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.     

Serological studies of two additional Australian blue-tongue virus isolates CSIRO 154 and CSIRO 156

Serological surveys revealed that some cattle in northern Australia possessed bluetongue virus (BTV) group-reactive (agar gel diffusion precipitin, AGDP, and complement-fixing, CF) antibodies, but not serum neutralizing (SN) antibodies, to BTV20, a new type previously found in Australia. Attempts were made during 1979 to isolate viruses causing these reactions. There was one isolate of a virus (CSIRO 154) and eight isolates of another virus (CSIRO 156) made from the blood of healthy cattle in the Northern Territory. These viruses could not be distinguished from BTV20 by AGDP, CF or fluorescent-abtibody tests and hence were designated members of the bluetongue serogroup. Serotyping was carried out using the plaque-inhibition and plaque-reduction SN tests. CSIRO 156 virus could not be distinguished from BTV1 by any of the SN tests and it was concluded that it was an Australian isolate of the BTV1 serotype. CSIRO 154 virus was found to be related to, but not identical with, BTV6. It is probably not one of the known 20 BTV serotypes and may represent a new BTV serotype. None of the three Australian BTV isolates is known to cause clinical disease in sheep or cattle under natural conditions, and biochemical comparisons with the African BTV serotypes may show differences not revealed by these serological studies.     

Bluetongue and epizootic hemorrhagic disease virus isolation from vertebrate and invertebrate hosts at a common geographic site

One serotype of bluetongue virus (BTV) and two serotypes of epizootic hemorrhagic disease virus (EHDV) were isolated from vertebrate and invertebrate hosts on a farm in Colorado. The isolations were from blood samples collected a week apart from a dairy heifer with stomatitis and laminitis; EHDV serotypes 1 and 2 were isolated from the first blood sample, and BTV serotype 13 and EHDV serotype 1 were isolated from the second. Antibodies to EHDV and BTV were detected in the serum from this heifer. Both EHDV serotypes and BTV serotype 13 were isolated from pools of female biting gnats (Culicoides variipennis) that had not had a recent blood meal. The BTV insect isolate was biologically transmitted by female gnats from an infected donor sheep to a recipient host sheep. Culicoides variipennis was the predominant insect collected during three nights of light trap captures at the farm.     

Recovery of dual serotypes of bluetongue virus from infected sheep and cattle

Dual serotypes of bluetongue virus (BTV) were recovered from field-collected samples of sheep and cattle blood. Two sheep, each infected with both BTV serotypes 10 and 17, were found in a flock with bluetongue disease associated with these two serotypes. One sheep infected with BTV serotypes 11 and 17 was found in a second flock; it was the only viremic sheep detected and was clinically ill. Dual serotype infections of one beef and two dairy cattle were found in three geographically separate herds; mixtures recovered were of BTV serotypes 10 and 17 and serotypes 11 and 17. Clinical signs of illness were absent in the cattle in two herds, but severe conjuctivitis was seen in several cows in a third herd, including the cow with a dual serotype infection (BTV 11 and 17). Two of the cattle with dual infections had no serological evidence of BTV as determined by the agar gel precipitin test; serum was not available from the other cow with a dual serotype infection. The significance of dual infections and immune tolerance are discussed.     

Bluetongue virus-induced interferon in cattle

Calves were inoculated IV with bluetongue virus (BTV), serotype 10. Titers of interferon (IFN) in serum and BTV in peripheral blood were determined. All inoculated calves produced circulating IFN that persisted for 2 to 4 days. Highest titers of BTV in peripheral blood were present after serum IFN was no longer detected. The persistence of BTV in peripheral blood, as compared with the transient IFN response, indicated that IFN was most important in the initial antiviral response of cattle to BTV infection. Bluetongue virus is probably not a suitable model inducer of circulating IFN in cattle because the profound neutropenia that accompanied BTV infection may predispose cattle to infections with other agents.     

Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1.

Dot immunobinding assay (DIA) was evaluated for the detection of bluetongue virus (BTV) antibodies in sheep experimentally inoculated with BTV 1. Serum samples collected on 14, 21, 28, 43 and 60 day post infection (dpi) were positive for precipitating antibodies by the agar gel precipitation test (AGPT) while antibodies could be detected as early as 7 dpi by DIA and ELISA. Virus neutralizing antibodies were detected first at 14 dpi. The sensitivity of the four tests was compared on the same serum samples collected at different intervals. The results indicated that DIA was more sensitive than AGPT and the serum neutralization test and as sensitive as ELISA. Thus due to sensitivity simplicity and economy, DIA could replace AGPT for diagnosis and serological survey for BTV infection in animals.     

Competitive ELISA for serodiagnosis of bluetongue: evaluation of group-specific monoclonal antibodies and expressed VP7 antigen.

The performance of 2 competitive enzyme-linked immunosorbent assays (C-ELISA) was compared with the reference C-ELISA I for the detection of antibodies to bluetongue virus (BTV). One of the assays (C-ELISA II) used a group-specific monoclonal antibody (MAb) to BTV, obtained from the American Type Culture Collection (8A3B-6) and tissue culture (TC)-derived BTV antigen (Ag), and the other assay (C-ELISA III) used BTV core protein VP7 (expressed in yeast) and the reference MAb (Pirbright Laboratory, 3-17-A3). Test sera were obtained by sequential blood samples from 22 calves, each inoculated with a different serotype (T) of BTV (South African [SA] T-1-T-16 and T-18-T-20 and USA T-11, T-13, and T-17). Sera were also obtained from 4 calves and 4 sheep inoculated with USA BTV T-10 and from several groups of calves exposed to single or multiple doses of epizootic hemorrhagic disease virus (EHDV) T-1-T-4 grown in TC (BHK-21) or suckling mouse brain (SMB). A total of 618 bovine and ovine field sera collected from BT-free and BT-endemic areas were also tested. The C-ELISA III was more sensitive than the C-ELISA II in the detection of anti-BTV antibody in sera from cattle and sheep early after infection with BTV. Seroconversion was demonstrated by the 3 C-ELISAs in all animals inoculated with BTV by 20 days postinfection (DPI), except in calves that received SA T-3 or USA T-13, which became positive at 40 DPI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of a fetal tongue cell line derived from bighorn sheep to five serotypes of bluetongue virus and its potential for the isolation of viruses

A cell line (BHFTE) was derived from a tongue explant of a bighorn sheep fetus (Ovis canadensis nelsoni). The cells have been maintained through 23 serial passages, and the modal number of chromosomes was calculated to be 55. Monolayer cultures were shown to be susceptible to various viruses, including bluetongue virus (BTV). Of 5 BTV serotypes (2, 10, 11, 13, and 17) tested, each produced a cytopathic effect (CPE) on initial passage at 33 C. A field isolate (serotype 10) of BTV from a black-tailed deer (Odocoileus hemionus columbianus) in its second passage in Vero-M cells also produced CPE when inoculated into BHFTE cells. Antigens of BTV were demonstrated by direct immunofluorescence in the cytoplasm of BHFTE cells inoculated with homogenates of chicken embryos injected with clinical specimens from a domestic sheep and an Arabian oryx (Oryx gazella leucoryx). A suspension of BTV-infected gnats (Culicoides spp.) produced CPE and BTV-specific fluorescence on the first passage in cells inoculated with a suspension of blood from sheep experimentally infected with BTV. Additionally, selected bovine viruses induced CPE in the cells. The cell line, which is free of mycoplasma and bovine viral diarrhea virus contamination, may be useful in diagnostic medicine and research involving the ruminant species.     

Epizootiologic study of bluetongue: virologic and serologic results

Heparinized blood and serum samples were obtained from 1,295 ruminants in herds or flocks with bluetongue virus (BTV) infection in 4 western states. Submissions were from herds or flocks with clinical bluetongue (BT), as well as from animals on premises with no history of BT disease. Insects, including Culicoides variipennis, were collected in areas enzootic for BT disease. Viral isolations were in 10-day-old embryonating chicken eggs that were then adapted to Vero cells for serotyping. Sera were tested from group-specific antibody to BTV by the micro agar gel precipitin (AGP) test. Viral isolations were from cattle (81), sheep (122), goats (9), antelope (2), and C varipennis (5). There were 7 isolates of serotype 120, 114 of serotype 11, 42 of serotype 13, and 56 of serotype 17. In herds or flocks from which BTV was isolated, 51% of cattle, 56% of sheep, 21% of goats, and 52% of antelope had AGP antibodies. Virus was isolated from 43% of the cattle and 23% of the sheep that had no demonstrable evidence of AGP antibodies. Viral isolations were seasonal, occurring from August until December. Approximately 30% of the herds or flocks from which virus was isolated had more than one serotype of virus causing infection.     

Experimental bluetongue virus infection of sheep; effect of previous vaccination: clinical and immunologic studies

Clinical and immunologic responses of sheep to vaccination and subsequent bluetongue virus (BTV) challenge exposure were studied and compared with those of non-vaccinated sheep. Sheep were vaccinated with inactivated BTV administered with aluminum hydroxide and cimetidine or levamisole. After sheep were vaccinated, precipitating group-specific antibodies to BTV were detected, but serotype-specific neutralizing antibodies were not detected. Cellular immune responses (lymphocyte blastogenesis) to BTV were not detected. After virulent BTV challenge exposure, vaccinated and nonvaccinated sheep developed acute clinical disease of similar severity. Clinical signs included hyperemia and petechiae of oral mucosa and coronary bands of the feet, excess salivation, nasal discharge with crusting, ulceration of the muzzle, and edema of lips and intermandibular space. Marked increases in serum creatine kinase activity were associated with stiff gait, reluctance to move, and vomiting. Fever and leukopenia were detected in most of the challenge-exposed sheep. Viremia and neutralizing antibodies were detected in vaccinated and nonvaccinated sheep after challenge exposure. Bluetongue virus-specific reaginic antibodies were not detected in sera from any of the sheep when the passive cutaneous anaphylaxis test was used.     

Temporal relationships of viremia, interferon activity, and antibody responses of sheep infected with several bluetongue virus strains.

Sheep had viremias that were first detected on day 3 (+/- 1) after infection with several strains of bluetongue virus (BTV) representing United States serotypes 10, 11, 13, and 17. Diphasic peaks of infectivity were attained on days 6 and 10 (+/- 2). Interferon (IFN) was first detected in serum samples on day 5 (+/- 1), and reached greatest concentrations on day 6 (+/- 2), which coincided with the first viremic peak; IFN concentrations then decreased toward zero by day 10 (+/- 2). Interferon peak concentrations induced approximately a 90% decrease in virus titer. The decrease in IFN concentrations by day 9 (+/- 2) corresponded with the second viremic peak on day 10 (+/- 2). Onset of the decrease in detectable concentrations of virus after the second peak of viremia corresponded to the initial detection of serum antibody to BTV by day 10 (+/- 2). Virus titer decreased and antibody production increased until approximately days 21 to 28, when the titers plateaued and virus was not detected. Febrile responses peaked on day 7 (+/- 1) during the peak viremic period. The WBC count was depressed at the time the virus titer increased, but returned to normal values while the sheep were still viremic. Diphasic viremias in BTV-infected sheep were attributed to induction of high concentrations of IFN concurrent with the first virus titer peak, followed by production of antibody to specific BTV strains and a subsequent reduction in viremia at the second virus titer peak.     

Bluetongue virus serotype 20: experimental infection of pregnant heifers

Three groups of 4 cows at 84 to 95 days, 100 to 160 days, and 170 to 180 days pregnant were inoculated both intradermally and subcutaneously with bluetongue virus serotype 20 (BTV20). Clinical observations and the viraemic and serological responses of the cows were followed for 9 to 17 weeks after inoculation. Viraemia developed in 9 of the 12 cows and was first detected 4 to 9 days after inoculation. Viraemia was detected for 4 to 21 days and in some animals only intermittently. The titre of the viraemia was obtained in 4 cows and ranged from detectable only, to 10(1) to 10(2.8) 50% tissue culture infecting doses per ml. Both serum neutralising and precipitating antibodies were detected in 11 of the 12 cows within 2 to 8 weeks after inoculation. No clinical responses were seen and one cow (516) did not develop a viraemia or produce detectable antibodies to the virus. The cows, calves and foetuses were necropsied following either parturition or slaughter between 200 and 270 days of pregnancy. No virus isolations were made from a wide range of tissues from the cows, calves or foetuses and no immunoglobulins or serum neutralising antibodies were detected in the serums of precolostral calves or foetuses at necropsy. No gross or histopathological lesions were seen in the cows, calves or foetuses, and there was no evidence that BTV20 crossed the bovine placenta or infected the foetus.     

Serological studies of Australian and Papua New Guinean cattle and Australian sheep for the presence of antibodies against bluetongue group viruses

Following isolation of a virus (CSIRO19) from insects in Australia and its identification as bluetongue virus serotype 20 (BTV20), a nationwide survey of antibodies in cattle and sheep sera was undertaken. Initial studies using the serum neutralization (SN) test showed that the distribution of BTV20 antibodies in cattle was confined to the northern part of Australia. Group-reactive antibody tests (agar gel diffusion precipitin, AGDP, and complement-fixation, CF) showed group-reactive cattle sera south of the BTV20 zone (northern Australia), and southwards from Queensland to New South Wales. Very few group-reactive sheep sera (45 out of 16213) were found and these were of doubtful epidemiological significance. Some of these BTV group-reactive, BTV20-negative, sera were tested in SN tests against BTV1 to 17 and Ibaraki (IBA) virus. The results indicated that BTV1, or a closely related orbivirus, was active in cattle in Queensland, northern Western Australia, and New South Wales, and that antibody to BTV15 was present in some of the cattle sera in northern Western Australia and the Northern Territory. Antibody to IBA virus was present in some cattle sera in Queensland, northern Western Australia and New South Wales. SN antibody titres ?60 were also found to a number of other BTV serotypes in cattle sera in northern Western Australia and Queensland (principally, BTV2 and BTV7). Low level reactions were commonly observed against these and a number of other BTV serotypes, often in the same serum samples. Further, 22% of the group-reactive cattle sera did not react with any of the viruses in the SN tests. Such results were difficult to interpret in terms of known Australian BTV or BTV-related isolates.     

Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.     

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.     

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.     

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.     

Serologic and virologic evidence of bluetongue virus infection in cattle and sheep in Mexico

Three independent 1-year studies were conducted during 3 consecutive years to better define the prevalence of bluetongue virus (BTV) infection in Mexico. Serologic data were obtained by use of agar-gel immunodiffusion for identification of BTV group-reactive antibodies, and virologic data were obtained by virus isolation. Samples were obtained from sheep in 6 states over a 1-year period, with 9% seropositive; samples were obtained from cattle in 11 states during the same 1-year period, with 35% seropositive. Two years later, samples were obtained from cattle in 4 additional states, with 69% seropositive. Virus isolation was conducted on pooled blood samples obtained from cattle in 7 states. Six virus isolates were recovered and included 2 isolates each of BTV serotypes 11 and 13 and 1 isolate each of serotypes 10 and 17. All virus isolates were partially characterized by electrophoretic analysis of genomic RNA migration profiles (electropherotypes) in polyacrylamide gels. All Mexican isolates of BTV differed considerably in electropherotype profile, as compared with their respective US prototype strain of the same serotype. Such differences appeared to be much more extensive than those described to exist between numerous California isolates of the same serotype.     

Virulence of bluetongue virus for British sheep

A South African isolate of bluetongue virus type 3 was inoculated intradermally into three different breeds of British sheep under conditions designed to test its virulence in animals under stress. All animals inoculated developed a pyrexia and viraemia followed by clinical evidence of bluetongue disease. Marked alterations in serum enzyme levels, in particular of creatine phosphokinase, lactate dehydrogenase and aldolase occurred in the more severely affected animals. Nine out of the 12 inoculated animals subsequently died. No major differences in response could be detected in the different breeds of sheep nor in the stressed compared with the unstressed groups. The virulence of this bluetongue virus isolate was thereby confirmed and its potential risk to the British sheep industry. Consequently, stringent import regulations must be maintained to prevent its entry into Britain.     

Bluetongue virus infection in sheep: haematological changes and detection by polymerase chain reaction

SUMMARY Eight sheep vaccinated with 10⁶ pfu of attenuated Australian bluetongue virus serotype 23 (BTV-free sheep were challenged with virulent BTV 23. There was little subsequent variation in the mean clinical score, or in the mean lymphocyte and platelet concentrations in the peripheral blood of the eight vaccinated sheep. There was a marked thrombocytopenia and lymphopenia in the naive sheep as the mean lymphocyte and platelet concentrations fell to a minimum at days 8 and 11 after inoculation, respectively. Similar changes were observed in three other naive sheep inoculated with field isolates of BTV 1, 9 or 23. BTV was detected by nested polymerase chain reaction in whole blood of these sheep between days 6 and 28, in mononuclear leukocytes between days 3 and 14, and in platelets between days 6 and 21.