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.     

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.     

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.     

Clinical and serological response of sheep to serial challenge with different bluetongue virus types

A group of British sheep was infected with bluetongue virus 5 (BTV5) and subsequently challenged with the same virus type. Protection from this challenge and a homotypic BTV neutralising antibody response were observed. A second group of sheep was infected serially with three different BTV types. Animals previously exposed to BTV4 and BTV3 were found to be resistant to challenge by BTV6. Animals infected with BTV4 and challenged with BTV3 were shown to produce a transient heterotypic neutralising antibody response to a number of types. Although the level of this heterotypic response diminished with time, after challenge with BTV6 these animals developed a similar broad heterotypic response. The nature of this response and its implications in terms of observed protection merit consideration in future vaccine design and evaluation of field survey work.     

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.     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

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。     

Bluetongue virus in the French Island of Reunion

This paper records the results of a bluetongue virus (BTV) serological survey and reports the first isolation of BTV on the French Island of Reunion. In January 2003, the French Island of Reunion, located off the coast of Madagascar, reported an outbreak of disease in cattle that resembled clinical bluetongue (BT) in sheep. The suspected causal agent was isolated and identified as epizootic haemorrhagic disease of deer virus (EHDV). However, because of the similarity in the clinical signs to those of BT, a retrospective survey against BTV was carried out using sera collected in 2002. Results revealed the presence of antibody in all sera tested indicating that BTV has been resident on the Island since 2002, and probably earlier. Although up to July 2003 no clinical BT had ever been reported in sheep, BTV viral RNA was amplified by RT-PCR from a single sheep blood collected in February that year, which strongly suggested that BTV was currently circulating on the Island. Following a second outbreak of disease in August 2003, this time involving a flock of Merino sheep, infectious BTV was finally isolated, and identified by both traditional and molecular techniques as serotype 3. The nucleotide and amino-acid sequences of the RT-PCR products amplified for BTV segments 7 and 10 from the sheep blood collected in February and August from different areas of the Island, were sufficiently diverse as to suggest that they were of different origins and/or different BTV serotypes.     

Immunodiffusion test antigen from bluetongue virus-infected newborn mouse brains

Three methods of extracting bluetongue virus (BTV)-infected newborn mouse brains to prepare immunodiffusion (ID) test antigen were used. The most readily readable and reproducible results were obtained with fluorocarbon-extracted brains homogenized in 8.5% sucrose. Mouse brain- and reference cell culture-derived antigens gave a line of identity with anti-BTV serum. Extracts of noninfected brains were nonreactive. ID tests on field-collected bovine sera, comparing the two types of antigen, resulted in only 73% agreement due to a greater sensitivity of cell culture-derived antigen. A 70.5% agreement resulted when comparing mouse brain-derived antigen in ID tests with complement fixation tests, the latter being least sensitive. ID test results with sera from experimental sheep gave 95.9% agreement between cell culture- and mouse brain-derived antigens. Between ID, which detects antibody to the BTV common or group antigen, and virus neutralization, which detects type-specific antibody, the agreement was 71.4% with postchallenge sera. Data from pre- and postinjection sera, however, indicate the possible activity in Texas of viruses other than International BTV Types 10, 11, 13, and 17.     

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)     

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.     

A serological comparison of the australian isolate of bluetongue virus type 20 (CSIRO 19) with bluetongue group viruses

Bluetongue virus serotype 20 (BTV20) (CSIRO 19 isolate) was compared with 17 other BTV serotypes using various serum neutralization (type antigen) tests to determine whether any serological relationships existed. Plaque-reduction neutralization tests employing 50% and 80% end-points could not clearly differentiate BTV20 from BTV4. Plaque-inhibition tests and quantal microtitre neutralization tests also showed a relationship between BTV20 and BTV4. Antisera against BTV20 and a Cyprus isolate of BTV4 (A SOT 1) showed a low level of cross-neutralization against BTV17. Investigation of plaque-reduction neutralization of virus—antiserum mixtures, by the calculation of regression curves and comparison of the area under the curves, showed that the BTV4 isolates studied could not be differentiated, and that BTV4 typing antiserum could not distinguish between BTV4 and BTV20, but that BTV20 antiserum could distinguish between BTV20 and BTV4. BTV20 did not show any significant type relationships with any of the other BTV types 1 to 17 using any of the neutralization tests. Our results suggest that BTV20 is closely related to, although not identical with, BTV4 and could be grouped as a subtype of BTV4. BTV17 appears to be distantly related to BTV20 and BTV4, but is clearly a distinct type.     

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.     

Prevalence of bovine herpesvirus-1, bovine viral diarrhea, parainfluenza-3, goat respiratory syncytial, bovine leukemia, and bluetongue viral antibodies in sheep

Sera from healthy sheep were collected in January and March 1982 from flocks of sheep located in southwestern and southeastern Louisiana. These sera were tested for bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), parainfluenza-3 (PI-3) virus, and goat respiratory syncytial virus (GRSV) antibodies by microtitration virus-neutralization test. The sera were tested also for bovine leukemia virus (BLV) and bluetongue virus (BTV) antibodies by immunodiffusion tests. The number of flocks with seropositive sheep for each virus were: 2/8 (25%) for BVDV; 8/8 (100%) for PI-3 virus; 7/8 (87.5%) for GRSV; and 6/8 (75%) for BTV. Seropositive rates for each virus for the individual sheep tested were: 4/158 (2.5%) for BVDV; 117/158 (74.1%) for PI-3 virus; 77/158 (48.7%) for GRSV; and 21/158 (13.3%) for BTV. All sheep were seronegative for BHV-1 and BLV.     

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.     

Involvement of the skin during bluetongue virus infection and replication in the ruminant host

ABSTRACT: Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence". BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/ macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.     

光生物素cDNA探针对体内外蓝舌病病毒感染的检测

蓝舌病病毒(BTV)基因的第三片段(RMA_3)在不同型间有较高的同源性.用光生物素标记的其cDNA重组体pC7,检测2～22型BTV BHK细胞培养物全部为阳性,而相关病毒EHDV_1、EHDV_2和Ibraki病毒为阴性;同一探针检测17个型BTV攻毒羊的全血样品均为阳性,未感染的正常羊血细胞和BHK细胞培养物样品均为阴性.     

No evidence for involvement of sheep in the epidemiology of cattle virulent epizootic hemorrhagic disease virus

Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus. While not previously considered as an important disease in cattle, several EHDV serotypes (EHDV-6 and 7) have recently been implicated in disease outbreaks. The involvement of sheep in the epidemiology of EHDV is still not understood. In this study we compared the prevalence of antibodies to EHDV and bluetongue virus (BTV) in sheep to their prevalence in cattle after an outbreak of EHDV that occurred in Israel during 2006. Sixty-six sheep and lambs scattered in seven herds were compared to 114 cows and calves scattered in 13 dairy cattle herds, matched to the sheep herds by location. While antibody prevalence to EHDV was high in cattle (35.2% within the outbreak zone) no evidence of exposure to EHDV was found in sheep (p<0.0001). Antibodies to BTV were apparent in both cattle and sheep though in the former it was significantly higher (63.2%, 16.7% respectively, p<0.0001), suggesting higher exposure of cattle to biting Culicoides midges. Taken together, these results imply that sheep have a negligible role in the epidemiology of EHDV.