Bluetongue

GENERAL INTRODUCTION: REOVIRIDAE

Introduction

Bluetongue (BT) is an arthropod-borne viral disease of domestic and wild ruminants, particularly sheep amongst domestic livestock. Clinical disease is characterized by haemorrhage, excoriations and erosion, and cyanosis of the mucous membranes of the oronasal cavity, coronitis and laminitis, oedema of the head and neck, and torticollis. The name of the disease derives from distinctive cyanosis of the tongue, which may occasionally be severe.

Bluetongue was first described in South Africa,¹⁴² where the infection (but not disease) has probably been endemic in wild ruminants from antiquity. The disease has been recognized since Merino sheep were introduced into the Cape Colony in the late eighteenth century. From the outset, BT was known to be most prevalent during the summer months, especially in wet seasons. The first detailed study of BT was made by Spreull,^{293, 294} but Theiler³¹² was the first to demonstrate the filterability of the aetiological agent, thus indicating its viral nature. He also introduced the first vaccination, making use of a virus strain (subsequently shown to be serotype 4) attenuated by passage in sheep.³¹³ This crude vaccine, which consisted of sheep blood, produced mild BT in recipient animals and, although it contained a single serotype, nevertheless induced remarkable protection.  Despite its obvious shortcomings it afforded sheep farmers a reasonably effective method of control, and was used for almost 40 years.

Bluetongue was initially thought to be confined to the African continent. In 1943 the first confirmed outbreak outside Africa was reported in Cyprus,¹⁰¹ but the disease may have occurred there as early as 1924.²⁸⁴ Outbreaks of BT or presence of the causative agent, Bluetongue virus (BTV), were subsequently reported in Israel,¹⁶² the USA,^{118, 207} Portugal¹⁹⁷ Spain,¹⁶⁷ Pakistan²⁷⁸ and India,²⁷⁷ and later in  the Middle East, China, Southeast Asia  and Australia.^{84, 185, 239, 287, 289, 345} With the notable exception of transient incursions of single virus serotypes into the Iberian Peninsula and Greece, Europe was historically free of BT  until 1998²⁴¹ when multiple serotypes of BTV spread throughout  extensive portions of the continent.^{86, 214, 256, 273} This recent epidemic initially involved only regions of Europe adjacent to the Mediterranean Sea but later, with the incursion of a novel strain of BTV serotype 8, spread to affect virtually the entire continent as well as portions of the United Kingdom and Scandinavia. In recent years live-attenuated vaccine-derived strains of BTV have also spread through much of Europe.^{67, 93, 233}

Early reports incriminating blood-sucking insects as vectors were based on circumstantial evidence. The first experimental proof that BTV is transmitted in South Africa by Culicoides imicola (= C. pallidipennis) was obtained by Du Toit in 1944.⁷⁷ Subsequently many other Culicoides spp. (Figure 1) were proved to be vectors of BTV in the Americas, Asia,  Australia, and Europe, indeed it is now clear that different vector species are responsible for dissemination of different constellations of BTV serotypes  in distinct global episystems.^{17, 31, 104, 108, 191, 252, 297, 309, 311, 339}

Figure 1 Culicoides zuluensis female

The existence of a plurality of BTV serotypes, which had long been surmised, was confirmed by Neitz in 1948 by means of cross-protection studies in sheep.²²⁶ It provided an explanation for the vaccination failures that had been experienced with the monovalent Theiler vaccine and also formed the basis for later studies on the in vitro serotyping of viral isolates.^{127, 128}

Further advances in the understanding of BT and BTV followed rapidly on the development of techniques for the study of viruses. Some of the important milestones were the isolation and propagation of BTV in embryonated chicken eggs (ECE) by yolk sac^{6, 201} and intravascular¹⁰⁹ inoculation, and its adaptation to the brains of suckling mice for antigen production.^{162, 318} This was followed in 1956 by the successful propagation of BTV in cell cultures by Haig et al.¹¹⁷

The availability of cell culture systems for the propagation of BTV led to its successful purification, to the demonstration that it is composed of a segmented, double-stranded RNA genome enclosed in a double-layered capsid,³²⁶ and to its classification in the genus Orbivirus in the family Reoviridae.^{37, 224, 327} Most recently, rapid sequencing procedures have facilitated the genetic characterization of strains and serotypes of BTV from different global ecosystems.^{17, 43, 169, 231}

The continued natural evolution of BTV and other orbiviruses poses a substantial challenge to research and regulatory institutions, stimulating worldwide interest and research activity. Comparative studies of the structural and functional relationships of these viruses have yielded much information, which has been reviewed elsewhere.³²⁹ Because of its economic importance and wide distribution, BTV, however, remained the focus of attention among orbivirus infections of ruminant livestock.^{139, 269, 295} The recent identification in Europe,¹⁷⁰ Africa and Asia of genetically distinct BTVs that infect small ruminants (goats and sheep), potentially without requirement for an insect vector (see Epidemiology), has further complicated the taxonomic classification of BTV and understanding of the epidemiology of BTV infection.^{25, 123, 183, 185, 281, 283, 331, 347} A number of reviews on both early and more recent aspects of BT research have been published.^{19, 42, 51, 110, 126, 137, 185, 187, 191, 255, 273, 320}

Aetiology

Based on its morphologic and molecular properties, BTV is classified as the typespecies of the genus Orbivirus in the family Reoviridae, subfamily Sedoreovirinae.^{37, 265, 327} Initially, BTV was classified as an arbovirus because of its transmission by biting midges, but its stability in the presence of lipid solvents, suggesting the absence of an envelope, as well as early electron microscopic studies³⁰⁴ subsequently led to its provisional classification as a reovirus.¹³ However, it differed in a number of respects from reoviruses (its smaller size, absence of a structured outer capsid layer and concomitantly greater sensitivity to extremes of pH and high temperature, and its requirement for an insect vector in the classical serotypes at least) and this resulted in it being classified in a separate genus.

Morphology

Negatively- stained BTV virions are non-enveloped, spherical and have an approximate diameter of 68 to 70 nm. When examined by electron microscopy, they have a ‘fuzzy’ appearance caused by a diffuse outer layer in the protein capsid. (Figure 2a) This layer, comprising two proteins (VP2 and VP5), can be removed enzymatically or by manipulating the salt concentration and pH of the environment^{220, 319} to reveal a highly structured core particle. (Figure 2b) The core particle, with a diameter of 54 nm, includes two structural proteins (VP3, VP7) and the transcriptase complex composed of VP1, VP4 and VP6. The name of the genus is derived from the capsomere structure (orbis = ring in Latin). Core particles can be further degraded by removal of VP7 to create subcore particles that include only VP3 and the transcriptase complex (Figure 2c).¹⁴¹ Using cryo-electron microscopy, diameters of approximately 80 and 70 nm have been obtained for virions and viral cores, respectively.¹²⁰ The atomic structure of the core particle of BTV has been determined by X-ray crystallography at a resolution approaching 3.5Å and is shown in (Figure 3). It explains how approximately 1 000 protein molecules self-assemble to form transcriptionally active viral core particles.¹¹³

Figure 2a Electron micrographs of negatively stained bluetongue virus core particles (Courtesy of Mr H.J. Els, Faculty of Veterinary Science, and University of Pretoria, South Africa)

Figure 2b Electron micrographs of negatively stained bluetongue virus subcore particles (Courtesy of Mr H.J. Els, Faculty of Veterinary Science, and University of Pretoria, South Africa)

Figure 2c Electron micrographs of negatively stained bluetongue virus x200 000. (Courtesy of Mr H.J. Els, Faculty of Veterinary Science, and University of Pretoria, South Africa)

Genomic structure

Bluetongue virus was the first virus of domestic animals to be shown to possess a double-stranded RNA genome, initially thought to be unique to reoviruses. This finding was based on heterochromatic staining of the viral inclusion bodies with acridine orange, resistance to degradation by pancreatic RNAse, a characteristic melting curve, and a base composition suggesting A-U and G-C base pairing.³²⁶ The genome consists of ten segments which can be separated electrophoretically after disruption of the virion. These segments vary in size from 0.5 kDa to 2.7 × 103 kDa,³²⁹ and function as genes (i.e. they are translated individually into functional viral proteins). The coding relationships between the genome segments and the viral proteins are shown in Table 1.

Genome segments are labelled 1 to 10 in descending order of size (S1 – S10), whereas viral polypeptides are divided into structural proteins (VP1–VP7) and non-structural proteins (NS1–NS5).³⁰⁰ Nucleotide sequences have been determined for all ten segments of a number of BTV serotypes. A schematic representation of the structural components of BTV is shown in Figure 3.

Table 1 Proteins and corresponding genome segments of bluetongue virus

Figure 3 A model for the bluetongue virion. (Adapted from Loudon, P.T. et al. 1992. In: Walton T.E. & Osburn, B.I., (eds). Bluetongue, African Horse Sickness and Related Orbiviruses. Boca Raton, Florida: CRC Press, Inc., pp. 383–389)

Structure and function of the viral proteins

The viral proteins range in size from approximately 25 000 to 149 000 Dalton.^{199, 220, 264, 265, 327} Four of the seven BTV proteins (VP2, VP3, VP5 and VP7) are major structural components and constitute about 94 per cent of the total virion protein. The other three (VP1, VP4 and VP6) that constitute the replicase complex are minor components, making up the other 6 per cent. Two of the major components (VP2 and VP5) form the diffuse outer capsid layer, whereas the other five are found in the core particle.^{111, 220, 327}

VP1 is a 149 kDa minor component of the subcore particle and functions as an RNA-dependent RNA polymerase. A comparison of the amino acid sequences derived from various serotypes indicates that it is a highly conserved molecule.

VP2 is the major constituent of the outer capsid and the main antigen responsible for the induction of serotype-specific neutralizing antibodies,^{136, 247} although VP5 also plays a role.²²¹ When these two proteins are removed, the infectivity of the virion for mammalian cells is greatly reduced, whereas it is essentially retained for the insect vector.²¹⁹ This reflects an essential role in binding to cell receptors and penetration into the host cell. Not unexpectedly, VP2 is the most variable of the BTV proteins, although conserved regions in genome segment 2 have also been identified.

VP3 proteins form the shell of the subcore particles and act as a scaffold for the surface layer of the core particle, which consists of VP7.¹⁴¹ It also binds to RNA and interacts with the replicase complex of VP1, 4 and 6, indicating a fundamental role in the early stages of assembly of the BTV particle. It has a sequence that is highly conserved among BTV serotypes and other orbiviruses.^{310, 342}

VP4 of BTV binds GTP and is a candidate guanylyl transferase of the virus.³⁵ It is a minor component of the subcore particle, but was shown to be involved in the encapsidation process in a dimerized form. A putative leucine zipper near the carboxy terminus of the protein was found to be essential for the dimerization of VP4.²⁵⁷

VP5 (together with VP2, as described above) is part of the outer capsid of intact BTV particles. In contrast to VP2, VP5 is a relatively conserved protein with no clear regions of variability.⁷⁹ In fact, comparing the complete nucleotide sequence of epizootic haemorrhagic disease of deer virus (EHDV) with those of various BTV serotypes, a 59 to 62 per cent homology was found.¹⁴⁸

VP6 can often be resolved into two closely migrating bands by SDS-PAGE. Expression of a cloned genome segment 9, which codes for this minor component of the BTV core particle, showed that the two forms of the protein are derived from the initiation of protein synthesis at two distinct sites.³³² Sequences within the protein are required for the retention of VP6 in the cytoplasm, and the protein was also shown to bind ATP and RNA and to possess an RNA-dependent ATPase function and helicase activity.^{60, 299}

VP7, a major structural protein, forms the surface layer of the orbivirus core particles responsible for the ring-like ‘capsomere’ surface structures. It is the immunodominant group-specific antigen and its crystal structure has been determined by means of X-ray crystallography.¹¹³ Full sequencing of cloned genome segments 7 of various orbiviruses revealed a remarkable homology between those of BTV, EHDV and African horse sickness virus (AHSV).^{61, 147, 268} This close relationship is probably explained by the recent demonstration that VP7 is intimately involved in the binding of BTV to membrane proteins of the Culicoides vector, common to all of them.³⁴⁴

The nonstructural proteins are produced in infected cells and probably play a role in the assembly of the virion. NS1 is formed in excessive amounts and in the form of characteristic tubules in the cytoplasm of infected cells that promote synthesis of other viral proteins. A high degree of homology was found between the nucleic acid sequences of segment 6 (which encodes NS1) derived from BTV and related EHDV.²²⁷ Extensive homology was also found in the nucleotide sequences of genome segment 8 of BTV and EHDV, which encode their respective NS2 proteins. It is the only orbivirus protein that is phosphorylated by means of a cytoplasmic protein kinase. It is the major component of viral inclusion bodies and also has the ability to bind single-stranded RNA.^{314, 323, 332}

The smallest RNA genome segment 10 encodes two closely related non-structural proteins, NS3 and NS3A. Both occur as glycosylated and non-glycosylated forms in infected cells.¹⁸ Glycosylation reflects their association with plasma membranes and suggests that they may be involved in the final stages of morphogenesis, i.e. the release of virions from infected cells.¹⁴⁵ The NS3 protein facilitates viral egress from infected cells and it may be involved in the host innate immune response by down regulating activation of the interferon (IFN-b) promoter.^{58, 188} A high degree of homology and conservation was found in a comparison of proteins derived from various BTV serotypes¹⁴⁴ but more variation between BTV, AHSV and Palyam virus.³²²

In addition to VP6, gene segment 9 encodes in another reading frame a small nonstructural protein (NS4) that can localize to the nucleolus of infected cells and which serves as an interferon agonist during virus replication.^{30, 258} Specifically, NS4 promotes replication of BTV in cells pre-treated with Type 1 interferon. An ORF in Seg 10 overlapping the larger ORF encoding NS3/3a expresses another non-structural protein S10-ORF2 (putative NS5 protein), which appears to function as an interferon antagonist.³⁰⁰

Physicochemical characteristics of the viral particles

The buoyant density of intact BTV virions is 1.36 g/ml, whereas the core particle has a density of 1.40 g/ml, reflecting the loss of the outer capsid proteins. When freeze-dried in buffered lactose-peptone, BTV can survive almost indefinitely at room temperature. In blood and tissue specimens it is very stable at 20 °C, 4 °C and −70 °C but not at −20 °C. The virus is unstable below pH 6.5,^{238, 308} and on purification, when all extraneous protein is removed, it is extremely unstable at high temperatures.¹²⁹ Because of the double-stranded nature of its RNA genome, BTV is relatively resistant to ultraviolet and gamma irradiation. It is also relatively resistant to lipid solvents such as ether and chloroform, but it is readily inactivated by disinfectants containing acid, alkali, sodium hypochlorite and iodophors.¹²⁶

Serological characteristics of bluetongue viruses

The existence of multiple BTV serotypes was first discovered in a series of tests in sheep when Neitz²²⁶ found that each of ten viral isolates from field cases of BT produced solid immunity against itself but only partial or no protection against heterologous strains. Although cross-protection tests in sheep proved the plurality of BTV strains they did not allow their classification into serotypes. Howell¹²⁷ devised an in vitro neutralization test by which 22 BTV isolates could be grouped into 12 distinct serotypes. A further four serotypes were discovered after the introduction of the plaque inhibition test.^{126, 128} A total of 24 distinct serotypes were subsequently identified using conventional virus isolation and serology but, with the advent of molecular detection and identification methods, at least 3 additional types (presumed serotypes) have been described. Some of these genetically novel BTVs have never been isolated in cell culture so their precise serological characteristics remain uncertain.^{29, 281} The 27 distinct serotypes of BTV are all interrelated in a complex network of cross relationships,⁹⁰ with common group antigens. However, analyses of the entire genomes of field strains of BTV from endemic regions of the world confirm widespread reassortment of viral genes, as well as remarkable genetic drift of individual BTV genes.^{17, 43, 231, 236}

Epidemiology

Repeated recent incursions of BTV into previously free regions confirm that the global range of the virus is expanding, and that the epidemiology of BTV infection is changing. Historically, the virus was thought to be restricted to a geographic band between the latitudes 40°N and 35°S, coinciding with the distribution of competent vectors, but it is now clear that the virus’ range extends beyond that limit in much of the Northern Hemisphere including North America, Europe and Asia, up to at least 50°N.^{3, 86, 104, 153, 180, 185, 191, 233, 263, 335, 340} Both anthropogenic and natural factors might have influenced the spread and transmission of BTV into previously free areas, specifically through movement of viraemic animals (domestic and wild ruminants) or through active (local) propagation following wind-borne dissemination of infected Culicoides spp. from infected regions.^{80, 104, 169} Other potential routes include the translocation of live-attenuated vaccine viruses or infective animal products, or infected midges within living (plants, animals) or inanimate (e.g. airplanes, ships) objects or products. However, it is increasingly apparent that climatic changes have facilitated recent expansions in the global range of BTV infection.^{86, 115, 191, 255, 256, 273}

With the notable exception of the recently identified, horizontally transmitted small ruminant-adapted BTVs (see Small ruminant-adapted bluetongue viruses), the establishment of BTV infection in previously virus-free areas is dependent on the presence of reservoir and amplifying hosts such as wild ungulates (game animals) and cattle, and on suitable species of Culicoides midges that serve as vectors.^{104, 185, 191, 309} Given that ruminant animals, wild and/or domestic, are abundant in many regions where incursions have occurred, it is the presence and activity of competent insect vectors that typically determines whether virus persists in a given area. The distribution of BT therefore closely follows the spatial and temporal distribution of its Culicoides vectors. The dynamics of vector–host relationships and their influence on the spread of the disease have been reviewed.¹⁰⁵ Factors such as flight periodicities, biting activities, host preferences, preferred habitats for oviposition and climate have a direct influence on the incidence and spread of BT.

Even in the very early descriptions of BT it was clearly recognized that the disease is geographically limited, seasonal in its occurrence in temperate zones, most common in areas of southern Africa with high rainfall and after good rains, and that sheep can be protected by being housed at night and dipped in an insecticide. These observations, as well as the disappearance of the disease after the first frosts, were convincing evidence for the role of an insect vector in the occurrence of the disease.^{143, 294}

In his classical experiments Du Toit⁷⁷ initially transmitted BT to susceptible sheep by inoculation of homogenized pools of wild-caught C. imicola, and subsequently by the bites of these midges 10 days after they had fed on a sheep infected with BTV. This finding was confirmed under controlled conditions in the USA, but with C. sonorensis (formerly C. variipennis).⁹⁸ Replication of the virus in the salivary glands of C. sonorensis (formerly C. variipennis) was demonstrated electron microscopically.⁴¹ Infected midges remain infective for the rest of their lives. Transovarial transmission of the virus from infected midge females to their F1 progeny has never been proven with any Culicoides species, rather variation in the distribution, activity and competence of the population of adult Culicoides midges can determine whether an area is endemic or BTV-free. The endemic areas for BT and BTV infection are largely defined by suitable climatological factors, which are mainly confined to the tropics and subtropics although temperate regions are increasingly affected.^{105, 108, 153, 285} Apart from certain high-altitude regions, such as parts of the north-eastern Eastern Cape Province in South Africa and Lesotho, the entire southern African region should be regarded as endemic. Bluetongue virus has been isolated in various parts of the world from a wide variety of Culicoides spp. (see Vectors: Culicoides spp.).  The most important and widespread Asian-African vector is C. imicola whose habitat includes most of Africa, the Mediterranean Basin including southern Europe, the Middle East, and portions of Asia including Laos, Vietnam and southern portions of of China. C. sonorensis and C. insignis are important vector species in the Americas, C. brevitarsis and C. wadai in Australia, and species of the Obsoletus (including C. obsoletus and C. scoticus) and Pulicaris (including at least C. pulicaris and C. newsteadi) complexes in Europe.^{31, 104, 105, 108, 214, 297, 301, 309, 311}

Laboratory infection of mosquitoes with BTV has been reported, and the disease itself has been transmitted experimentally by the sheep ked, Melophagus ovinus,¹⁷⁵ and a soft tick, Ornithodoros coriaceus,³⁰³ but a biological cycle was not demonstrated in these vectors, suggesting mechanical transmission. Blood-sucking flies such as Stomoxys and Tabanus spp. should also be considered as potential mechanical vectors. However, compared to biological transmission, mechanical transmission – whether by insects or fomites - is probably of minor significance in the spread of BTV. Interestingly, transstadial and transovarial passages of BTV have been observed in hard and soft ticks, but the biological significance (if any) of this observation remains uncertain in naturally-occurring virus transmission.³⁸

Vector independent transmission of BTV clearly can occur, although its significance is largely unknown. Although BTV infection is not generally contagious and very little virus is found in the secretions and excretions of infected animals, oral BTV infection of both ruminant livestock and wild and zoo carnivores has been described, including infection of calves via the feeding of infective colostrum.^{187, 203} Direct contact transmission of certain recently identified novel “small ruminant” BTV serotypes has been demonstrated in livestock, likely by aerosol, and at least some recent European strains of BTV can be transmitted directly between ruminants in close contact.^{25, 45, 291, 292} The mechanisms of contact transmission are uncertain but might include shared feed and water troughs, contamination of wounds with blood during fighting among red deer, and contact with infected placentas in a case in cattle. Seropositive dogs, cats and wild carnivores in Africa were thought to have eaten tissues from infected animals,⁵ and Eurasian lynx in a zoo had been fed ruminant foetuses and stillborn animals from outbreak areas.¹⁵⁰

Vertical transmission of BTV in animals certainly does occur, notably with live-attenuated vaccine strains of BTV and European BTV-8, but this route is considered unlikely to be important to the epidemiology of natural BTV infections.^{187, 191, 274} However, some of these animals can be born infected and could therefore introduce the virus to new areas if the dam is transported during gestation. Transplacental transmission has been demonstrated in dogs, at least for serotype 11.³³⁷

BTV-8 has been detected in the semen of naturally infected bulls by both PCR and virus isolation, indicating that BTV-8 may contaminate semen in a similar way to tissue culture passaged viruses. Further work is required to investigate the duration of BTV-8 presence in semen.¹⁶¹ Apart from BTV-8, field/wild strains of BTV have only been identified in the semen of older bulls, most likely due to blood contamination.³⁹ It has been demonstrated that females inseminated with cell-culture adapted BTV-contaminated semen can become infected. The vast majority of evidence shows that isolation of BTV in semen coincides with the viraemic period in bulls/rams. However, one study showed that a tissue culture passaged strain of BTV-1 was detected in semen for approximately 10 days beyond the period of detectable viraemia in the bull.  No evidence was found for congenital transmission when cows were infected by various routes.^{187, 191, 244}

The role that embryo transfer might play in the transmission of BT seems to be negligible. Embryos recovered non-surgically from infected donor cattle with high BTV titres at the time of collection failed to infect any of 39 recipients, as judged by the absence of seroconversion and the inability to isolate virus from them.⁴⁰ It was also found that BTV does not infect bovine zona-intact oocytes or embryos during in vitro maturation and culture.³¹⁶ In sheep, embryos from BTV-infected ewes also failed to transmit the disease.¹¹⁹ A quantitative risk analysis indicated that the risk of transmission of BT by embryos is 1:30 000 during the vector season and 1:1 million out of season.³⁰⁶ In general, embryo transfer can be regarded as a safe procedure as far as accidental BTV transfer is concerned, provided the donor animals are not viraemic at the time of semen and embryo collection,¹⁰⁶ and that the embryos are washed extensively.²⁹⁰

Human activities may in some instances be important in mediating the introduction of BTV, rather than movement of either infected animals or vectors.^{67, 259} For example, the recent appearance of at least 3 (BTV-6, BTV-11, BTV-14) different South African live-attenuated BTV vaccine strains amongst livestock in Europe (most recently BTV-14 in western Russia, Poland and Lithuania).⁸⁶ Interestingly, live attenuated vaccine strains of BTV-6 and BTV-11 first appeared in the same general region of northern Europe as did BTV-8 in 2006, again suggesting an as yet unexplained anthropogenic phenomenon. Similarly in India, strains of BTV that are genetically similar or identical to live-attenuated vaccine viruses used elsewhere in the world have also been identified as infecting local livestock, raising further concerns regarding the unauthorized international movement of vaccine viruses.¹⁸⁴

Contamination of biological products, especially those that utilize foetal bovine serum or primary ruminant cell lines, is also well described as, for example, in canine, sheep and cattle vaccines.^{4, 49}

Although traditionally regarded as a sheep disease because of its economic importance in this species and the occurrence of major epidemics that cause heavy economic losses in sheep-rearing countries,⁸⁷ BT affects a wide range of species. The outcome of infection, however, varies among different species and breeds as well as among individuals of the same species. Indigenous breeds of sheep are generally less severely affected by BT than are exotic breeds. In sheep, the viraemia reaches a relatively high level (about 10⁵ sheep ID₅₀/ml) and is usually associated with severe clinical disease in fully susceptible animals. South American camelids (llamas, alpacas etc.) also may develop severe disease following BTV infection.^{103, 222, 234} With the notable exception of especially virulent viruses such as European BTV-8, clinical disease is rare in cattle and goats, and, when present, is much milder than in sheep.¹²⁵

During a season of exceptionally high rainfall and Culicoides activity in South Africa, cattle with typical signs of stomatitis and coronitis were shown to have a double infection with BT and EHD viruses, apparently exacerbating the clinical signs.¹⁰²

The susceptibility of wild ruminants was first established by experimental infection of blesbok (Damaliscus albifrons).²²⁵ Evidence of inapparent infections have subsequently been found in many other species^{111, 164} and it is now accepted that potentially all ruminants and camelids are probably susceptible to infection.²¹ African antelopes do not develop clinical disease, whereas white-tailed deer (Odocoileus virginianus), pronghorn antelope (Antilocapra americana) and desert bighorn sheep (Ovis canadensis) of the North American continent may develop severe clinical disease.¹²¹ The European strain of BTV-8 caused severe disease in all non-African ungulates, whereas infection was invariably asymptomatic in ungulates of African origin.¹⁹⁶

Canine fatalities and abortion were recognized in pregnant dogs immunized with a canine vaccine contaminated with BTV.^{4, 337} Similarly, fatal infections occurred in Eurasian lynx that consumed BTV-8-infected cattle foetuses aborted during the recent European epidemic.¹⁵⁰

Bluetongue virus may be transmitted throughout the year in areas where the winter is mild. However, overwintering of the virus in areas with long, cold winters is more difficult to explain, as transovarial transmission of BTV in Culicoides spp. apparently does not occur.^{157, 235} It is possible that cattle are able to act as reservoirs,²²⁸ although recent investigations only partially support this contention as viraemia in experimentally infected livestock is typically less than 60 days.^{187, 191} Furthermore, BTV replication in tissues is transient and, with the exception of some recently described small ruminant BTVs, truly persistent infection does not occur.¹⁸⁹ It has also been postulated that in southern Africa the primary replicative cycle involves one or more species of African antelope and Culicoides midges, and that the role of antelopes has been largely supplanted by cattle in areas where agricultural development has displaced wild animals.⁷⁸ Outbreaks of the disease in sheep usually occur simultaneously in widely separated localities in late summer or autumn, suggesting that populations of BTV-infected midges build up in the primary cycle involving cattle or wild animals during spring and early summer, and that sheep become infected in a secondary cycle as a result of ‘spill-over’.⁸⁹ Importantly, recent studies in North America, and subsequently in South Africa, confirm the presence of BTV-infected adult midges during winter months in endemic temperate regions, suggesting that the virus overwinters in long- lived vector midges.^{202, 204, 301} It is likely that these infected midges that survive during winter could initiate a low level cycle of infection amongst domestic and/or wild ungulates, an infection cycle that would further facilitate virus overwintering.²²⁸ Other less likely mechanisms of over-wintering include vertical transmission of the virus in animals or ticks, although biological significance of these to occur is likely minimal.

In contrast to epidemic areas where incursions occurred, such as much of Europe currently, BTV serotypes are distributed apparently randomly throughout endemic areas like South Africa and North America, and the dominant serotypes at any given time are largely determined by herd immunity. Monitoring for BTV of Culicoides trapped at various sites in South Africa over a period of six years (1979 to 1984) revealed that 14 to 18 different serotypes were encountered every season, albeit at markedly varying frequencies. Usually, three to five serotypes were isolated predominantly and these were often responsible for more than 60 per cent of the total number of isolates for that particular season. These dominant serotypes were largely replaced by others the following season, only to become dominant again three to four years later. Such serotypes obviously possess a high epidemic potential. In this category were types 1 to 6, 8, 11 and 24. A second group of BTV serotypes was present at much lower levels but was encountered every season. In this category were types 9, 10, 12, 13, 16 and 19. A third group represented by types 7, 15 and 18 appeared sporadically and probably has a low epidemic potential.⁹²

The epidemiology of the small ruminant-adapted BTVs that recently have been identified amongst goats and sheep in Europe, Africa and Asia may be quite different than that of the “conventional” 24 serotypes.^{25, 123, 183, 185, 281, 283, 331, 347} For some of them transmission apparently occurs without any requirement for an insect vector and may therefore be independent of climatic conditions.  In further contrast to other BTV serotypes, infection of goats with BTV serotype 25 is persistent (> 2 years) (see section: Small ruminant- adapted BTVs).

Small ruminant-adapted bluetongue viruses

In the past decade, new serotypes of BTV have been described in Europe, Asia and Africa.^{49, 123, 170, 183, 198, 281, 305, 347} These viruses represent a novel group of BTVs that differ in their biological and genetic properties from the historical 24 serotypes (BTV 1 – 24).  These novel BTVs exhibit minimal virulence or pathogenicity to ruminants; they have all been isolated from apparently healthy goats or sheep, and at least some of them can be transmitted by contact without requirement for vector insects. Although  little as yet is known about these new BTV types, some have already been classified as additional serotypes (specifically BTV  25, 26, 27),^{123, 183, 347} whereas other apparently new serotypes have not been assigned a numerical serotype designation, only an identification designation.^{49, 170, 198, 281, 305}

BTV-25 was initially identified as a new member of the genus Orbivirus and named as Toggenburg Orbivirus (TOV) after the region of its first detection in goats in Switzerland. The sequences of several genome segments (2, 5, 6, 7, 8, 9, and 10) of TOV clearly confirmed that it was a new BTV serotype, specifically BTV-25.¹²³ Although first detected in 2008, subsequent retrospective analyses confirmed positive samples from Swiss goats dating back to 1998, indicating that BTV-25 has circulated in that part of Europe for at least a decade and likely considerably longer.  BTV-25 produces subclinical infections in goats and is only mildly pathogenic to sheep.⁵⁶ Although BTV-25 infection is highly prevalent amongst goats in certain areas of Switzerland, infection was not found in either sheep or cattle cohabiting with virus-positive goats, nor in wild animals, suggesting a highly restricted host range.⁵⁴ The high prevalence of infection in goats is in part likely a consequence of the ability of BTV-25 to induce persistent infection in goats, as BTV-25 infected goats remain infective for up to 19 months and RNA-positive up to 25 months.³³¹ Direct contact transmission of BTV-25 might also contribute.⁵⁶ Experimental studies are difficult with BTV-25 because the virus is yet to be propagated in vitro using systems that are reliably used for propagation of BTV 1-24.²⁵⁰ A probable variant of BTV-25, BTV-Z ITA2017, was recently identified in a goat flock from North Western Italy.¹⁹⁸ This strain is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to BTV-25. No clinical signs were observed in infected animals, which showed only low levels of RNA-aemia.  Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV.

BTV-26 was first isolated in 2010 from a blood sample of a sheep in a mixed goat and sheep flock in the Abdali region of Kuwait.¹⁸³ The RNA extracted from this isolate was not amplified by a segment 2 (Seg-2) specific RT-PCR for each of BTV1-25, indicating it was a novel serotype. The sequence of Seg-2/VP2 of BTV-26 differs from all other known serotypes, but is more closely related to BTV-25 than other serotypes with a nt/aa identity of 63.9% and 61.5%, respectively. These results were then confirmed by the virus neutralization assay as none of the antisera against the existing 25 BTV serotypes neutralized BTV-26. Unlike BTV-25, BTV-26 can be propagated in vitro although its growth capability is limited to mammalian cell lines and the virus does not replicate in Culicoides-derived cell lines (Kc). Experimental BTV-26 infection results in a higher and longer-lasting RNA-aemia in goats than in sheep (28 vs. 21 days); whereas BTV-26 resulted in subclinical infection of goats, it produced mild disease in sheep.^{26, 27} BTV-26 can be experimentally spread between goats by direct contact transmission, with both nasal and ocular secretions as possible sources of infection.²⁵ Horizontal transmission of BTV-26 requires close contact among animals. Serological evidence of BTV-26 infection subsequently was shown amongst cattle and dromedaries in Mauritania and in Libyan sheep.^{168, 195}

BTV-27 was first detected among goats in Corsica in 2014.³⁴⁷ The infected goats were positive by BTV group-specific PCR but negative to Seg 2 specific assays specific for each of the other 26 BTV serotypes.  Sequence analyses of Seg-2/VP2 of this virus showed a high identity with both BTV-25 and BTV-26, sharing 73% nt and 75% aa identity with BTV-25 and 65% nt and 60% aa identity with BTV-26. Furthermore, the virus was not neutralized by antisera raised against BTV-1 to BTV-26. Like BTV-26, in vitro propagation of BTV-27 is limited to selected cell lines; specifically, the virus was not isolated in ECE or on cell lines such as Kc, Vero and BHK that are commonly used for isolation of “traditional” BTV serotypes. Indeed, only a single isolate was obtained and that was accomplished using BSR cells. Three different variants of BTV-27, with small but detectable nt and aa differences (<8%nt/<10%aa) were described subsequently.²⁸³ All were found in healthy goats. Cattle appear to be refractory to infection as neither RNA nor antibodies were detected following experimental infection with a variant of BTV-27. It appears that BTV-27 is poorly immunogenic as virus - specific antibodies and RNA were detected at low levels and for a short period in infected goats. Analogous to BTV-26, recent studies suggest BTV-27 also can be spread by direct contact transmission.⁴⁵

There are several other recently identified BTVs that likely constitute new virus serotypes and, of these, X ITL2015 is best characterized.²⁸¹ X ITL2015 was identified in healthy goats in Italy, and has yet to be detected in either sheep or cattle. Although clearly a BTV in terms of reaction with group-specific RT-PCR and serological testing of infected animals, the virus is virologically and serologically distinct from the 27 recognized BTV serotypes. The Seg-2/VP2 sequences of this virus cluster with the other new BTVs (BTV-25, 26, 27) with the highest identity (75.3–75.5% nt/77.4–78.1% aa) with the recently isolated BTV-27s from Corsica and with recently discovered XJ1407 from China (75.9% nt /78.2% aa), whereas it is less related to BTV-25 (73.0% nt/75.0% aa) and BTV-26 (62.0% nt/60.5% aa).  Serum of X ITL2015 infected goats failed to neutralize any of the other virus serotypes but, like BTV-25, all attempts to isolate X ITL2015 using ECE or cell cultures have been unsuccessful. Serological studies suggest that this virus, like BTV-26 and 27, may be spread by direct contact transmission.

XJ1407 was first detected in clinically normal goats and sheep in China.³⁰⁵ The virus is most related to BTV X ITL2015, and Seg-2/VP2 of XJ1407 shares high sequence identity with BTV-27 (75% nt/78% aa) and BTV-25 (73% nt/75% aa).^{123, 183, 283} The virus has not yet been serologically characterized, although it can be propagated in ECE and several mammal and insect cell lines.

Another possible new BTV serotype is a virus designated as SP vaccine-derived BTV, which was identified in 2014 in batches of sheeppox (SP) vaccine purchased in Israel.⁴⁹ Sequence analyses of the genome segments 1, 4, 7 – 10 confirm that this virus is closely related to BTV-26. Like BTV-26, the virus was easily propagated in ECE and several mammal cell lines. A possible variant of SP vaccine- derived BTV (SPV BTV) was found in healthy sheep in Tunisia that were imported from Libya.¹⁷⁰ The sequences of genome segments 4, 9, 10 of this new virus, designated BTV-Y TUN2017, have high nt sequence identity (99.3%, 96.9% and 91.1%, respectively) with SPV BTV. However, unlike the SPV BTV, attempts to isolate BTV-Y TUN2017 using ECE or several mammalian cell lines have been unsuccessful. The sequences of Seg-2 of this virus are only distantly related to other BTVs, including the small ruminant-adapted BTVs ( <62.3% nt identity), and serum from BTV-Y TUN2017 infected sheep failed to neutralize other BTV serotypes, suggesting that it is a novel serotype.

As the small ruminant- adapted BTVs cause subclinical or very mild infections and their presence currently does not lead to restrictions on animal movement or trade (like those associated with the presence of BTV 1 – 24), these viruses currently are not problematic to the livestock industries of countries in which they are endemic. However, the biological characteristics of novel and classical BTV serotypes are remarkably different,^{29, 281} and reassortment events between them could potentially result in progeny viruses with different properties such as the ability for contact transmission. Reassortment between classical and novel serotypes has been demonstrated to occur in vitro but has not been shown to date in animals or nature.^{232, 254, 321}

Pathogenesis

The pathogenesis of BT reflects virus tropism and both direct cytopathic effect of the virus on vascular endothelium as well as the activities of proinflammatory and cytokine mediators produced by infected cells.  Bluetongue virus first multiplies in the regional lymph nodes after virus inoculation by the bite of a midge before spreading to the rest of the body.^{22, 23, 24, 248} Viral replication occurs primarily in dendritic cells, macrophages and endothelial cells lining capillaries and small blood vessels.^{130, 165, 187, 216, 296, 336} Cytopathic changes in endothelial cells include degenerative and necrotic lesions such as cytoplasmic vesicles, nuclear and cytoplasmic hypertrophy, pyknosis and karyorrhexis. These changes, often accompanied by hyperplasia of the endothelium, lead to vascular occlusion, stasis and exudation, which eventually gives rise to hypoxia, oedema and haemorrhage as well as to secondary lesions in the overlying epithelium.^{165, 296}

In a study of BTV infection in white-tailed deer it was concluded that viral-induced endothelial damage is the primary triggering mechanism for the disseminated intravascular coagulation that is responsible for the haemorrhagic diathesis characteristic of the disease in these animals.¹³⁰ However, similar studies in sheep confirmed marked coagulopathy but without occurrence of disseminated intravascular coagulation, suggesting that the vascular injury in fulminant BT of sheep is analogous to that which occurs in human viral haemorrhagic fevers such as Ebola.¹⁸⁶ Specifically, in these diseases, rather than direct virus-mediated endothelial injury, cytokine mediators released from virus-infected dendritic cells and macrophages cause endothelial cell retraction that results in vascular leakage and hypovolemic shock.^{70, 71, 72, 75, 76, 132, 186, 187, 271, 276}

The severity of the secondary lesions in BTV-infected sheep is influenced by mechanical stress and abrasion, and severe lesions develop mainly in tissues exposed to the environment, such as the oral mucosa and skin of the coronary border of the hooves. There is probably a correlation between the distribution of the lesions and temperature gradients within the host because the most severe lesions occur in areas where the temperature is lower than that of circulating blood.²⁹⁶ There is also evidence for a highly selective involvement of endothelial cells in certain blood vessels (such as the pulmonary artery and those where the most severe lesions are found), the mechanism of which is not understood although it is abundantly clear from in vitro studies that endothelial cells from different levels of the pulmonary vascular system differ in their response to BTV infection. Exposure of affected animals to sunlight exacerbates the severity of the disease. The mortality rate can escalate dramatically when infected sheep are exposed to cold, wet conditions as often happens in late autumn.⁹⁰

The innate immune response to BTV infection begins at the site of virus deposition in skin via the bite of an infected midge, and it is now clear that components of midge saliva impact this initial response.²⁴⁰ Infection of ruminants with most (but not all) serotypes of BTV leads to a highly blood cell-associated viraemia that may be prolonged but not persistent. Recovered animals are resistant to reinfection with the homologous virus serotype, which is the basis for vaccination strategies to prevent BTV infection and clinical disease in domestic livestock. Replication of BTV in target cells, notably mononuclear phagocytic cells (dendritic cells and macrophages) and endothelium, leads to the generation of the innate and adaptive immune responses that mediate both initial virus clearance and subsequent resistance to infection with the homologous virus serotype.^{53, 57, 73, 188, 262, 271, 330} The Type 1 interferon response is induced soon after infection, followed by both humoral and cellular immune responses.  The non-structural NS3 and NS4 proteins of BTV have been identified as suppressors and/or agonists of the Type I interferon response.^{58, 258} Furthermore, there is recent evidence that BTV infection of follicular dendritic cells compromises transiently the host’s ability to develop an effective adaptive immune response.²¹⁶

Following initial replication of the virus in lymphoid tissues and endothelial cells, BTV appears in the circulation three to six days after infection. The viraemia reaches a peak about seven to eight days after infection and accompanies or precedes a febrile reaction that normally lasts about four to eight days. In sheep the viraemia rarely persists for longer than 14 days^{7, 8, 36} and is usually present for six to eight days. Results that conflict with this statement were reported by early workers, but can probably be explained by natural reinfections with different serotypes that were unknown at that time. Biphasic viraemia has been observed in BT-infected sheep with viraemic peaks at day 5 (±1) and day 10 (±2). It was attributed to the induction of high concentrations of interferon concurrent with the first viral peak, followed by production of antibodies and a subsequent reduction in viraemia at the second peak.^{99, 189, 243}

Despite a robust immune response, viraemia in BTV-infected cattle is often prolonged for several weeks^{23, 36, 191, 192} and RNA-aemia is even more extended (several months). It was found that in vitro infection of bovine erythrocytes and non-replicating lymphocytes did not progress beyond adsorption after which virus particles persisted in invaginations of the cell membrane. This could explain both the prolonged viraemia and the absence of clinical disease.^{22, 46} It has also been demonstrated that infected ovine endothelial cells bind more BTV initially and produce more virus than their bovine counterpart,²⁷² although this finding was not reproduced in more extensive studies with endothelial cells cultured from different levels of the pulmonary vasculature of sheep and cattle.

Bluetongue virus in the blood is primarily associated with erythrocytes and, to a lesser extent and particularly during acute infection, with the buffy-coat fraction,^{122, 172} while only a small fraction is found free in the plasma.^{22, 23, 63, 165} Panleukopenia preceding the viraemia and a febrile reaction is consistently found in BT.¹⁶⁵ Changes in the packed cell volume and the concentration of aspartate transaminase and creatinase in the plasma have also been observed.⁹⁶

Experimental infection of dogs was only successful in pregnant bitches, leading to abortions and death from severe pulmonary oedema. Viral nucleic acid was mainly detected in lung endothelial cells.⁴⁸

In cattle there is evidence of a rapid accumulation of IgE antibodies directed against the virus after infection, which may result in immediate hypersensitivity reactions involving the release of mediators such as histamine, prostaglandins and thromboxane A2^{11, 85} However, the importance of this phenomenon in disease expression in cattle naturally infected with BTV remains uncertain as cattle infected with especially virulent strains of the virus, such as European BTV-8, may develop clinical signs similar to those in sheep.^{64, 82, 196, 346}

Abortion or malformation, particularly of the central nervous system (CNS), of lambs may follow vaccination of ewes with attenuated live vaccines during the first half of pregnancy.^{114, 237, 260, 282} Similarly, infection of bovine foetuses during the first trimester of pregnancy with laboratory-modified strains of BTV results in severe teratogenic defects in the central nervous system.^{20, 176, 179, 191, 193, 194, 261} The natural occurrence of similar congenital teratogenic defects in the progeny of unvaccinated animals was rare until the emergence of BTV serotype 8 in Northern Europe in 2006, when large numbers of ruminants were born with these same defects in the central nervous system (e.g. hydranencephaly) (Figures 4a and b) following infection of their dams in early gestation.^{196, 325, 343}

Figure 4a Hydranencephaly in a calf experimentally infected with modified bluetongue vaccine virus. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 4b Hydranencephaly in a calf following infection of the dam with bluetongue virus. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Clinical signs

The extreme variability in the clinical manifestation of BT, not only between different ruminant species but also between different breeds of sheep, is a feature of the disease. Individual idiosyncrasies and environmental conditions affect the severity of the disease, as does the virulence of the infecting virus strain. Many infections in sheep are clinically inapparent and, even in fully susceptible animals, it is difficult to reproduce severe clinical signs under laboratory conditions where there is limited exposure to sunlight or ultraviolet light.

The course of the disease in sheep can vary from peracute to chronic, with a mortality rate of between 2 and 30 per cent. Peracute cases usually die within seven to nine days of infection, mainly as a result of lung oedema and eventual asphyxia. They may show very few signs of illness prior to death. In chronic cases death can result from secondary bacterial pneumonia and exhaustion, or recovery can be very protracted. Mild cases usually recover rapidly and completely.

The incubation period, following natural infection, is about seven days (judging by the length of the period during which clinical cases continue to appear after frost). Following experimental infection the incubation period is usually four to six days but may vary between 2 and 15 days. The first clinical sign is a rise in temperature over a period of 48 hours, reaching a peak of 41 to 42 °C. The febrile reaction usually lasts about six to eight days and its termination is determined by the course of the disease and the extent of secondary infection. Generally there is a fair correlation between the duration of the fever and the severity of the disease.⁹⁰

Other clinical signs may appear within one or two days of the onset of fever. Hyperaemia of the buccal and nasal mucosae usually occurs first and may also develop on more exposed parts of the skin around the muzzle and eyes and on the ears (Figure 5) increased salivation and lachrymation as well as a serous nasal discharge may soon follow. Licking movements of the tongue and smacking movements of the lips may result in froth formation at the corners of the lips.

Oedema of the tongue, lips, face, eyelids and ears develop about 48 hours after the onset of fever. Submandibular oedema (Figure 6) may be marked and occasionally extends down the neck to the axillae. Petechiae may be observed on the muzzle, papillae of the lips and mucous membranes of the mouth and conjunctivae. At this stage mild cases may recover uneventfully.

Figure 5 Hyperaemia of the exposed parts of the skin of the face. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 6 Severe oedema of the face, particularly of the submandibular area. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

In severe cases the lesions progress and result in excoriations and erosions on the muzzle and nostrils and in the mouth (Figures 7 to 11) particularly at sites subject to friction, such as parts of the cheeks and tongue adjacent to the molar teeth and the lower lip opposite the corner incisors (Figures 12, 13a to d). Necrotizing mouth lesions result in foetid breath, which is sometimes the first clinical sign observed. They also induce pain, which may cause the animal to submerge its mouth and lips in drinking water for prolonged periods. Anorexia is common and may be exacerbated in some cases by severe swelling of the tongue, which may become cyanotic (‘blue tongue’) (Figure 14) and even protrude from the mouth. Progressive weakness and emaciation follows, accompanied by rumen stasis and occasionally haemorrhagic diarrhoea before death.

The watery nasal discharge frequently becomes mucopurulent and eventually forms crusts that interfere with breathing and cause panting. In peracute cases, severe oedema of the lungs leads to dyspnoea, frothing from the nostrils (Figure 15) and death by asphyxiation.

Figure 7 Hyperaemia, haemorrhages and erosions on the muzzle and nostrils. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 8 Haemorrhages and erosions at the mucocutaneous junction of the upper lip. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 9 Severe erosions, haemorrhage and congestion of the muzzle. Note oedema and hyperaemia of the lips. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 10 Severe ulcerative stomatitis and gingivitis in a sheep. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 11 Severe necrotizing gingivitis and stomatitis. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 12 Severe swelling and protrusion of the tongue. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 13a Multifocal erosive glossitis and oedema of the tongue in a sheep. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 13b Severe swelling of the tongue. Note numerous petechial haemorrhages and ulceration at the base of the tongue. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 13c Ulcerative glossitis, particularly adjacent to molars. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 13d Secondary infection of the ulcerative legion at the base of the tongue. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 14 Cyanosis of the mucosa of the mouth and tongue. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 15 Frothy discharge from the nose as a result of severe lung oedema. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Foot lesions usually develop towards the end of the febrile reaction. Hyperaemia of the coronary bands and petechiae under the periople, which later become streaky in appearance as a result of haemorrhage into the fine medullary canals of the growing horn, give rise to a red zone or band in the horn of the hoof (Figure 16). As this lesion persists for some weeks after others have disappeared, it may constitute valuable evidence of BT during the recovery period. The lesion is most pronounced on the bulbs of the feet, and particularly of the lateral digits. The hind feet are most frequently affected. The feet are warm and painful, and affected animals are reluctant to move and often stand with arched backs or are recumbent. In affected sheep the gait is often stiff with varying degrees of lameness. Sometimes severely affected animals try to walk on their knees. In animals that recover, the bands of discoloration in the hooves grow out and a ‘break’ in the hoof (Figure 17) may develop (‘slipper formation’), with the old horn eventually sloughing off after three to four months. Inability to move and recumbency may also be exacerbated by emaciation and muscle lesions. Severe muscle degeneration and cachexia are sometimes seen in cases where buccal lesions are mild and the appetite undiminished.⁹¹ Degeneration and necrosis of skeletal muscles in the neck may lead to torticollis (Figure 18). Hyperaemia of the skin is usually most severe in those areas that are exposed to sunlight (such as, in Merino sheep, the face, ears and legs, which are not covered by wool), but may involve the whole body (Figures 19 to 21). In severely affected sheep, exanthema develops on the legs and other parts of the body subject to trauma. In such instances the slightest abrasion or handling may immediately result in extensive cutaneous haemorrhage, particularly in the groin and axilla (Figures 20 and 21). In most affected animals the wool fibres ‘break’ within the wool follicle (Figures 22 and 23) and the fleece may be shed three to six weeks later.^{90, 126} Normal new wool soon appears in recovered animals.

Figure 16 Acute bluetongue: hyperaemia and petechiation of coronary band and adjacent hoof. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 17 A ‘break’ in the hoof of a recovered animal. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 18 Torticollis (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 19 Severe subcutaneous hyperaemia. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 20 Severe hyperaemia of the skin. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 21 Severe hyperaemia and haemorrhage in the skin in the inguinal region of a sheep. (Courtesy of Drs Massimo Scacchia and Giovanni Savini, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise "G.Caporale" Via Campo Boario, 64100 Teramo, Italia)

Figure 22 Recovery stage of bluetongue. Note break in the wool. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 23 Recovery stage of bluetongue. Note break in the wool. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Clinical signs in cattle are rare and are usually limited to a transient febrile response, increased respiratory rate, increased lachrymation and salivation (Figure 24), stiffness and inflammatory changes in the skin.^{11, 155, 177, 178} Calves that were infected experimentally remained healthy throughout the experiment.¹⁸⁹ In contrast, some cattle infected with BTV-8 during the recent European epidemic developed signs similar to those in BTV-infected sheep, with oral erosions/ulcers, conjunctivitis, nasal discharge, coronitis, and lesions of the mammary glands.^{64, 82, 196, 346}

Recently, fatal cases of BT of camelids have been described in both Europe and the Americas. Affected llamas and alpacas developed a peracute disease course and quickly succumbed to fulminant pulmonary oedema, with few other signs or lesions at post-mortem.^{103, 222, 234}

Figure 24 Catarrhal nasal discharge and salivation in a bovine with bluetongue. (Courtesy of Prof Christian Griot, Institut für Virologie und Immunologie IVI, In Kooperation mit der Vetsuisse-Fakultät, Universität Bern, Germany)

Pathology

The clinical pathology of BTV infection has been studied and characterized in white-tailed deer,¹³¹ and progressive prolongation of activated partial thromboplastin time and prothrombin time as well as progressive reduction of Factors VIII and XII plasma activities were observed. Haematologic changes included leukopenia, lymphopenia, and neutrophilia and low total plasma protein concentration. Mild to severe increases in serum aspartate transaminase activity were accompanied by more marked increases in creatinine kinase activity. These changes were associated with the development of disseminated intravascular coagulation, which was regarded as being important in the development of haemorrhagic diathesis — a characteristic of BT in white-tailed deer. Interestingly, however, disseminated intravascular coagulation did not occur in sheep inoculated with a highly virulent South African strain of BTV- 4, although the inoculated sheep did have marked coagulopathy and developed severe and fatal disease.¹⁸⁶ Indeed, there is compelling evidence that the pathogenesis of fulminant BT in domestic livestock is typical of viral haemorrhagic fevers in that it reflects cytokine-mediated vascular leakage and hypovolemic shock, with or without accompanying disseminated intravascular coagulation.¹⁸⁷ Many of the gross pathological changes, described in detail by early workers such as Spreull,²⁹⁴ Theiler,³¹² Thomas and Neitz,³¹⁵ and Moulton,²²³ correspond largely with the clinical signs.

Oral lesions initially consist of hyperaemia, oedema, cyanosis and haemorrhage of the mucous membrane (Figure 14). Destruction of epithelial cells gives rise to excoriations and erosions on the inside of the lips, dental pad, cheeks and tongue (Figures 10 to 14). Many of these lesions are transient and usually disappear within days. Secondary bacterial infection is often responsible for the diphtheric necrosis of the erosions.^{28, 221} Microscopically, there is mononuclear cell infiltration and degeneration and necrosis of epithelial cells in which large acidophilic intracytoplasmic masses accumulate.

Hyperaemia, petechiation and erosions of the mucosa of the forestomachs, particularly of the papillae, rumenal pillars, reticular folds and oesophageal groove are common (Figures 25 and 26). Oedema and haemorrhages are often present in the submucosa, especially in the vicinity of the pylorus and the anterior third of the omasum. Lesions in the small intestines vary from mild localized areas of hyperaemia to severe catarrhal or haemorrhagic lesions extending into the large intestine.⁹⁰

Figure 25 Necrosis, congestion and haemorrhage of the mucosa of the oesophageal groove and parts of the omasal folds. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 26 Multifocal necrosis and haemorrhage of the ruminal pillars in a sheep with acute bluetongue. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Hyperaemia, oedema and petechiae occur in the mucosae of the nasal cavity, pharynx and trachea, as well as in the lungs. Severe hyperaemia and oedema of the lungs accompanied by copious amounts of froth in the trachea and hydrothorax occur, especially in acute fatal cases. Aspiration pneumonia may occasionally be found.¹⁷⁴

The widespread, but often localized, occurrence of hyperaemia, oedema and haemorrhages is evidence of damage to the vascular system, likely as a consequence of the activity of proinflammatory and vasoactive mediators in conjunction with virus infection of endothelium.^{72, 75, 132, 186, 187, 276} Petechiae, ecchymoses or even larger haemorrhages in the tunica media of the pulmonary artery near its base are characteristic of BT (Figures 27 and 28). These lesions are sometimes described as being pathognomonic for BT, but they have also been seen on rare occasions in other infections such as Rift Valley fever, heartwater and pulpy kidney disease in sheep. These haemorrhages may be difficult to see and it may be necessary to stretch the pulmonary artery and to hold it against the light. Epicardial and endocardial haemorrhages (Figure 29), as well as focal necrosis (c. 0,5 to 1,0 mm in diameter) of the papillary muscle are sometimes found in the left ventricle. Hydropericardium may be present (Figure 29), as may accumulation of oedema fluid around the nuchal ligament and in the fascial planes of the abdominal musculature (Figure 30).

Figure 27 Haemorrhages in the wall of the pulmonary artery. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 28 Haemorrhages in the wall of the pulmonary artery as seen from the inside of the artery. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 29 Small haemorrhages in the epicardium and hydropericardium containing a fibrin clot. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Pharyngeal, cervical and thoracic lymph nodes are commonly swollen and oedematous and the spleen may be slightly enlarged with subcapsular haemorrhages.

From an economic point of view, in addition to loss of wool, the pathological changes in the skeletal musculature are probably the most important lesion in BT, as they are usually associated with gross loss of condition, weakness, and a slow, protracted recovery period. Macroscopically, the muscle lesions comprise petechiae, ecchymoses and gelatinous oedema of the intermuscular connective tissues (Figures 30 and 31), particularly in the neck and the dorsal thoracic region.

Small localized areas of degeneration and necrosis appear as greyish-white foci. Extensive degeneration and necrosis involving entire muscles is occasionally seen (Figures 32 and 33). Microscopically, hyaline degeneration, necrosis and mineralization of individual muscle fibres occur together with intermuscular oedema and haemorrhage. Infiltration by neutrophils, macrophages and lymphocytes as well as evidence of regeneration of muscle fibres are present in the lesions of acute cases. In sheep that develop torticollis, the damage is most extensive on the side of the neck towards which the head is turned. At one time it was held that fibrous replacement was the cause of torticollis³¹⁵ but more recent observations suggest that torticollis occurs in some cases in which there is no evidence of fibrosis.⁶²

Figure 30 Intermuscular oedema of dorsal thoracic region. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 31 Multifocal haemorrhages in skeletal muscles in the shoulder region of a sheep. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 32 Subacute bluetongue in a sheep. Greyish-white discolouration of the shoulder muscles as a result of extensive degeneration and necrosis. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Figure 33 Subacute to chronic bluetongue in a sheep. Note greyish-white discolouration of the muscles in the back as result of degeneration, necrosis and mineralisation. (Courtesy of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa)

Diagnosis

A presumptive diagnosis of BT based on clinical signs, lesions and epidemiological assessment requires laboratory confirmation.¹ Rapid detection, reliable identification and serotyping of BTV are all crucial in designing and implementing appropriate control measures, and to limiting the spread of infection.

Samples to be examined in the laboratory for virus detection should include blood (10 to 20 ml) collected as early as possible during the febrile reaction in anticoagulants such as heparin, sodium citrate, EDTA or OCG;⁹⁰ in fatal cases, specimens of spleen, lymph node, and lung are useful for virus detection, and from foetuses, specimens of brain and spleen. These tissues should be collected as soon as possible after death and kept at 4°C.²⁴³ For transport, these samples should be kept on ice. Whole blood samples can be stored at 4°C for a long time. Red blood cells washed and resuspended in phosphate- buffered saline and stored at 4°C can be used for virus detection.⁹ Alternatively they may be frozen at −70°C in 10% dimethyl sulphoxide for storage prior to testing.

Various techniques have been used to detect the presence of infectious virus, viral antigen, or viral RNA in infected samples. The most commonly used are: real time RT-PCR, conventional RT-PCR with agarose gel electrophoresis (to identify specific amplicons), capture ELISA, viral isolation from embryonated chicken eggs (ECE),  and viral isolation from insect (e.g. Kc and C6/36 cells derived from Culicoides sonorensis and Aedes albopictus, respectively) and/or mammal cells. Traditional RT-PCR and real time RT-PCR assays provide a versatile system able to give information on virus serogroup and serotype within a few hours. They are also highly sensitive and capable of detecting very low concentrations of viral RNA. In addition, the real time PCR assay is able to quantify the viral genome. Among the several existing RT-PCRs, real time RT-PCR assays based on detection and quantitation of the conserved and group-specific S10 gene (which encodes NS3) are now the most widely used¹⁴ as they are able to detect both traditional and small ruminant-adapted BTVs. Blood samples positive by this assay then can be subjected to serotype-specific real time RT-PCR assays to determine the specific virus serotype(s) involved.^{181, 205} Due to all these advantages, in most laboratories these new techniques are preferred to classical viral detection techniques, which require three to four weeks to be completed. However, RT-PCRs are unable to distinguish whether the RNA detected in the animal is contained in an infectious virus or merely RNA from a degraded particle no longer able to infect the vector. In other words, the mere identification of BTV nucleic acid by real time RT-PCR is not proof of disease causality, as BTV nucleic acid persists in the blood (RNA-aemia) and tissues (e.g. spleen) of infected animals for several months, long after clearance of infectious virus.^{74, 191, 192}

The classical virus isolation techniques (inoculation of ECE or mammalian or insect cells) remain the only viable method to confirm the presence of infectious virus in an animal.

Bluetongue virus can most readily be isolated by the intravascular inoculation of 10- to 12-day-old ECE or by infection of susceptible cell cultures with processed blood or clarified tissue suspensions.⁹⁷ Primary isolation can only be achieved with difficulty by intracerebral inoculation of new-born mice or in monolayer cell cultures (BHK-21, mouse L-cells, Vero, Kc or C6/36 cells). However, after a single passage in ECE or insect cells (Kc) the virus can readily be cultivated in mouse brain or in a variety of cell cultures.

Isolated BTV is now typically characterized by sequence analysis, increasingly by whole genome sequence analyses^{17, 29, 43, 123, 169, 182, 183, 231, 236, 281, 283, 347} that can also facilitate analyses to map the origins, movement and spread of individual virus strains. In the past, techniques like complement fixation, ELISA and immunofluorescence or immunoperoxidase staining that detect group-specific antigens were used to distinguish BTV from other orbiviruses.^{33, 34}

Several techniques can be used to detect the presence of BTV-specific antibodies in either infected or vaccinated animals. The methods to detect serogroup- and serotype-specific antibodies in serum and milk samples of infected or vaccinated animals most commonly used are: competitive ELISA (c-ELISA), milk ELISA and serum-virus neutralisation (SN). Previously, a complement fixation test using antigen prepared from infected mouse brains^{160, 318} was used, and this was later replaced by the gel immunodiffusion test.²⁴⁵ Cross-reactions between different orbiviruses were a significant problem with these early serological techniques, but less so with more recently developed c-ELISA.⁵² The OIE has prescribed the c-ELISA test for serological diagnosis of BTV infection for international trade.¹⁴ Over time, the accuracy of this assay has been progressively improved through the use of antigens obtained from recombinant structural proteins of BTV. The c-ELISA incorporates a monoclonal antibody to an epitope on the core protein VP7 that is common to all BTV serotypes; thus, the assay detects the presence of antibodies to all BTV serotypes. The technique is highly sensitive and specific as the monoclonal antibody used detects an epitope common to BTV serogroup viruses that is not present in other orbiviruses. The test is relatively inexpensive, commercially available, and the preferred diagnostic assay. However, the available c-ELISA tests currently do not discriminate BTV-infected from vaccinated animals (DIVA).

Milk ELISAs are successfully used to evaluate BTV-infection in a herd.  Since these tests are applied on bulk milk samples, they have the advantage of testing groups of animals in one step.¹⁶³ Its specificity was recently improved by precipitating the milk protein prior to carrying out the ELISA.⁵⁶

As c-ELISA does not identify which BTV serotype induced virus-specific antibodies in any given animal, other assays are required to determine serotype-specificity. Type-specific antibodies are best detected by means of virus neutralization tests, such as SN, plaque reduction, or by haemagglutination inhibition tests.¹⁷³ However, because of cross-reactions between serotypes and heterotypic antibody responses, the significance of type-specific antibody titres in animals exposed to multiple serotypes may be difficult to interpret and require skilled personnel.

Differential diagnosis

In southern Africa, the clinical signs of acute BT in sheep should be differentiated from early signs of hepatogenous photosensitivity caused by a variety of plant and mycotoxin poisonings.¹⁵⁹ In areas where the disease does not normally occur, it should be differentiated from vesicular diseases such as foot-and-mouth disease. The clinical signs could also be confused potentially with those seen in polyarthritis, foot rot and white muscle disease, orf (contagious pustular dermatitis), acute haemonchosis or liver fluke (with depression and submandibular oedema), peste des petits ruminants, laminitis and clostridial diseases.³³⁸ In sheep that die after the typical lesions of BT (oedema of the lips and tongue, and mouth and tongue erosions) have regressed, it may be difficult to differentiate BT from diseases such as heartwater and pulpy kidney disease.³¹⁷

In cattle, clinical BT can easily be confused with clinical signs and lesions of epizootic haemorrhagic disease of deer and Ibaraki disease. In this species differential diagnosis should also include malignant catarrhal fever, pododermatitis, infectious bovine rhinotracheitis, bovine viral diarrhoea, bovine papular stomatitis, bovine herpesvirus 2 infection (bovine herpes mammillitis) and parainfluenza-3 infection.^{210, 279, 338}

The teratological defects (eg. hydranencephaly and porencephaly) of the central nervous system of the foetus in sheep and cattle caused by European BTV-8 and modified BTV strains are similar to the defects caused by teratogenic bunyaviruses (Aino, Akabane, Cache Valley, Schmallenberg viruses) as well as Wesselsbron disease and palyam viruses.^{187, 193, 194}

Control

Strategies for the control of BT differ according to whether outbreaks of the disease occur in endemic regions or in areas where the disease is not usually present.¹⁹⁰ In the latter case the usual goal is eradication, whereas in endemic areas attempts can only be made to limit the occurrence of the disease and its economic impact. However, it is increasingly obvious that eradication of a vector-borne disease like BT is unrealistic, thus control of outbreaks, if attempted, is usually based on animal movement restrictions and vaccination. Ongoing surveillance is required to monitor the effectiveness of any control programme. In any attempt to control arthropod-borne diseases three factors should be taken into account: possible virus reservoirs, the vector, and the target animal. Where possible control actions should be integrated⁸⁸ and directed towards protecting animals from insect attack and/or BTV infection.

Vector control

Bluetongue virus has now been isolated from many species of Culicoides, and the Culicoides species involved vary by region of the world.^{80, 105, 108, 153, 185, 211, 301, 309, 339} Elimination of Culicoides midges from the environment is generally impractical, particularly in extensive pastoral settings. Air-borne transport of infected Culicoides midges over long distances cannot be prevented and is likely to occur. The important determinant of virus spread is whether or not the midges resident in any invaded area can then acquire, spread and maintain the incursive BTV – clearly this was the case during the recent epidemic in Europe where strains of BTV vectored by C. imicola in Asia and Africa were introduced into Europe and then successfully transmitted by native C. imicola and species of midges within the Obsoletus complex (involving at least C. obsoletus and C. scoticus).

Control of Culicoides populations, or, alternatively, protection of target animals from contact with them, can make a significant contribution towards control of the disease. Methods used to control Culicoides include the use of insecticides, larvicides, and sterilization of males by irradiation,¹²⁴ as well as the subcutaneous administration of ivermectin to cattle in an attempt to break the primary cycle before the infection spills over into sheep.²⁹⁷ Protecting sheep from exposure to Culicoides is a more practical approach and can be achieved by avoiding low-lying wet pastures, shearing in early summer to allow some wool growth before the onset of the BT season in late summer, and even the use of insect repellents.⁸⁸ Screening of access points (eg. windows, doors) of stables using fine mesh capable of excluding midges, or coarser mesh impregnated with a suitable insecticide such as a synthetic pyrethroid can be used to further reduce the attack rate.²¹⁵ Keeping cattle in close proximity to sheep is also effective in some areas, apparently because C. imicola, the most prevalent vector in South Africa, has a preference for cattle.²²⁹ The overall efficacy of many of these strategies is uncertain as the endo/exophagy behaviour and circadian host-seeking activity vary remarkably between individual midge species. Repellents are difficult to apply and typically have only a short duration of efficacy.¹⁹⁰

Immunization

Vaccination remains the most effective and practical control measure against BT in endemic regions, and also to control BTV incursions into previously BT-free areas. Effective vaccination requires products that rapidly induce protection against the specific virus serotype or serotypes, especially in incursions. Vaccination of livestock in endemic areas should induce long- lasting and broad protection against all relevant virus serotypes.  The main objectives of BT vaccination strategies are to (i) prevent clinical disease, (ii) reduce the spread of BTV, (iii) eradicate BT from the country or the region, and (iv) permit the safe movement of susceptible animals between BT-affected and BT-free zones.

In endemic African regions,¹⁰⁵ a wide variety of domestic and wild ruminants (including cattle, goats and various species of antelope) can act as sources of BTV. Immunization of cattle should therefore theoretically help to limit the incidence of BT, but the presence and ubiquitous distribution of other reservoirs, both known and unknown, makes eradication of the disease impossible.

After recovery from natural infection, animals have a solid, life-long immunity to the homologous serotype but only partial or no protection against heterologous types. Protective immunity is generally associated with the presence of type-specific neutralizing antibodies that may persist for years, but it is not associated with the group-specific antibodies that are detected in standard serological tests (e.g. c ELISA).  However, infection or immunization with more than one virus type usually results in protection against a wider range of serotypes, even types against which no neutralizing antibodies are present. This suggests that cellular immunity may contribute as it is less type-specific than is the humoral response.^{151, 152, 154}

A wide variety of vaccine types have been developed for prophylactic immunization of ungulates against BTV, including inactivated, live-attenuated, recombinant and a variety of engineered molecular constructs.^{50, 55, 94, 190, 191, 208, 230} Currently, live-attenuated and inactivated constructs remain the only BTV vaccines produced commercially. These inactivated and live-attenuated vaccines are available in only some regions of the world.

Vaccines comprised of attenuated virus strains are highly effective, especially in epidemic situations where only one serotype of BTV is involved. Historically, vaccine strains were attenuated by serial passage in embryonated chicken eggs. These vaccine strains, now grown in BHK-21 cells, have been in use for 40 years.¹²⁶ In endemic areas such as South Africa, the situation is complicated by the existence of multiple serotypes. This necessitates the use of polyvalent vaccines with attendant problems resulting from interference between virus strains, differences in immunogenicity and growth rates between various strains, as well as differences in the response of individual animals to the components of such vaccines. To overcome these problems, three pentavalent vaccines have been developed in South Africa. They are administered to sheep at three-week intervals and repeated annually. Although 21 BTV serotypes have so far been shown to occur in South Africa (types 17, 20 and 21 have not yet been isolated) only 15 serotypes are included in the present vaccine. Types 15, 16, 18, 22, 23 and 24 are not included because of their low pathogenicity for sheep and low prevalence in Culicoides.

It is anticipated that types 16 and 24 may be included in future on the basis of their increased prevalence, whilst type 7 may be omitted because of its low pathogenicity for sheep. After two or three annual immunizations, most sheep are immune to all the serotypes in the vaccine. Experience in the field has shown that the three pentavalent vaccines are far more effective when administered properly than are previous BT vaccines, which at times contained as many as 14 serotypes in a single dose.

In addition to the inconvenience of repetitive inoculations of a multivalent vaccine there are some risks involved in the use of live attenuated vaccines and some important precautions must be observed when using them. Pregnant ewes should not be immunized during the first half of pregnancy, as brain defects may result in the foetus.⁹⁵ Secondly, because lambs born to immune ewes usually acquire effective colostral immunity that may last up to six months,¹²⁶ during which time lambs are refractory to immunization, losses from BT may occur in spring-born lambs during the following autumn. There are also indications of temporary infertility in both ewes and rams vaccinated for the first time, necessitating immunization well before or after the mating season.⁸ However, this should not pose a problem if sheep are vaccinated before they reach breeding age.

It is increasingly clear that there are serious potential complications to the use of live attenuated vaccines. It is now well established that these viruses can be acquired and spread by insect vectors and reassort their genes with other vaccine or field viruses to create genetically novel reassortants with potentially deleterious properties (e.g. reversion to virulence, transplacental transmission etc.).^{208, 231, 233, 236, 279} In turn, these reassortant viruses are then spread by vector midges in the same manner as authentic “field strains” of the virus.  The fact that animals vaccinated with live vaccines cannot be distinguished serologically from those recovered from disease poses a real problem in the international regulation of animal movement for those countries normally free from the disease.

Inactivated vaccines also suffer from the fact that vaccinated animals cannot be distinguished serologically from naturally infected animals (DIVA). However, inactivated vaccines are safe and if produced properly, they can be highly efficacious, as shown during the recent European epidemic.¹⁶ Their inherent potential disadvantages include high costs of production as vaccination requires large amounts of antigen, and the need for booster immunizations as inactivated vaccines generally induce a relatively transient immunity.

To overcome these problems, much research on new generation vaccines has been carried out during recent years aimed at the development of effective inactivated or subunit vaccines, or of constructing recombinant viruses that would combine the advantages of live vaccines with the safety of inactivated products.^{55, 94, 190, 191, 208, 230, 320}

The demonstration that VP2 can be isolated and used to induce protective immunity¹⁴⁰ paved the way for various studies in which the gene (segment 2) encoding VP2 was cloned, sequenced and expressed using a variety of vectors.^{138, 146} Again, the problem of producing sufficient quantities of the protein precluded its use as a subunit vaccine. In addition, it was soon discovered that other viral proteins also played a role in eliciting a protective immune response. A variety of recombinant vaccines have been produced and partially characterized, although none have yet been developed commercially. These include baculovirus expressed virus-like particles, recombinant VP2 expressed in plants and by various heterologous expression vectors including pox and adeno viruses, and various genetically modified or deleted BTVs that are replication defective.

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