Dr. 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 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.
Live vaccine 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.
Inactivated
vaccine 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.