsoletus complex is widespread in Europe and Asia. I have asked Pirbright for a map of its range but this has not been forthcoming. C obsoletus may not extend into the high arctic but it occurs across the whole of Europe, Scandinavia, Russia and into Japan. C obsoletus lays her eggs in leaf litter, grass and damp hay, and is favoured by a landscape with trees. C dewulfi lays her eggs in dung, sheep, cattle and horse dung. C obsoletus may also lay eggs in dung. Both species of midge can be caught in traps set-up in animal housing. Whilst the midge may bite at dawn and dusk in the main, they have been caught in broad daylight biting cattle. The life of the female midge may be prolonged beyond 3 or 4 weeks in cold weather in association with housed cattle over-wintered in barns. C obsoletus midges can be caught all winter in small numbers in barns in Holland- this is Rudi Meiswinkels work in 2006. In the vector free period no mature female midges were caught, only young newly emerged females rather than the mated and mature, the gravid and blood sucking older females. Of course the vast majority of the adult midge population dies off over winter.
Once BTV-8 reaches Southern Europe there is nothing to confine it to C obsoletus or C dewulfi, the resident C imicola will be able to act as vector as well when infected ruminants stray into her territory. I would like to point out that France is the only European country to have ordered sufficient vaccine to vaccinate all the domestic ruminants and to have a plan in place to do so, in order to protect the Mediterranean countries if possible against infection with BTV-8 as well as saving her own ruminant husbandry.
Midge species occur worldwide, they are 100 million years old, and there are 1000s of species adapted to their particular environment. There are a number of bluetongue virus serotypes on 3 continents, Africa, North America and SE Asia and Australia, each group of BTV serotypes adapted to certain midge species that are the most important vector on each continent, for Africa this is the C imicola midge. Midges over-winter as larvae. There are a number of stages in the midge life cycle, from egg through 4 larval stages, to a pupa and then and adult; the adults emerging when conditions are favourable. Their numbers are so great, and their faunal home so widespread there is no way to kill them off. As North European farmers have found to their cost they cannot be repelled either.
Bluetongue virus has not been thought to over-winter by vertical infection of the egg, so that an infected female midge eventually develops from an infected egg (as can happen with different viruses in mosquitoes or ticks). It is widely held that the female midge can only be infected by taking a blood meal from an infectious ruminant. A period of time must elapse after feeding until her salivary glands become infected, shortened from 16 days at 150C to 2 days at 300C, and the virus is shed in her saliva thereafter for the rest of her life. She is then a vector for bluetongue infection. Thus the common trait of midges implicated as vectors in the transmission of bluetongue is that the females lay eggs in batches so must take several blood meals. A female midge infected as a result of her 1st or 2nd blood meal can infect ruminants she bites subsequently until she expires. There are a number of species of midge in the UK that do not take blood meals at all, or that take only one blood meal like the fierce Scottish midge (C impunctatus), these cannot transmit BTV. The male midges feed on nectar from flowers and do not bite animals.
For the virus to over-winter a ruminant host should be infectious for at least 3 months so that when female midges emerge and mate in Spring (April in N Europe) there are infectious ruminants to hand for blood meals. A recent hypothesis has been forwarded that some adult female midges do not die but survive the winter presumably hibernating near buildings and when the ambient spring temperature reaches 150C virus replication resumes by which time the midge is already active again. Cattle are the favoured host of the midge species C obsoletus and C dewulfi, bitten far more frequently than sheep for example, perhaps because of their large size and the amount of CO2 on their breath that acts as a powerful midge attractant. [In N Europe recent sero-surveillance has shown cattle over 24 months to be between 90 and 100% immune (infected in 2006 or 2007) whilst infection rates may be just 30% in sheep in the same area.] In experiments with other BTV sero-types cattle have in general been observed to be infectious for longer than sheep - a sheep not more than 60 days if that, but cattle for up to 90 days or longer. Vertically infected young ruminants may be infectious for considerably longer that 3 months. Apart from remaining infectious through the winter, cattle are also important for the amplification of the infection. From July through to end of October at the peak of the BTV season in N Europe each infected bovine infects 1000s of female midges, that can go on to bite and infect 1000s of other susceptible ruminants- a phenomenon called amplification.
You cannot vaccinate midges (though many prove to be resistant to BTV infection - there are thought to be internal barriers to prevent infection passing from the stomach via the haemocoel to the salivary glands so that only 1 in 1000 female midges of the vector species were found to be infected in summer 2006 in N Europe). One can vaccinate the ruminant host and there is a long history of doing so. By preventing the infection of the biting female midges, spread to other susceptible ruminants is prevented. Vaccination is also highly desirable because the BTV-8 sero-type in Northern Europe is relatively virulent (pathogenic) to cattle as well as sheep which is unusual for BTV. Many of the incursions into Southern Europe of different BTV sero-types have been with relatively non-virulent strains, so that infection may not be noticed even in infected sheep. Not so BTV-8, it can cause disease in cattle. It can also cross the placenta to infect the foetus, reduce or terminate milk yield, and render males temporarily or permanently sterile as we have heard.
All infections are generalised whether there is disease or not
The disease BTV-8 causes gives rise to much suffering, death peaking at the 6th day of disease. Deaths follow even upon recovery, due to 2ary bacterial infection and hemorrhage. Even when there is no obvious disease in the pregnant cow or ewe, the foetus can be infected and milk yield fall, and similarly bulls or rams rendered sterile after an unnoticed infection. This is because in every single case of infection of the ruminant host with BTV there is a generalized infection, whether disease occurs or not. Virus is delivered into the bloodstream, small capillaries, either from the saliva of an infected female midge when she bites sawing through the skin with her proboscis, or from a needle used previously on an infected ruminant when an invisibly small amount of blood, 1 or 2 micro-litres, containing infectious virus can be transferred. The virus infects cells of the immune system, the cells lining small blood vessels called endothelial cells and young red blood cells in the marrow or spleen. Other cell types can also be infected, pneumo-cytes in the lung for instance. The virus is carried round the body in the bloodstream, in immune cells and in red blood cells and perhaps free in the plasma, and after several days many millions of cells are infected throughout the animals body. The virus present in the bloodstream (called viraemia) can cross the placenta and infect the foetus.
What a virus is and how it takes over an infected cell
Viruses are obligate intra-cellular parasites. This means they must enter a living cell in order to come alive and multiply, reproduce themselves. The virus must key onto a receptor on the cell surface, like a key inserted into a lock, so the cell will take the virus in, otherwise the virus cannot gain admittance. Once inside the virus hi-jacks the cell in order to reproduce itself making use of every facility. It makes the cell manufacture 100, 000 new viruses. These newly formed viruses must exit the cell, they do not always kill it in the process. (Imagine this school building is an animal cell. Imagine this football to be a virus. These are roughly their relative sizes. What is a virus? Simply a protein shell protecting the genome, in the case of BTV the genome consists of 10 different RNA segments and these are hidden inside the outer shell. The virus is inert. Imagine this room has a small round hemispherical window. The ball is kicked up against this small round window from the playground and it gets stuck because it fits perfectly. The window is opened because the cell thinks it is something useful, the post perhaps or a message, and lets the football, the virus, in. The virus this football puts a stop to all lessons and multiplies so that 100, 000 of these footballs fill several rooms of the school. Finally they must be let out of the doors and windows or the walls will be broken down. In reality this whole cycle of infection may only take half a day.)
The battle between host and virus
How do animals fight off virus infections? The animal immune system has evolved to beat the virus invader and prevent a re-infection in a way unique to viruses. This is different from bacteria and parasites such as worms).
The animal immune system has 2 ways of responding to the virus invader. The initial response is innate - natural killer cells and interferon immediately start to contain the infection.
This is quickly augmented by a specific immune response focused against the virus proteins or antigens. The generation of killer T-cells (cytotoxic T-cells) by the immune system occurs within days of the invasion. These multiply in number into an army actively seeking out virus infected host cells. The killer T-cell can detect a virus infected cell because they see a little bit of virus protein on the cell’s surface. They signal to the cell that it must die by committing suicide, unfortunate for the cell, but also for the virus multiplying within, all the progeny and any viral proteins are degraded and so lost as well. This attack focuses specifically on the invader BTV-8 for example, the foreign virus proteins triggering it.
Neutralising antibodies, how they work
A slightly later response of the animal’s immune system is to make antibody against the foreign virus proteins. There may be a role for antibody in mopping up the results of the battle of the killer T-cells, but the most important and unique consequence of the secretion of billions of antibody molecules directed at the virus comes later. As the immune response matures a small proportion of the antibody formed is focused on the proteins protruding from the surface of an intact virus. This has the unique property of inactivating the virus, what we virologists call neutralising antibody. The BTV an orbivirus is round and can be imagined to look like a football (demonstrate a football). The antibody molecules are in reality only about an inch in relation to the size of the football (demonstrate with hazel twigs Y-shaped). Each antibody is Y-shaped and the 2 arms bind at their tips tightly and formed to fit perfectly the shape of the protein on the outside of the virus (hold up football with Y twig applied). Only a few such antibodies, a few Yes, are needed to bind to the surface to inactivate the virus. Even if the virus still attached to the neutralising antibody enters a cell it is in effect locked in, irreversibly inactivated, and harmlessly degraded and the cell escapes infection. These protective antibodies only form in response to virus infection. The neutralising antibodies protect against re-infection with the same virus because they are constantly circulating in the blood ready and waiting to bind to a similar virus, of the same serotype (the same surface protein type) to which they were initially raised. As there are billions of neutralising antibodies secreted into the bloodstream for the lifetime of the animal following an infection, they are more than a match for the few hundreds or thousands of viruses reintroduced into the host in re-exposure. The 24 serotypes of BT virus all require their own distinctly different neutralizing antibodies- hence for immunization against BTV-8, BTV-8 must itself be used in the vaccine.
Reinfection is usually completely prevented. If there is a large inoculum and the virus not all neutralised before it can infect a cell then re-infection can occur but it is limited and localised, serving as a strong reminder to the immune system and boosting neutralising antibody production. Any re-infection is inconsequential as the level of virus in the bloodstream is not likely to be high enough to infect a female midge, let alone cause any disease in the host. Nor will the level of virus in the blood be high enough to cross the placenta. Memory immune cells were formed in the first infection and are able immediately to regenerate killer T-cells and antibodies, all this within days of re-exposure.
The best vaccine is a modified or attenuated live virus vaccine because this most closely resembles a natural infection- it does give rise to a generalised infection with the vaccine virus but without any serious disease. The immune system is thus fully engaged and generates memory immune cells that ensure long lasting, life-time immunity and neutralising antibody. [There is no rational way to change the virulence factors of the BT virus because they are not known at present so there is a hit and miss method which involves passaging wild virus in tissue culture cells and checking it out in experimentally infected animals. Eventually a vaccine virus is produced, with a number of mutations fortuitously introduced that make it unlikely to cause any disease. This virus is stored] as a seed virus and then grown up to create doses of vaccine that each contain about 1 000 infectious viruses, this small dose is all that is needed to produce an infection and so result in vaccination. Modified live BT vaccines (MLV) have been generated and used in South Africa for about 50 years- bluetongue virus was isolated in 1902 and it has been worked on in S Africa for a century as domestic european ruminants particularly sheep could not be farmed. The S African BTV serotypes are endemic in wild ruminants such as wildebeast. The vaccine used in South Africa consists of 3 shots of pools of BTV modified live vaccine virus each pool containing 4 different serotypes of BTV so that the animal is immunised against 12 different serotypes of BTV. The BTV-8 MLV is too virulent (pathogenic) for N European sheep according to Pirbright when it is given as a mono-valent vaccine.
Advantages and disadvantages of live vaccines
A modified live vaccine for BTV-8 surely cannot be too long in coming, perhaps in 2009, but there are disadvantages to its use. The vaccinated ruminant can infect midges with the vaccine virus, [which could recombine with the wild BTV-8 or another BT virus. This happened when Israel was routinely using MLV to BTV-16 and when BTV-16 appeared in Italy it was found to be the same MLV as used in Israel brought over to Italy in a plume of infected C imicola midges.] BTV viruses grown and passaged in tissue culture have the property of crossing the placenta to infect and possibly damage or kill the foetus even if this does not usually occur as a property of the wild virus (the source of the vaccine). In the very young animal any maternal antibody still in circulation could neutralise the small vaccine dose of live virus so it does not take. Ideally an MLV would be given to animals greater than 3 months old, not pregnant and in a vector free period, say during the European winter. The advantage of a live vaccine virus is that only one dose would be needed to immunise an animal for life. I think we should look forward to using live vaccines if they can be attenuated to safe levels.
The quickest method to come by a vaccine is to make an inactivated vaccine. The virulent virus is grown up in culture then inactivated, by chemical means, whilst retaining its ability to induce protective neutralising antibody. The BT virus is very tough, having a double protein coat (the skin of the football) so it must be completely inactivated, whilst remaining undamaged so that it is still immunogenic. Both the inactivation of each vaccine batch and its immunogenicity must be checked out. This is the type of vaccine that should be ready to come on line in May this year produced by Intervet. However I believe it has been unnecessarily delayed. As soon as BTV-8 resurfaced in ruminants widely in Europe in July 2007 the race should have started to make a vaccine then. Pirbrights studies had shown the MLV serotype 8 to be too pathogenic to use. Inactivated vaccine has been used already in Europe for BTV-2, 4, 9 and 16. Unfortunately no government in Europe funded development of an inactivated BTV-8 vaccine and the EU itself did not because it was ‘old unexciting technology’, not sexy. Dr Laddomada, a commissioner and vet explained in Brussels that the EU had a policy not to fund this long established method of vaccine production. Hence the vaccine manufacturers refused to make any until orders were placed by governments as they had been caught out before over classical swine fever vaccine that they developed and was never bought or used. As it would be illegal to use BTV-8 vaccine unless it was specifically authorised in each country they could be caught out again. Countries placed orders so late that the 6 month lead up time to release will be difficult to meet even in May.
How do inactivated vaccines work? It is seemingly miraculous that a specific and focused immune response can be generated by introducing foreign protein from BTV-8 for example, by injection into the animals tissue at a point source even on a single occasion. In reality a sizeable amount of virus protein must be introduced, with adjuvents to enhance the immune response. Several billions of BTV-8 viruses must be inactivated to produce a single dose, hence the vaccine is more costly and it takes considerable manufacturing capacity to generate many millions of doses. The immune response to an inactivated virus is more limited than in the case of infection but the all important neutralizing antibody is made in response and some immune memory is generated. The neutralising antibody confers protection against infection with the wild BTV-8 virus.
Advantages and disadvantages of inactivated vaccines
The disadvantage of inactivated vaccines (apart from the danger of incomplete inactivation) is that booster doses are needed to jog the immune memory and sustain a protective level of neutralising antibody. This would probably be annually in the case of BTV-8. Also large animals such as cattle especially bulls will need large amounts of virus protein and at least 2 doses, to prime and boost antibody to attain a protective level of neutralising antibody that would last as long as a year. It seems that a single dose will be enough to vaccinate sheep. There are several advantages in using inactivated vaccines. Normally neonatal and young animals respond well to inactivated vaccines even before all maternal antibody has waned. Inactivated vaccines are safe to use in pregnant animals (they don’t of course give rise to an infection so cannot infect and damage the foetus) and they can be used when the vector is active without danger of infecting the vector.
What should be vaccinated and where
It will be necessary for us to vaccinate both sheep and cattle and all other susceptible ruminant species against BTV-8, both to prevent spread and to prevent disease so that all domestic ruminants are immune either as the result of infection or vaccination.
In Britain we have ten times as many sheep as any other North European country whilst having equal numbers of cattle to France or Germany.
England and Wales have ordered insufficient vaccine to vaccinate all susceptible domestic ruminants in 2008 and Scotland and N Ireland have ordered none at all.
Even in N Europe where so many adult cattle and somewhat less sheep have been infected, without vaccination, BTV-8 infection during 2008 will be little different to 2007 and continue to spread. This is because each year the number of new susceptibles added to the domestic ruminant population is about the same as the number of adults - these will be more than enough to sustain BTV-8 infection unless they are vaccinated. The level of immune ruminants, domestic and wild, should exceed 80% to stop the spread of BTV infection. In order to achieve this in the UK we would need to vaccinate I estimate at least 95% of all domestic ruminants, to take account of the unvaccinated wild ruminants and the vaccinated animals where the vaccine has not been successful.
The rules and why we should change them
We shall be playing catch-up all through 2008 as vaccination will only begin in May at the earliest in the protection zone, and vaccination will be confined to the protection zone according to the rules. It is forbidden in the surveillance zone - so during 2008 vaccination will follow infection instead of preceding it. Vaccination will be incomplete because there is no mechanism to ensure all stock keepers will vaccinate all their stock; will you vaccinate all your lambs as well as ewes? It will take several weeks in cattle before vaccine is fully protective, perhaps 10 days in sheep, meanwhile infectious female midges will be actively spreading infection further afield. Infected herds and animals are much more widespread than realised from documenting only those herds with barn door clinical cases of infection.
As the supply of BTV-8 vaccine will be limited during the summer of 2008 we should aim to vaccinate all domestic ruminants in Wales, Scotland, N Ireland and those parts of England not yet covered in autumn 2008 and winter 2009. The vaccination should be completed by Spring 2009 as there should be limitless supplies of vaccine by then. The extent of BTV-8 infection should be documented by testing animals for infection, the presence of virus RNA in the blood. Virus RNA remains present in blood long after infectivity has ceased because the virus is sequestered in red blood cells. Red blood cells have a lifespan of about 180 days, 6 months, in the blood stream before they are removed by the spleen. Sampling for antibody in vaccinees when they are vaccinated would be another way to document the extent of infection (before they could have responded to vaccination). There should be an ongoing statistical surveillance of this type to determine the extent of the protection zone.
It is ridiculous to a virologist to make rules about not vaccinating in the surveillance zone or outside the surveillance zone in uninfected areas to prevent and block the spread of infection. We are back in the mid 20th century if we continue to act as though we are unable to diagnose virus infection unless we wait for a clinical case to take samples to the virology laboratory. That is the reason for the redundant rule that vaccination is not allowed in the surveillance zone. Why should we be afraid of using vaccination to prevent virus infection from spreading and establishing itself in new areas? Is this because of an unfounded suspicion that somehow vaccination allows animals to be infectious whilst concealing signs of clinical disease? I hope I have explained clearly to you how vaccination works against viruses and the powerful properties of neutralising antibody. Jenner who was a country doctor, a general practitioner, was the first person to use a live vaccine virus to protect humans against smallpox in 1876. The modified live vaccine virus was thought originally to be cowpox but mysteriously evolved into Vaccinia virus over the next two hundred years of medical use. The original live virus vaccine was taken from the udder of a cow called Blossom whose hide is framed and hangs on the library wall at St George’s Hospital where Jenner trained. Jenner foresaw and wrote that it would be possible to eradicate smallpox entirely from the world one day, and he was proven correct in 1977 when global eradication was declared complete. Global eradication would never have happened if each person had had to pay for their vaccine and their tests for smallpox.
Ruth Watkins - 22nd February 2008