The immunisation properties of an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were evaluated in sheep and cattle. Five sheep were vaccinated with one dose of vaccine and five cattle were vaccinated with two doses 28 days apart. Six sheep and five cattle served as unvaccinated controls. All animals were subjected to a virulent BTV-8 challenge, and safety and antibody responses were monitored. All control animals developed disease and viraemia, while vaccinated animals were clinically protected and viraemia was completely prevented.
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BLUETONGUE virus (BTV) is an arbovirus and a member of the Orbivirus genus, family Reoviridae (Murphy and others 1971, Taylor 1986). It is spread by biting midges of the Culicoides genus (Mellor and others 2000). BTV is the causative agent of bluetongue disease, which can affect all ruminants, although the disease is normally most evident in sheep (Verwoerd and Erasmus 2004). There are 24 different serotypes, BTV-1 to BTV-24 (Mertens and others 1984, 2009), with little crossreactivity between them. Consequently, a different vaccine is required for each serotype.
Bluetongue was originally described in southern Africa (Spreull 1905), but since 1998 several BTV serotypes have spread into southern Europe (Baylis and others 2001, Mellor and Wittmann 2002, Calistri and Caporale 2003) and circulated in the Mediterranean area. In 2006, BTV-8 emerged in northern Europe and has spread remarkably rapidly throughout Europe (van Wuijckhuise and others 2006, Wilson and others 2007, Saegerman and others 2008, Gloster and others 2008). It is speculated that the BTV-8 epizootic was due to a new introduction of the virus, possibly from Africa (Darpel and others 2007, European Food Safety Authority [EFSA] 2007). Without having been previously exposed to BTV, all ruminant populations of northern Europe were sensitive to infection. During 2007 the disease recurred and large numbers of BTV-8 cases were reported (European Commission 2007a, 2008, Szmaragd and others 2007). It now appears that some Culicoides species indigenous to northern Europe are highly competent vectors (Gerbier and others 2006, Gomulski and others 2006, Meiswinkel and others 2007) for BTV. This implies that BTV-8 (and perhaps other BTV serotypes) is adapted to 'northern' Culicoides vectors and that it can potentially spread throughout the northern hemisphere. The economic significance associated with bluetongue infection should not be underestimated as it can cause death of animals and affect production over large geographical areas. In addition, international restrictions on the trade of animals and animal products from infected areas (European Commission 2007a, c, 2008) have the potential to cause significant economic losses for livestock owners and national economies.
When BTV was introduced into southern Europe in 1998, no vaccine was available in the region. Until 2003, live attenuated vaccines from South Africa were used to control the spread of BTV disease (Savini and others 2009). Because of the risks associated with the use of live vaccines, which include transmission among non-vaccinated ruminants, reversion to virulence or reassortment of the genes of the attenuated vaccine virus with wild-type virus (Venter and others 2004, Ferrari and others 2005, Monaco and others 2006), research into safer vaccines began quickly. There are currently a number of approaches aimed at developing safer vaccines, using recombinant technology to produce virus-like particles, recombined vaccinia virus (Huismans and others 1987, Roy and others 1990, Lobato and others 1997) or canarypox vector systems (Boone and others 2007). However, the quickest and most straightforward approach in the face of the epidemic was to develop an inactivated vaccine. Since 2005, a number of inactivated BTV vaccines have been developed and employed successfully in many countries in Europe (Savini and others 2008). In this paper, the safety and efficacy of an inactivated, adjuvanted BTV-8 vaccine (BTVPUR Alsap 8; Merial) in sheep and cattle are reported.
Materials and methods
The BTV-8 strain was isolated from a sick sheep in the Ardennes region of France in 2006 and used to prepare a controlled master seed on BHK-21 cells, after embryonated chicken egg passages and cell adaptation. The virus culture was grown on BHK-21 cells in a 50 l bioreactor for two days, then inactivated by the addition of binary ethyleneimine and subjected to purification and concentration procedures. This inactivated and purified BTV-8 antigen was used to formulate a vaccine. The vaccine was prepared on a pilot scale (Merial SAS; France) and contained a low dose (25 per cent) of the commercial antigen payload with aluminium hydroxide and saponin as adjuvants.
Vaccination of sheep
Eleven four- to five-month-old Ile-de-France crossbred sheep were included in the study. The sheep, confirmed to be seronegative for BTV-8 by serum neutralisation at the beginning of the study, were housed in a high containment facility. on day 0, five sheep were inoculated subcutaneously with a single 1 ml dose of BTV-8 vaccine. Six control sheep were not vaccinated. During the vaccination phase, the health of the sheep was monitored (rectal temperature, clinical signs and injection site) for 14 days. Blood samples were taken on days 0, 14, 21, 31 and 45 and serum neutralisation antibody titres were determined in sera.
Vaccination of cattle
Ten six- to seven-month-old Holstein, Montbeliarde, Charolaise and crossbred cattle were included in the study. They were housed in a high containment facility and were confirmed seronegative for BTV-8 by serum neutralisation at the beginning of the study. on days 0 and 28, five cattle were inoculated subcutaneously with 1 ml of vaccine. The other five served as control animals. During the vaccination phase (42 days), the health status of the cattle was monitored (rectal temperature, clinical signs and injection site). Blood samples were taken on days 0, 14, 28, 42, 51 and 79 and sera were titrated for serum neutralisation antibodies.
Antibody titration by serum neutralisation
Briefly, sera that had been inactivated at 56°C for 30 minutes were tested in three-fold dilutions starting at 1/3 in microtitre plates. The sera, diluted in MEM (supplemented with L-glutamine), were incubated with a reference BTV-8 suspension, titrating 500 50 per cent cell culture infective dose (CCID50)/ml. A fixed number of Vero cells were added to the mixture and the plates were incubated for seven days. Reading of the plates was based on cytopathic effect. Serum titres, expressed in log10 50 per cent protective dose (PD50) were calculated by regression after angular transformation (Snedecor and Cochran 1971). A titre of more than 0·48 was considered to be positive.
BTV challenge and monitoring of sheep
On day 31, the rectal temperatures of all sheep were recorded and the animals were challenged intradermally with 3 ml (approximately 30 separate injection points) of a virulent BTV-8 virus strain originally obtained from Belgium after cell passages. This inoculum titrated approximately 7·0 log10 copy genome number/ml. Sheep were monitored daily from days 5 to 14 for rectal temperature and for clinical signs (general and body condition, congestion and/or oedema, hypersalivation, nasal discharge/crusts, plaintive bleating, swollen lymph nodes, locomotion difficulty, respiratory problems and digestive problems). A clinical score was assigned for each: between 0 and 3 for general condition, 0 and 1 for body condition, and 0 and 4 for hyperthermia. Any other sign scored 1. A daily score was calculated for each animal by adding these scores. In addition, a global clinical score (GCS) was calculated for each sheep by adding the individual daily scores. Blood samples were taken into EDTA tubes on days 5, 7, 9, 12 and 14 after the challenge for quantitative RT-PCR testing.
Quantitative RT-PCR testing
For the one-step real-time RT-PCR assay, primers and TaqMan MGB Probe were designed with Oligo 5 software (MedProbe) to hybridise nucleic acid sequences within segment 10, at positions that are conserved among all known BTV serotypes. Blood samples were extracted and treated for RNA extraction using the QIAamp Viral RNA Mini Kit according to the manufacturer's instructions (Qiagen). The onestep RT-PCR was performed using TaqMan EZ RT-PCR core reagent (Applied Biosystems). Serial ten-fold dilutions of RNA standard in vitro (transcribed from the complete NS3 gene), from 107 to 101 copies per reaction, were generated to establish a standard curve. The method was demonstrated to be specific for BTV, and reference strains of serotypes 1 to 24 that were provided by the French National Reference Laboratory (Afssa Lerpaz) were amplified successfully, indicating a wide reactivity among BTV. The detection limit has been determined at 3·14 log10 BTV copies per ml of blood. During its validation, this technique was compared with viral titration on Vero cells (data not shown) and the techniques had similar sensitivities.
All statistical analyses were performed with Statgraphics Plus 5.1 suite (Statistical Graphics). Fisher's exact test was used to compare the number of sheep or cattle from the vaccinated and control groups that were positive for BTV detection by RT-PCR. The student's t test (when normality of distribution was verified) or Mann-Whitney U test were used to compare the control and vaccinated groups for maximal rectal temperatures and global clinical scores after challenge. Statistical significance was defined as P≤0·05.
Safety and immunogenicity of BTV-8 inactivated vaccine in sheep
All vaccinated sheep remained healthy and no adverse clinical signs were seen. only small local reactions were observed immediately after vaccination; these had mostly disappeared by day 14. The control sheep remained seronegative until challenge (Fig 1a), but all vaccinated sheep seroconverted against BTV-8 and titres increased gradually from 14 days after vaccination. Fourteen days after challenge, all controls had developed high antibody titres against BTV-8. The challenge also induced a strong booster effect in the vaccinate levels (mean >3·2 log10 PD50) in both groups.
Protection of sheep following virulent BTV-8 challenge one month after immunisation with BTV-8 inactivated vaccine
In the control sheep, mean peak rectal temperatures reached 41·0°C by day 7 after challenge (Fig 1b). In the vaccinated group, the mean rectal temperatures remained below 40°C and so maximal hyperthermia was significantly reduced (P<0·01) in those vaccinated (mean [sd] 39·9 [0·3]°C) compared with the controls (41·2 [0·6]°C). The clinical signs most frequently observed in the controls were congestion and/or oedema of the ears, conjunctivae, nostrils and lips, and respiratory signs. The average daily clinical scores are shown in Fig 1b. Mean scores for the controls peaked six days after challenge and moderately high scores persisted for at least 13 days. Very few clinical signs were seen in the vaccinated animals and no animal presented with any sign typical of bluetongue. There was a marked difference in the clinical scores of the control and vaccinated groups for a duration of nine days. The GCS was significantly (P<0·01) reduced in the vaccinated animals (1·2 [1·6]) compared with the controls (28·8 [9·4]).
The primary indication that the BTV-8 inactivated vaccine protectively immunised sheep was the comparison of the presence of BTV-8 RNA in the blood of controls and vaccinated animals. High titres of virus RNA were detected in all control sheep for up to 14 days after challenge while no viral RNA was detected from any of the vaccinated sheep (Fig 1c). The mean maximum viraemia titre observed in the controls was 7·5 [0·4] log10 BTV copies per ml of blood. The vaccinated group was significantly protected from viraemia (P<0·01).
Safety and immunogenicity of BTV-8 inactivated vaccine in cattle
All vaccinated cattle remained healthy and no adverse clinical signs were seen. only limited local reactions were observed in two of the animals, which had disappeared by 14 days after vaccination. All control cattle remained seronegative until challenge (Fig 2a) while all vaccinates seroconverted against BTV-8 by 14 days after the second vaccination.
Protection of cattle following virulent BTV-8 challenge 23 days after the second immunisation with BTV-8 inactivated vaccine
Eight days after the challenge, an increased rectal temperature was recorded for all controls and elevated temperatures persisted for at least eight days (Fig 2b). There was no rise in rectal temperatures in the vaccinated group. Maximal hyperthermia was significantly (P<0·01) reduced in the vaccinates (39·0 [0·2]°C) compared with the controls (39·6 [0·2]°C).
The clinical signs most frequently observed in the control group were congestion and/or oedema of the conjunctivae, nostrils and lips, respiratory signs and nasal or oral ulcers. The average daily clinical scores are shown in Fig 2b. Mean clinical scores for the controls peaked 14 days after challenge and high or moderately high scores persisted for at least a further 14 days. Very few clinical signs were seen in the vaccinates and no animal presented with any signs typical of bluetongue. There was a marked difference in the clinical scores of the control and vaccinated groups for a duration of nine days. The GCS was significantly (P<0·01) reduced in the vaccinated animals (11·6 [3·9]) compared with the controls (68·0 [28·3]).
As with sheep, the primary criterion to assess the protective immunisation of cattle was the comparison of the presence of BTV-8 RNA in the blood of control and vaccinated animals. High titres of virus RNA were detected in all controls from seven to 28 days after the challenge. The maximum viraemia titre observed in the controls was 7·7 [0·2] log10 BTV copies per ml of blood. No viral RNA was detected in the blood of any of the vaccinates at any time (Fig 2c). The vaccinated group was significantly protected from viraemia (P<0·01).
The challenge models used to assess the efficacy of the vaccine consistently reproduced hyperthermia, clinical disease and viraemia in the controls of both species. This confirms the clinical pathogenicity of BTV-8 for sheep and cattle, and also that the challenge methodology was appropriate to assess vaccine efficacy.
Different types of vaccine have been developed to prevent BTV infection in sheep and cattle, ranging from conventional or inactivated vaccines to recombinant or vectored vaccines, as well as subunit vaccines (Roy 2004, Boone and others 2007). The live attenuated vaccines have been used with considerable success in Italy (Patta and others 2004) but there have been reports of live attenuated viruses shedding and spreading by natural infection. Furthermore, there is potential for reversion to virulence and for reassortment of the vaccine strains with field viruses. Clinical disease due to under attenuation of live attenuated vaccines has been confirmed (Savini and others 2008). There is a critical need for vaccines that will be safer but still effective in preventing BTV infection and spread. Some of the high technology virus-like particles (Roy and others 1990, 1992, 1994) and canarypox-vectored vaccines (Boone and others 2007) may be of value in the future, but further developments are needed for commercial production.
It has been suggested that, while inactivated vaccines are very safe, they may be of limited efficacy and typically require two doses to induce a good immunological response (Di Emidio and others 2004). However, the data described in this paper indicate that it is possible to induce a strong serological response in sheep with a single dose of this inactivated, adjuvanted BTV-8 vaccine. The results also indicate that in cattle, two doses of the BTV-8 vaccine achieves a comparable level of serological response to one dose in sheep. It is further demonstrated that the tested BTV-8 vaccine provides very significant protection against clinical signs caused by BTV-8 in sheep and cattle and greatly reduces or even prevents viraemia in both species. Further studies are required to assess the duration of immunity induced by the vaccine.
The anamnestic antibody response observed following challenge demonstrates contact with BTV-8 antigen. In the BTV-8 vaccinated sheep and cattle, this occurred in the absence of any detectable viraemia. This could be due to a local antigenic stimulation at the challenge administration site, with or without viral multiplication, which prevented further dissemination in the host.
With the BTV-8 serotype currently spreading rapidly through Europe, safe and effective vaccines against BTV-8 are required quickly at an industrial level. Scale-up of production of the vaccine described in this report has been achieved rapidly and the BTV-8 antigen is now produced and formulated on an industrial scale.
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