Experimental infection of camels with bluetongue virus

Three camels aged 4–5 years were experimentally infected with Bluetongue virus serotype 1 (BTV-1) and were observed for 75 days. No clinical signs of disease were observed throughout the experiment, however all three animals seroconverted and developed BTV-1 specific neutralising antibodies after challenge. All three camels developed a viraemia from 7 days post infection albeit at a lower level than that usually observed in experimental infections of sheep and cattle. Virus was isolated from the blood of all three animals suggesting that camels may act as a reservoir for BTV and play an important role in its transmission.     

Bluetongue virus serotype 26: infection kinetics and pathogenesis in Dorset Poll sheep

Bluetongue virus serotype 26 (BTV-26) has recently been isolated from sheep in Kuwait. The aim of this study was to assess the pathogenicity and infection kinetics of BTV-26 in Dorset Poll sheep. Six sheep were experimentally infected with BTV-26 and samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and two group specific ELISAs. Five of the six sheep showed mild clinical signs characteristic of bluetongue including conjunctivitis, reddening of the mouth mucosal membranes, slight oedema of the face and nasal discharge. Viral RNA was detected in 5 of the 6 sheep by real time RT-PCR, however the levels of viral RNA detected in the samples were lower and of shorter duration than seen with other field strains of BTV. Virus was isolated from the blood of infected animals at the peak of viraemia at around 9 dpi. Antibodies against BTV were first detected by 7 dpi using the early detection BTV ELISA and a little later (7-14 dpi) using a BTV specific competitive ELISA. Four of the five remaining sheep developed neutralising antibodies to BTV-26, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 1.40 to 2.08.     

Immunologic response of sheep to inactivated and virulent bluetongue virus

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.     

Precipitating antibodies in experimental visna and natural progressive pneumonia of sheep

Serological responses of Icelandic sheep experimentally infected with visna virus (vv) were contrasted with responses in American Targhee sheep naturally infected with progressive pneumonia virus (PPV). Precipitating antibodies assayed by immunodiffusion were compared with the neutralising and complementing fixing antibody response. In experimental infections with vv, complement fixing and neutralising antibodies appeared early after infection and rose to high levels in all sheep, while precipitating antibodies were detected only at minimal titre. In natural infections with PPV, immune responses were less consistent and precipitating antibodies were detected more frequently than complement fixing or neutralising antibodies against PPV. These results may suggest important biological differences between the lytic fibroblast-tropic virus strains used for experimental infection of Icelandic sheep and the nonlytic macrophage-tropic strains of PPV circulating in nature. Lytic strains evoke a brisk response against the viral glycoprotein with high titre neutralising antibody while nonlytic strains induce a less consistent response to the glycoprotein.     

Temporal development of bluetongue virus protein-specific antibody in sheep following natural infection

Humoral immune responses of sheep to natural bluetongue virus (BTV) infection were studied on a temporal basis. The temporal development of viral protein-specific IgG was determined by western immunoblotting; virus neutralization and agar gel immunodiffusion (AGID) were conducted for comparative purposes. Prior to the emergence of the arthropod vector and the associated transmission of BTV, virus-neutralizing antibody was absent from all sentinel sheep; 3 sheep had pre-existing AGID antibody and all sheep had IgG, specific for 4 viral proteins, as determined by immunoblotting. Following emergence of the BTV vector, 9 of 11 sheep became infected, as determined by virus isolation, with BTV. All sheep developed virus-neutralizing and AGID antibody. However, only those sheep with a demonstrable viremia experienced an increase in viral protein-specific antibody. Development of viral protein-specific IgG varied with the individual animal and no obvious correlation between a specific response and protective immunity or viral clearance was noted. From a diagnostic viewpoint, the immunoblotting procedure was superior in identifying past exposure to BTV, as compared with neutralization and AGID. In addition, the application of immunoblotting to paired serum samples appeared to be a sensitive indicator of viremia.     

Bluetongue virus serotypes 20 and 17 infections in sheep: Comparison of clinical and serological responses

The clinical, virological and serological responses of sheep infected with an Australian bluetongue virus (BTV) isolate (serotype 20) were compared to responses in sheep inoculated with an American bluetongue isolate (serotype 17) with which it had shown cross-reactions in serum neutralization tests. In sheep inoculated with BTV 20, clinical signs were very mild and viremia was first detected by day 5; virus was isolated intermittently for a further 2 to 3 days. Neutralizing and precipitating antibodies were first detected in the serum of the sheep between 2 to 3 weeks following inoculation. In contrast, sheep inoculated with BTV 17 showed pyrexia and severe hyperemia of the nasolabial area and oral mucosa from day 7 to 17. Viremia was first detected on day 3 and extended to day 20, while the appearance and titers of serum antibodies was similar in both groups.After challenge with BTV 17 the sheep in both groups remained clinically normal, and virus was not detected in the blood; however, serum neutralizing antibody titers to both viruses increased 2 weeks after challenge and the mean titer of the two groups ranged from 1:250 to 1:640.     

Effect of malignant catarrhal fever virus infection on the immune response of rabbits to sheep red blood cells

Rabbits infected with African Malignant catarrhal fever virus mounted a depressed antibody response to sheep red blood cells compared to the antibody response of uninfected rabbits. Immunodepression was observed in rabbits immunised 0, 3, 5, 7 and 10 days after infection. Antibody response was not depressed in the rabbits immunised 1 day after infection. Despite the depressed antibody response to SRBC, the rabbits died with rising virus neutralising antibody to the virus. The possible causes of the immunodepression are discussed.     

A two year BTV-8 vaccination follow up: molecular diagnostics and assessment of humoral and cellular immune reactions

The compulsory vaccination campaign against Bluetongue virus serotype eight (BTV-8) in Germany was exercised in the state of Bavaria using three commercial monovalent inactivated vaccines given provisional marketing authorisation for emergency use. In eleven Bavarian farms representing a cross sectional area of the state the immune reactions of sheep and cattle were followed over a two year period (2008-2009) using cELISA, a serum neutralisation test (SNT) and interferon gamma (IFN-γ) ELISPOT. For molecular diagnostics of BTV genome presence two recommended real time quantitative RT-PCR protocols were applied. The recommended vaccination scheme led to low or even undetectable antibody titers (ELISA) in serum samples of both cattle and sheep. A fourfold increase of the vaccine dose in cattle, however, induced higher ELISA titers and virus neutralising antibodies. Accordingly, repeated vaccination in sheep caused an increase in ELISA-antibody titers. BTV-8 neutralising antibodies occurred in most animals only after multiple vaccinations in the second year of the campaign. The secretion of interferon gamma (IFN-γ) in ELISPOT after in vitro re-stimulation of PBMC of BTV-8 vaccinated animals with BTV was evaluated in the field for the first time. Sera of BTV-8 infected or vaccinated animals neutralising BTV-8 could also neutralise an Italian BTV serotype 1 cell culture adapted strain and PBMC of such animals secreted IFN-γ when stimulated with BTV-1.     

Vaccination of sheep with cell culture grown orf virus

Orf virus, derived from contagious pustular dermatitis (scabby mouth) lesions in sheep, was adapted to cell culture and subsequently evaluated as a potential vaccine for sheep. The traditional vaccine virus, prepared from the infected scabs of orf virus lesions in sheep, was used to vaccinate sheep by scratching with an applicator (mounted pins) dipped in virus. Less than 10 TCID50 (50% tissue culture infectious doses) of virus was required to produce large lesions (greater than 5 mm diameter) which developed during a period of 10 to 14 d prior to onset of healing which was complete by 28 to 30 d. A serum neutralising antibody response was also detected and protection against challenge by application of virulent virus to abraded skin was demonstrated in that challenge lesions developed and healed more quickly (14 d against 30 d). However, cell culture-adapted virus required more than 10(5) TCID50 to induce even small lesions (less than 2 mm diameter). An antibody response could not be detected and no evidence of protection against challenge with virulent virus was demonstrated. In contrast, a recent field isolate has yielded a cell culture-adapted virus preparation that readily infects sheep, produces large lesions, detectable antibody and protects against challenge. This isolate is distinct from the traditional vaccine strain on the basis of restriction enzyme analysis but provides cross-protection in sheep inmmunisation and challenge studies. These results demonstrate that a cell culture produced scabby mouth vaccine is feasible.     

感染16型蓝舌病病毒后绵羊部分细胞因子消长特点研究

本试验旨在探明绵羊感染16型蓝舌病病毒(Bluetongue virus type 16,BTV16)后细胞因子IFN-γ、IL-2、IL-4和IL-10的消长特点。用实时荧光定量PCR检测方法对感染BTV16后3只绵羊上述4种因子mRNA进行检测,同时设立阴性对照绵羊,并以0 d mRNA为基准,计算mRNA的相对表达量,同时检测病毒抗体效价、测量绵羊体温。结果显示,接种BTV16的3只绵羊均不同程度产生抗体和体温症状,4种细胞因子的mRNA在接种病毒2~4 d内均出现显著上升,其中IFN-γ峰值在2.58~27.84倍之间,IL-2峰值在5.24~17.19倍之间,IL-4峰值在2.16~3.43倍之间,IL-10峰值在15.78~48.77倍之间,个体上升幅度存在显著差异,4种细胞因子均在高水平持续6 d左右后逐渐下降。对照绵羊上述参数在正常范围内波动。本研究阐明了接种BTV16后绵羊细胞因子IFN-γ、IL-2、IL-4、IL-10在转录水平上的消长特点,为进一步深入开展BTV感染特征、宿主机体免疫机制研究提供参考。     

感染16型蓝舌病病毒后绵羊部分细胞因子消长特点研究

本试验旨在探明绵羊感染16型蓝舌病病毒（Bluetongue virus type 16,BTV16）后细胞因子IFN-γ、IL-2、IL-4和IL-10的消长特点。用实时荧光定量PCR检测方法对感染BTV16后3只绵羊上述4种因子mRNA进行检测,同时设立阴性对照绵羊,并以0 d mRNA为基准,计算mRNA的相对表达量,同时检测病毒抗体效价、测量绵羊体温。结果显示,接种BTV16的3只绵羊均不同程度产生抗体和体温症状,4种细胞因子的mRNA在接种病毒2~4 d内均出现显著上升,其中IFN-γ峰值在2.58~27.84倍之间,IL-2峰值在5.24~17.19倍之间,IL-4峰值在2.16~3.43倍之间,IL-10峰值在15.78~48.77倍之间,个体上升幅度存在显著差异,4种细胞因子均在高水平持续6 d左右后逐渐下降。对照绵羊上述参数在正常范围内波动。本研究阐明了接种BTV16后绵羊细胞因子IFN-γ、IL-2、IL-4、IL-10在转录水平上的消长特点,为进一步深入开展BTV感染特征、宿主机体免疫机制研究提供参考。     

Evaluation of a hemolysis-in-gel test for detection and quantitation of antibodies to bluetongue virus

A hemolysis-in-gel (HIG) test was developed to detect and quantitate antibody to bluetongue virus (BTV). The HIG test was sensitive and accurate when applied to sera from sheep and cattle infected with BTV. Sensitized sheep RBC were prepared by adsorption of partly purified BTV to the cells. Regression analysis of data showed a linear relationship between the diameter of the hemolytic zone and the log of the antibody concentration. The HIG test did not differentiate among antiodies to four serotypes of BTV, but did differentiate between antibody of BTV and antibody to epizootic hemorrhagic disease virus.     

SIMULATED NATURAL INFECTION OF CHICKENS WITH AUSTRALIAN LENTOGENIC NEWCASTLE DISEASE VIRUS AND SUBSEQUENT CHALLENGE WITH VIRULENT VIRUS

A total of 291 eight-week-old chickens were exposed to chickens infected with either of two Australian lentogenic strains (V4 and AVL NDV-1) of Newcastle disease virus (NDV). At 3 weeks after exposure, all chickens exposed to V4 infected chickens had developed haemagglutination-inhibition (HI) antibody. All chickens exposed to AVL NDV-1 virus infected chickens had developed HI antibody 5 weeks later. This sudden late appearance of HI antibody, to titres higher than those observed with V4 chickens, was explained by V4 virus being introduced to the AVL NDV-1 group of chickens. When groups of these chickens were challenged with Roakin virus (mesogenic NDV) at 3 weeks and Fontana 1083 virus (viscerotropic velogenic NDV) and Texas GB virus (neutrotropic NDV) at 3, 5, 10 and 21 weeks only three chickens developed clinical illness one of which died. These chickens were one AVL NDV-1 chicken contact challenged with Fontana 1083 virus at 3 weeks, one V4 chicken oronasally challenged with Texas GB virus at 5 weeks and one V4 chicken challenged oronasally with Fontana 1083 virus at 10 weeks. Susceptible non-vaccinated chickens died soon after challenge. Challenge by oronasal infection with 10(7.0) ELD50 of virus or contact with susceptible infected chickens enabled virulent virus to be isolated from most chickens and was accompanied by a large anamnestic increase in serum HI antibody.     

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

Serial inoculation of sheep with two bluetongue virus types

Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.     

Late immune responses in experimental maedi

Thirteen sheep were inoculated with maedi-visna virus by respiratory or intra-cerebral (i.c.) routes and were kept in close contact with 7 uninoculated control sheep. Immune responses and virological events were followed in all 20 sheep for periods up to 3 years post-inoculation.Three out of 7 i.c. infected sheep produced no complement fixing (CF) or serum neutralising (SN) antibody, whilst all 5 infected by the respiratory route and the hyperimmunised sheep developed CF and SN antibody which persisted with fluctuating titre over the period of study.Ten out of 11 sheep, which were followed from 3 months after experimental inoculation to 16–21 months (5 sheep) or 3 years (6 sheep) after inoculation, developed transient late virus-specific cell-mediated responses with intermittent virus isolation from peripheral blood leucocytes (PBL) over the period of study.In the contract control sheep, virus was isolated from PBL of 2 sheep during the 2nd year of contact, and transient virus-specific cell-mediated responses, but no antibody responses, were detected.     

Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle

The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV-2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow-up, and challenge. Thirty-two 4-month-old calves were randomly allocated into 2 groups of 16 animals each. One group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged with 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in the study and used as sentinels to confirm that no BTV was circulating locally. At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4. However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response in terms of both the number of virus neutralization (VN) positive animals and antibody titres. After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in the circulating blood of all unvaccinated animals, commencing on day 3 post-challenge and lasting 16 days. It is concluded that administration of the bivalent BTV-2 and BTV-4 inactivated vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged with high doses of BTV-2 or BTV-4.     

LABORATORY AND FIELD STUDIES WITH A BOVINE EPHEMERAL FEVER VACCINE

Bovine ephemeral fever (BEF) virus vaccines, prepared from the brains of suckling mice infected with strain 525 BEF virus, were evaluated in housed cattle and in the field. The virus in lyophilised preparations was stable for 6 months at -50 degrees C. Thirty-four calves, 5 to 18 months old, were used in laboratory vaccination trials. An increase in serum neutralising antibody was detected in 13 of 14 calves initially free of serum antibody, and all 13 failed to develop clinical illness following challenge with virulent BEF virus. Vaccination resulted in no detectable serum antibody increase in 4 calves, 5 months old, with pre-existing antibody of presumed maternal origin. Seven animals, 18 months of age with serum antibody presumed due to previous BEF infection, developed increased antibody titres following vaccination. In 3 animals vaccinated but not challenged, vaccine-induced antibodies decreased to low levels over 5 months. In contrast, the antibody titres following infection with virulent virus in 2 calves were maintained over 5 months. Field trials, involving 236 animals initially free of serum antibody, were conducted on 5 properties near Mackay and 4 properties near Brisbane. Most of 164 animals were vaccinated with a single dose of lyophilised vaccine containing aluminium hydroxide adjuvant. Only 4 animals failed to develop serum antibody and no adverse reactions to vaccination were reported. Natural infection with BEF occurred in 4 herds at Mackay and clinically mild BEF occurred in 3 of 109 vaccinated and 3 of 46 control animals. On the basis of measured serum antibody titres it was assumed that 8 of 53 animals receiving full vaccine volume, 20 of 40 animals receiving half vaccine volume and 18 of 40 control animals became infected with BEF virus. Two dairy herds in Brisbane became naturally infected with virulent BEF virus 7 months after vaccination. Clinical BEF was observed in 8 of 11 control animals and in 3 of 26 animals which received 2 doses of vaccine. Two strains of BEF virus were isolated from unvaccinated animals that developed clinically mild BEF in the field. These strains either failed to infect, or produced subclinical or very mild BEF, when inoculated intravenously into susceptible calves. The anitbody response to natural infection with apparently mild viruses was short-lived, similar to that produced by vaccination.     

Epizootic hemorrhagic disease virus serotype 7 in European cattle and sheep: Diagnostic considerations and effect of previous BTV exposure

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants that is closely related to bluetongue virus (BTV). The present study examines the outcome of an experimental EHDV-7 infection of Holstein cattle and East Frisian sheep. Apart from na?ve animals that had not been exposed to BTV, it included animals that had been experimentally infected with either BTV-6 or BTV-8 two months earlier. In addition, EHDV-infected cattle were subsequently challenged with BTV-8. Samples were tested with commercially available ELISA and real-time RT-PCR kits and a custom NS3-specific real-time RT-PCR assay. Virus isolation was attempted in Vero, C6/36 and KC cells (from Culicoides variipennis), embryonated chicken eggs and type I interferon receptor-deficient IFNAR(-/-) mice. EHDV-7 productively infected Holstein cattle, but caused no clinical signs. The inoculation of East Frisian sheep, on the other hand, apparently did not lead to a productive infection. The commercial diagnostic kits performed adequately. KC cells proved to be the most sensitive means of virus isolation, but viremia was shorter than 2 weeks in most animals. No interference between EHDV and BTV infection was observed; therefore the pre-existing immunity to some BTV serotypes in Europe is not expected to protect against a possible introduction of EHDV, in spite of the close relation between the viruses.