Bluetongue Virus

Also known as: BTV, Bluetongue.

Description

Bluetongue is a non-contagious, arthropod-borne disease of ruminants, caused by bluetongue virus (BTV). The clinical severity of disease is variable, but is characterised by inflammation of mucous membranes, haemorrhages and oedema¹. Although cattle are the main reservoir of infection, sheep are more severely affected and often suffer a cyanotic tongue, lending the disease its name. The virus has been isolated from hosts and vectors on all continents(excluding Antartica)², despite being initially recognised in Africa in the late 19th and early 20th centuries³. Originally thought to be a disease of tropical and sub-tropical regions, bluetongue has shown a propensity to become established in temperate areas, and in recent years has spread North, through the Mediterranean Basin, to become endemic in many European countries including the UK. Although BTV's transmission and epidemiology is dependent on insect vectors, bluetongue greatly influences the global trade of ruminants as it is included on the Office International des Epizooties List A of animal diseases⁴.

Aetiology

Bluetongue virus particle. Source: Wikimedia Commons; Author: CDC (2007)

Bluetongue virus is a species of the genus Orbivirus, within the Reoviridae family. The Reoviridae are non-enveloped and possess a double-stranded RNA genome contained in an outer-shelled icosohedral capsid. The BTV genome is arranged into 10 segments and encodes 7 structural and 4 non-structural viral proteins². The BTV receptor is currently unknown, but is proposed to included sialic acid and junctional adhesion molecules. After interaction with this receptor, the virus enters an endolysosome where the capsid is partially digested to allow the genome into the cell. Replication begins at this partially uncoated stage since the virus particles contain all the necessary enzymes⁵. First, the dsRNA is transcribed to form positive sense RNA, of which some is delivered to cytoplasm for ribosomal translation and the remainder is packaged into partially assembled virions. Complementary negative sense RNA is then formed in the virions, to give a dsRNA genome. Complete virus particles are released from the cell.

All BTV’s share group antigens, which can be demonstrated by agar gel diffusion tests, fluorescent antibody tests and the group reactive ELISA¹. There are 24 distinct serotypes, which are are distinguished by epitopes on the outer capsid protein VP2⁴, encoded by L2, the only serotype-specific BTV gene. Serotypes are differentiated using serum neutralisation tests, although there is some degree of cross-reactivity between serotypes¹. Numerous strains of bluetongue virus also exist, and these are characterised by molecular analysis.

Hosts

All ruminants are susceptible to bluetongue virus infection, including sheep, goats, cattle, deer, buffaloes, camels and antelopes. Sheep are most severely affected, and disease is occasionally seen in goats. Although cattle BTV infection is significant in the epidemiology of disease, the condition is generally subclinical in this host. Mortalities in white-tailed deer due to bluetongue¹.

Pigs and horses do not become infected with BTV, but may act as a food source for the Culicoides midges that transmit bluetongue virus to ruminants. Their habitats may also provide areas suitable for vector breeding.

Transmission and Epidemiology

BTV is transmitted by biting insects. Although vertical and venereal transmission between ruminant hosts can occur, it is insignificant in the overall epidemiology of bluetongue.

Vectors

The arthropod vector for bluetongue virus is the Culicoides biting midge. These are biological vectors of BTV, so the virus replicates in insect tissue after feeding on an infected host⁶. It takes 10-14 days for the virus to dissminated from the insect's gut to its salivary glands, after which bluetongue virus may be transmitted to a new, susceptible ruminant host. This incubation period may be reduced when ambient temperatures are higher², and midges maintaine a life-long infection.

These insects take blood meals from vertebrate hosts and breed in damp, dung-enriched soil enriched, and so are abdunant in the vicinity of domestic livestock. Once eggs are laid in soil, Culicoides progresses through four larval stages and pupates before becoming an adult midge. The lifecycle is greatly influenced by temperature: in temperate regions such as Britain, the adult midge population declines in October and is absent by December. The fourth larval stage overwinters, and adults re-appear the following April. The environment affects the activity of adult midges in a variety of ways. Culicoides survive around 10 days in warm weather but up to one month when conditions are cooler and are most active at night, from an hour before sunset to an hour after sunrise. Activity is decreased by windy conditions, and increased during the day when the weather is dull. Bluetongue virus replicates more rapidly in vector species when temperatures are warmer.

Classically, the major vector for BTV is Culicoides imicola. This midge is found throughout Africa, the Middle East, southern Asia, Portugal, Greece, Corsica, Sardinia, Sicily and areas of Italy¹, and its distribution appears to be extending northwards. However, C. imicola has not yet been demonstrated in the United Kingdom. The Culicoides species found in the British Isles are C. pulicais and C. obsoletus, which are also common across central and northern Europe. Knowledge of the distribution of these species in the UK is incomplete but the insects tend to gather where breeding sites and hosts occur in tandem, with the highest midge concentrations in areas containing cattle, horses and pigs. Removal of livestock decreases populations of Culicoides by a factor of 10 to 20¹, but some persist by feeding on wild animals and man. Hill sites have fewer midges as climatic conditions are less favourable, and sheep have a less positive influence on distribution.

In Britain, studies are ongoing to determine midge distribution, seasonal incidence and the competency of the various Culicoides species to act as BTV vectors.

Vector Competence

Certain information can help inferences be made regarding BTV vectors in Britain. Both C. obsoletus and C. pulicaris have been implicated in transmission before. Previously, BTV has been isolated from C. obsoletus in Cyprus, and African horse sickness virus (another Orbivirus) in Spain. C. obsoletus and C. pulicaris were also the most abundant Culicoides species detrected in the 1999 BTV epizootic in Greece and Bulgaria, and so are strongly suspected of acting as vectors in this case. They may also have mediated outbreaks in Serbia, FYR Macedonia, Croatia and Bosnia in 2001-2002, where C. imicola has not been recorded. Both species are therefore contenders to transmit bluetongue virus in the UK.

A British population of C. obsoletus has been shown to have and oral susceptibility rate of less than 2%¹, suggesting that C. obsoletus is likely to be an inefficient or minor vector of BTV in the UK. However, it is possible that a high abundance or survival rate may compensate for this low vector competence. Indeed, C. brevitarsis, the major Australian vector of BTV, has an extremely low experimental competency yet is an effective vector in the field.

Two other Culicoides species, C. nuberculous and C. impunctatus, exist in mainland Europe and the UK, and have been experimentally infected with bluetongue virus.

Epidemiology

Althbough bluetongue virus is capable of infecting any ruminant, cattle are the main amplifying and maintenance hosts and are most abundantly fed on by Culicoides vectors. Infection of sheep with BTV is therefore usually preceded by widespread infection of cattle and an increase in vector density¹.

Although vertical and venereal transmission of bluetongue is possible, only to the presence of competent insect vectors influences the epidemiology of BTV². This is illustrated by the fact that bluetongue virus is limited to geographical areas where competent vectors are present and that transmission only occurs at times of the year when conditions are favourably for vector activity¹. In Britain, transmission occurs mainly in late summer and autumn. Once bluetongue virus is transmitted to a vertebrate host, there are two possible outcome: either the host may die, or an immune response is mounted against the virus and renders the host resistant to re-infection. Either way, animals quickly become "unavailable" for BTV infection as the virus spreads, particularly where livestock populations are small. This presents a hurdle that must be surmounted if bluetongue virus is to persist in an area. By movement of infected vectors or viraemic animals, BTV can become established in new locations with naive hosts in order to overcome this obtascle. This means that even in zones where bluetongue virus is endemic, persistence dynamic and comprises perpetually shifting "hot spot" of infection¹. Creation of an enzootic zone is only possible in locations where adult midges are present throughout the year since bluetongue cannot be maintained through vertebrate-vertebrate or vector transovarial transmission. Any points where vectores are absent from the system must not exceed the maximum duration of viraemia in the ruminant host, otherwise the last infected vertebrate will have died or recovered by the time new vectors are availble for onwards transmission.

In some areas, bluetongue can occur in annual bouts. This may be due to new introduction of virus each year from adjacent areas where the disease is endemic, via the transportation of Culicoides on the wind for up to 100 kilometres. Alternatively, this could be the manifestation of low-level persistence.

Introduction of bluetongue virus to a new area has the potential to occur in several ways. Firstly, infected animals may be transported to the region, and local insect vectors could spread and perpetuate BTV infection within naive animals. It is also possible that local vectors could acquire BTV from infected animals in neighbouring areas, where there is a cross-over in the distribution of Culicoides species. Finally, infected vectors can be acquired from areas where bluetongue infection exists. Culicoides can be transported considerable distances on the wind, and it is also conceivable that the distribution of competent vectors may expand to colonise previously unpopulated areas under the influence of climate change.

Bluetongue virus infection was first confirmed in the UK in September 2007 when a veterinarian spotted suspicious clinical signs on a cattle holding near Ipswich⁷. It is believed that BTV-laden vectors were dispersed to the UK on the wind, since meteorological conditions on 4th August 2007 were capable of carrying midges from northern Europe to East Anglia. This would be expected to produce disease at a point that would coincide with the first case, given the time necessary for clinical detection to occur.

Pathogenesis

The pathogenesis of BTV infection has been shown to be similar in sheep and cattle, and is assumed to be similar in other species of ruminants^{8, 9, 10}. However, the severity of disease varies greatly with species and cattle in particular express very few signs.

When a BTV-infected midge takes a blood meal from a ruminant host, innoculated virus spreads from the skin to replicate in the regional lymph nodes, tonsils and spleen¹¹. A secondary cell-associated viraemia then carries the virus to many tissues where further replication occurs in macrophages and endothelial cells. In the process of reproducing, bluetongue virus causes endothelial cell injury and necrosis¹⁰ which can increase vascular permeability to cause oedema. Endothelial damage can also give thrombosis, leading to tissue infarction. In sheep and deer a consumptive coagulopathy may occur².

Several factors can influence the presentation of disease. Firstly, each virus strain is associated with its own particular virulence and thus clinical manifestation¹⁰. Host factors are also important: breed^{3, 12}, stress, nutritional status and age^{12, 13} can all affect bluetongue presentation.

Diagnosis

Where animals present with clinical signs of bluetongue a presumptive diagnosis may be made, especially in regions where bluetongue is endemic. Post-mortem examination can be used to confirm the diagnosis. However, many cases of bluetongue are mild or subclinical and so laboratory confirmation of disease is required. Cattle in particular show few clinical signs.

Clinical Signs

Bluetongue is primarily a disease of sheep, and in the face of infection these animals can display clinical signs ranging from acute to subclinical^{1, 14}. Acute disease follows an incubation period of about one week, which may depend on the infectious dose of virus received. Signs begin with pyrexia of around 40.5-42°C, and hyperaemia of the oral and nasal mucosa is seen 24-36 hours later accompanied by hypersalivation and a serous nasal discharge. The nasal discharge quickly becomes mucopurulent and potentially blood-tinged, and dries to froms a crust around the nostrils. Oedema of the head occurs, which particularly affects the lips and tongue but may also spread to include the ears and submandibular areas. In a few cases the tongue becomes severely swollen and cyanotic, lending the disease its name. Petechial haemorrhages appear on the still-hyperaemic mucosae, and areas of necrosis appear on the gums, cheeks and tongue 5-8 days after the onset of fever. Covered by a diptheritic membrane, these necrotic lesions heal slowly and contribute to inappetance, dysphagia and hypersalivation. In some cases, profuse bloody diarrhoea is seen. Erythema and petechiation of the coronary band can cause lameness, and sheep stand with an arched back, reluctant to move. In advanced disease, skeletal muscle is necrosed and contributes to rapid weight loss, along with inappetance. Animals in the late stage may also suffer torticollis. In pregnant ewes, infection with BTV may lead to abortions, foetal mummification, or the birth of stillborn or weak lambs, which may suffer congenital defects.

Considering all cases, including those which are subclinical, mortality due to bluetongue in sheep ranges between 2% and 30%¹⁴. Death can occur up to a month after the onset of clinical signs, or a protracted recovery may follow acute infection. Recovery in mild cases is often much more rapid.

Disease is most often sub-clinical in cattle despite its epidemiological significance in this species: it has been reported that only 0.01% of infected cattle show signs of bluetongue¹. Clinical signs, when seen, can include inflammation or erosions of the oral and nasal mucosae and a stiff gait. Similar signs to sheep may also occur: pyrexia, tachypnoea, lacrimation, salivation and an ulcerative dermatitis are all possibilities. In cattle in early gestation, embryonic death and resorption can result from BTV infection.

Laboratory Tests

In the United Kingdom, bluetongue is a notifiable disease and so samples from suspected cases should be submitted to the Institute for Animal Health (Pirbright) for laboratory diagnosis. Samples are collected from sheep with raised temperatures and include jugular blood collected into a plain tube to provide serum for an antibody test, and a heparinised blood sample to be used for PCR. Serum from in-contact ruminants is also submitted, as well as spleen and lymph node from all post-mortem cases. All samples should be stored at 4°C and never frozen. Paired serology may be necessary.

There is an array of laboratory tests available for the diagnosis of bluetongue, and a table published by DEFRA¹ summarises these:

Test	Specimen Required	Test Detects	Timescale
Virus Isolation	Whole EDTA blood	Virus	1 - 3 weeks
Antigen Detection Sandwich ELISA PCR Serum Neutralisation	Whole heparin or EDTA blood; tissues Whole EDTA blood; tissues Whole heparin or EDTA blood; tissues	Antigen - group specific Viral RNA - group specific Serotype	Blood: 5 - 14 days; Tissues: 4 hours 2 days 2 - 4 weeks
Antibody Detection Competition ELISA Serum Neutralisation Pathogenicity Testing in Sheep	Serum Serum Virus isolate	Antibody - group specific Antibody - serotype specific Virulence	3 hours 2 - 4 weeks 2 weeks

Pathology

A haemorrhagic gross pathology of BTV infection reflects the endothelial damage responsible for disease pathogenesis^{1, 4, 10, 12}. Certain lesions have been describes as "pathognomic" for bluetongue: these include necrosis of the papillary muscle in the left ventricle, and haemorrhage in pulmonary arterial wall. However, these lesions may be difficult to visualise in mild or recovering cases and may occasionnally occur in other diseases such as pulpy kidney disease or Rift Valley fever.

In addition to these characteristic lesions, the oral mucosa is found to be hyperaemic and oedematous and occasionally cyanotic on post-mortem examination, and petechial or ecchymotic haemorrhages may be present. The ruminal pillars and omasal folds can also appear hyperaemic, and abrasions may be seen on the lips, dental pad, tongue and cheeks. These are sometimes covered by grey necrotic material. Moderate lymphomegaly and splenomegaly are apparent, and there are areas of necrosis in the skeletal musculature. Pulmonary oedema and catarrhal inflammation of the upper respiratory tract is seen in some cases.

Histologically, endothelial damage in capillaries and minor arterioles causes thrombus formation and vascular occlusion, leading to tissue infarction. Haemorrhage, necrosis and mononuclear cell infiltration may be seen in the myocardium.

Control

There is no efficient treatment for bluetongue, and so the emphasis is on prophylaxis and control.

When bluetongue is present in the UK, there are several broad principles to disease control. Firstly, premises suspected of having the disease are inspected by a veterinary surgeon, and a ban is placed on moving animals on and off the site. Once it has been confirmed that bluetongue is circulating (i.e. there is not a single, isolated case of disease, due to importation for example), a Restricted Zone is imposed around the infected premises. A Restricted Zone is composed of a Protection Zone of at least 100km radius, and a Surveillance Zone of at least a further 50km radius. These sizes ae dictated by EU legislation for bluetongue control.

•The Restricted Zone is the overall area where restrictions apply and comprises the Surveillance Zone and the Protection Zone, and if required, the more tightly restricted disease control zone around infected premises. •The Surveillance Zone forms the outer area of the Restricted Zone and is at least 50km wide. •The Protection Zone forms the inner area of the Restricted Zone and is at least 100km radius surrounding infected premises. •Member states may demarcate part of a Protection Zone as a Lower Risk Zone. This allows livestock holders to continue vaccinating against bluetongue in an area without bluetongue virus circulation.

Prophylactic immunization of sheep remains the most effective and practical control measure against bluetongue in endemic regions. Three polyvalent vaccines, each comprising 5 different bluetongue virus serotypes attenuated by serial passage in embryonated hens’ eggs followed by growth and plaque selection in cell culture, are widely used in southern Africa and elsewhere, should epizootics of bluetongue occur. A monovalent modified live virus vaccine propagated in cell culture is available for use in sheep in the USA. Live-attenuated vaccines should not be used during Culicoides vector seasons because they may transmit the vaccine virus(es) from vaccinated to nonvaccinated animals, eg, other ruminant species. This may result in reassortment of genetic material and give rise to new viral strains. Abortion or malformation, particularly of the CNS, of fetuses may follow vaccination of ewes and cows with attenuated live vaccines during the first half and the first trimester of pregnancy, respectively. Passive immunity in lambs usually lasts 4-6 mo. The control of bluetongue is different in areas where the disease is not endemic. During an outbreak, when one or a limited number of serotypes may be involved, vaccination strategy depends on the serotype(s) that are causing infection. Use of vaccine strains other than the one(s) causing infection affords little or no protection. The vector status, potential risk from vaccine virus reassortment with wild-type viral strains, virus spread by the vectors to other susceptible ruminants, and reversion to virulence of vaccine virus strains or even the production of new serotypes also should be considered. Although a number of noninfectious vaccines are in development, they are not yet commercially available. Control of vectors by using insecticides or protection from vectors by moving animals into barns during the evening hours lowers the number of Culicoides bites and subsequently the risk of exposure to bluetongue virus infection.

Current UK Situation

The last BTV-8 infected premise was confirmed in the UK in November 2008, and since this date the situation has remained unchanged. No imported ruminants have tested positive for BTV-8 and there is no evidence that the disease has been circulating since this point. On 12th June 2010 the Uk's status was changed from a protection zone for BTV-8 to a lower risk zone (LRZ). This status allows EU member states that do not have circulating bluetongue disease to vaccinate against BTV, and also imposes stricter restriction on importing animals from zones with the same BTV serotype. The aim of this is to help prevent bluetongue re-entering the country.

Up-to-date information on the current UK bluetongue situation can be found on the DEFRA Bluetongue: Latest situation webpage.

Links

• Defra - Bluetongue
• Office International des Epizooties (World Organisation for Animal Health) - Bluetongue
• BTV Control in Cattle and Sheep (Intervet)
• Institute for Animal Health - Bluetongue
• Bluetonguevirus.org - BTV information and resource portal

References

1. DEFRA (2002) Technical Review - Bluetongue : The Virus, Hosts and Vectors.
2. Gibbs, E P J and Geiner, E C (1994) The Epidemiology of Bluetongue. Comparative Immunology, Microbiology and Infectious Diseases, 17(3-4), 207-220.
3. Spreull, J (1905) Malarial catarrhal fever (bluetongue) of sheep in South Africa. Journal of Comparative Pathology and Therapeutics, 18, 321-337.
4. MacLachlan, N J (2004) Bluetongue: A Review and Global Overview of the Only OIE List a Disease that is Endemic in North America. Proceedings of the 55th Annual Meeting of the American College of Veterinary Pathologists (ACVP) and 39th Annual Meeting of the American Society of Clinical Pathology (ASVCP), p1237.
5. Carter, G R and Wise, D J (2005) A Concise Review of Veterinary Virology, IVIS.
6. Mellor, P S (2000) Replication of arboviruses in insect vectors. Journal of Comparative Pathology, 123, 231-247.
7. IAH (2008) Institute for Animal Health - Bluetongue Research Programme
8. Barratt-Boyes, S M and MacLachlan, N J (1995) Pathogenesis of bluetongue virus infection of cattle. Journal of the American Veterinary Medical Association, 206(9), 1322-1329.
9. MacLachlan, N J (1994) The pathogenesis and immunology of bluetongue virus infection of ruminants. Comparative Immunology, Microbiology and Infectious Diseases, 17(3-4), 197-206.
10. Mahrt, C R and Osburn, B I (1986) Experimental bluetongue virus infection of sheep; effect of vaccination: pathologic, immunofluorescent, and ultrastructural studies. American Journal of Veterinary Research, 47, 1198-1203.
11. Pini, A (1976) Study on the pathogenesis of bluetongue: replication of the virus in the organs of infected sheep. Onderstepoort Journal of Veterinary Researh, 43, 159-164.
12. Parsonson, I M (1991) Overview of bluetongue virus infection of sheep. Bluetongue, African Horse Sickness and Related Orbiviruses, CRC Press.
13. Thomas, A D and Neitz, W O (1947) Further observations on the pathology of bluetongue in sheep. Onderstepoort Journal of Veterinary Science and Animal Industry, 22, 27-40.
14. Mullens, B A et al (1995) Effects of temperature on virogenesis of bluetongue virus serotype 11 in Culicoides variipennis sonorensis. Medical and Veterinary Entomology, 9, 71-76.
15. Gould, A R and Hyatt, A D (1994) The orbivirus genus: diversity, structure, replication and phylogenetic relationships. Comparative Immunology, Microbiology and Infectious Diseases, 1, 163-188.
16. Merck & Co (2008) The Merck Veterinary Manual (Eighth Edition), Merial.
17. Dal Pozzo, F et al (2009) Bovine infection with bluetongue virus with special emphasis on European serotype 8. The Veterinary Journal, 182(2), 142-151.
18. MacLachlan, N J et al (2009) The Pathology and Pathogenesis of Bluetongue. Journal of Comparative Pathology, 141(1), 1-16.
19. Afshar, A (2004) Bluetongue: Laboratory Diagnosis. Comparative Immunology, Microbiology and Infectious Diseases, 17(3-4), 221-242.
20. Gould, E A and Higgs, S (2009) Impact of climate change and other factors on emerging arbovirus diseases. Transactions of the Royal Society of Tropical Medicine and Hygiene, 103(2), 109-121.