The immunological response to intact and dissociated blue-tongue virus in mice.

Antigenic fractions of bluetongue virus were separated by ultracentrifugation in Tris-buffered CsCl gradients at pH 6, 7 or 8 and the bluetongue virus polypeptide composition of the bands isolated from these gradeints was monitored by polyacrylamide gel slab electrophoresis. The immunological response to these fractions in mice was determined by a haemolytic plaque-forming cell assay, using sheep erythrocytes onto which intact bluetongue virus was adsorbed as lytic indicator cells. Isolated outer layer bluetongue virus polypeptide 2, from gradients at pH 6, and polypeptides 2 and 5, from gradients at pH 7, produced a strong primary IgM plaque-forming cell response. The subviral particles of density 1, 39 g.cm-3 and the bluetongue virus core particles of density 1,42 g.cm-3 also stimulated an IgM response at least as strong as that to intact bluetongue virus of density 1,38 g.cm-3. The isolated bluetongue virus fractions therefore appear to maintain their immunogenic integrity as effectively as those of intact bluetongue virus. The pattern of the immune response to bluetongue virus type 4 is similar to that of type 10.     

Kinetics of the IgM and IgG immunological response to sheep erythrocytes and bluetongue virus in mice.

The IgM and IgG response of mice to sheep erythrocytes (SRBC) and bluetongue virus (BTV) was determined by means of haemolytic plaque assays. Maximum primary IgM response to SRBC occurred after 4 days but declined rapidly to 4% of the maximum by Day 9. A lag period of about 2 days was observed in the appearance of IgG haemolytic plaque-forming cells (PFC) but they reached a maximum after 6-9 days. Secondary immunization resulted in the stimulation particularly of IgG PFC and from Day 6 onwards IgG predominated in the immunological response. The IgM response to BTV was remarkably similar to that observed when SRBC were used as antigen. IgG PFC, however, appeared within a day of the IgM, reaching a peak on Days 4-5. From then onwards, IgG PFC predominated in the response. At BTV concentrations of up to 10 mug per mouse, the virulent strain of BTV type 3 produced the weakest response. At higher antigen concentrations there was very little difference in the response to the serotypes tested, although the virulent strain of BTV type 4 tended to produce the strongest response.     

Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies

The immune response to bluetongue virus in sheep and cattle was studied by applying a newly developed indirect enzyme-linked immunosorbent assay (ELISA). Purified virus obtained by sucrose gradient centrifugation was used at a concentration of 0.01 optical density units (formula: see text) to coat individual wells (200 microliter) of a microtitration plate. Dilution of antigen was performed in 0.05 M carbonate buffer, pH 9.6, and adsorption lasted for at least 16 hours at 4 C. Coated plates retained their activity for 10 weeks when stored at 4 C. Sera recovered from experimentally infected sheep and cattle were tested together with known negative sera. A good correlation between results was obtained with the modified complement-fixation test and the ELISA; however, the ELISA proved to be more sensitive. The group specificity of the ELISA was proven by testing various type-specific sheep and cattle immune sera. The ELISA has potential for the detection of group-specific antibodies to bluetongue virus infection.     

Primary in vitro stimulation of antibody production by lizard splenocytes

Single-cell suspensions of adult lizard (Chalcides ocellatus) spleen have been induced, in vitro, to produce a primary immune response. Using rat red cells (RRBC) as antigen and the culture conditions normally used in most vertebrate species but new for reptilia, it has been found that, in vitro at 37 degrees C, lizard spleen cells produce an antibody-forming response optimal at day 10. The response depends on the number of cultured cells and the dose of antigen, and parallels that obtained in vivo. Leibovitz (L-15) medium supplemented with 10% normal adult lizard serum was a satisfactory culture medium. 2-mercaptoethanol (2-ME), an ingredient used in mammalian cell culture, enhanced antibody production in lizard cells.     

Presence of bluetongue virus in the marginal zone of the spleen in acute infected sheep

Bluetongue virus, a member of the genus Orbivirus of the family Reoviridae, is the causative agent of bluetongue, which is a non-contagious Culicoides mediated blood-borne disease. The present study characterizes the pathogenicity of a Taiwan prototype BTV2/KM/2003 in Corriedale sheep inoculated subcutaneously into the ear pinna. Histologically, multifocal petechiated hemorrhage, with mild to moderate inflammation and edema, were present in the contralateral ear pinna, tongue, and facial skin, without remarkable lesions in lymphoid organs. By days post-infection (DPI) 7, viral VP7 antigen, detected by immunohistochemistry, presented in the spleen, chiefly located in the outer rim of <3 cell thickness of marginal zone macrophages bordering the marginal zone and red pulp, and T lymphocytes of the red pulp. By DPI 11, viral signals shifted from the marginal zone to macrophages and small lymphocytes within follicles of the spleen. In situ hybridization with VP7 gene probe detected strong signals in the spleen, chiefly spanning the whole width of 5-10 cell thickness of the marginal zone, including the marginal zone macrophages and marginal zone B cells, as well as macrophages of sheathed capillaries in the red pulp. This study demonstrates molecular as well as morphologic evidence of the presence of bluetongue virus in the marginal zone of the spleen, most likely associated with viremia in acute infection, as previously demonstrated by the authors.     

A soluble immunosuppressor substance in spleen in deer mice infected with Trypanosoma brucei.

A soluble substance that blocks B-cell response was extracted from spleen of deer mice (Peromyscus maniculatus) chronically infected with Trypanosoma brucei. Immunosuppressor activity of this substance was demonstrated by plaque-forming cell assays of spleen cells from Swiss/Webster mice inoculated with sheep red blood cells and simultaneously given the suppressor substance, and of spleen cell cultures treated with sheep red blood cells and suppressor substance. Studies by light and electron microscopy of spleen of immunosuppressed Swiss/Webster mice showed that the suppressor substance blocks germinal center formation and prevents plasma cell differentiation in the red pulp cords.     

Immunization and culture of rainbow trout organ sections in vitro

Splenic and anterior kidney sections or whole organs were excised from large (1 kg) or small (200 g) rainbow trout (Salmo gairdneri) and placed in sterile 60 mm plastic plates containing 10 ml of Eagle's minimal essential medium (EMEM) supplemented with normal or fetal calf serum for in vitro culture. The organ samples were immunized in vitro by direct injection or by mixing in the medium Yersinia ruckeri O-antigen or dinitrophenyl-Ficoll. The medium was changed once during the 10-day incubation at 15 C. The passive hemolytic plaque assay demonstrated antibody production from the plaque-forming cells (PFC); passive hemagglutination was used to measure antibody titers in the media. High numbers of PFC occurred in cultures of either kidney or spleen, demonstrating that these organs can function independently for antibody production. Splenic sections from large fish produced more PFC than comparable whole organs from small fish. EMEM supplemented with 2% normal calf serum was a satisfactory culture medium. 2-hydroxyethyl-mercaptan an ingredient used in mammalian cell culture, inhibited antibody production in trout cells. These techniques are being used in the culture of organs and cells to elucidate pathways and sequences of antigen uptake and delivery of the immunopoietic tissues in trout.     

Isolation of bluetongue vaccine virus from infected sheep by direct inoculation onto tissue culture.

A technique is described by which the vaccine strain of bluetongue virus (BTVV) may be isolated from infected fetal, neonatal, and adult sheep tissues utilizing tissue culture. The data from these studies provides evidence that 1) BTVV can be readily isolated from infected fetal and newborn tissues by tissue culture, 2) mild treatment of tissues and utilization of lysed cells as inoculum an effective means of recovering vaccine virus, 3) BTVV can be isolated with equal efficacy from mononuclear fractions and from erythrocyte granulocyte fractions of viremic blood, and 5) the brain of fetal lambs and the spleen and liver of neonatal lambs appear to be the tissues from which vaccine can be consistently isolated.     

Bluetongue virus in bovine semen: viral isolation

Vero cell cultures and embryonating chicken eggs were used for direct isolation of bluetongue virus from cattle blood and from semen samples. Cell culture and embryonating chicken eggs each were more effective than was the blood autograft inoculation of susceptible sheep with selected blood and semen samples. Evaluation of the cell culture technique indicated that the quality of the distilled water was the primary factor responsible for the increased sensitivity of the Vero cell cultures for the present blue-tongue viral isolations. Test results showed that urine was a poor specimen for viral isolation when assayed in chicken eggs. A comparison of tests for precipitating and complement-fixing antibodies to bluetongue virus indicated that the precipitin test was the more accurate of the two tests.     

Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.     

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.     

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep

Objective To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.
Design A clinical and pathological study.
Procedure Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies.
Results All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection.
Conclusions The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.     

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.     

Anti-idiotype technique: an alternative approach for immunodiagnosis of bluetongue

In development of a bluetongue alternative immunodiagnostic rest, the polyclonal anti-idiotypic antibodies were generated by the sequential immunization of rabbits with three monoclonal antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies recognize the idiotypes that are located within or near the antigen-combining sites and are associated with both heavy and light chains of the antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies mimic the VP7 antigen by recognizing the anti-VP7 antibodies from cattle and sheep that were infected with various serotypes of bluetongue viruses. The results indicate that the rabbit anti-idiotypic antibodies may be used as surrogate antigen in serological assays to detect the antibodies from different species of animals infected with various serotypes of bluetongue viruses.     

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment and characterization of a chicken mononuclear cell line

A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.     

Toggenburg Orbivirus, a new bluetongue virus: Initial detection, first observations in field and experimental infection of goats and sheep

A novel bluetongue virus termed “Toggenburg Orbivirus” (TOV) was detected in two Swiss goat flocks. This orbivirus was characterized by sequencing of 7 of its 10 viral genome segments. The sequencing data revealed that this virus is likely to represent a new serotype of bluetongue virus [Hofmann, M.A., Renzullo, S., Mader, M., Chaignat, V., Worwa, G., Thuer, B., 2008b. Genetic characterization of Toggenburg Orbivirus (TOV) as a tentative 25th serotype of bluetongue virus, detected in goats from Switzerland. Emerg. Infect. Dis. 14, 1855–1861].In the field, no clinical signs were observed in TOV-infected adult goats; however, several stillborn and weak born kids were reported. Although born during a period of extremely low vector activity, one of these kids was found to be antibody and viral genome positive and died 3.5 weeks postpartum.Experimental infection of goats and sheep, using TOV-positive field blood samples, was performed to assess the pathogenicity of this virus.Goats did not show any clinical or pathological signs, whereas in sheep mild bluetongue-like clinical signs were observed. Necropsy of sheep demonstrated bluetongue-typical hemorrhages in the wall of the pulmonary artery. Viral RNA was detected in organs, e.g. spleen, palatine tonsils, lung and several lymph nodes of three experimentally infected animals.Unlike other bluetongue virus serotypes, it was not possible to propagate the virus, either from naturally or experimentally infected animals in any of the tested mammalian or insect cell lines or in embryonated chicken eggs.In small ruminants, TOV leads to mild bluetongue-like symptoms. Further investigations about prevalence of this virus are needed to increase the knowledge on its epidemiology.     

Isolation and characterization of epizootic hemorrhagic disease virus from white-tailed deer (Odocoileus virginianus) in eastern Washington.

A virus was isolated from the spleen of a white-tailed deer (Odocoileus virginianus) that had died during an epizootic in Washington state in 1967. Inoculation of a 10% spleen suspension from the deer caused hemorrhagic disease in normal white-tailed deer. Studies were conducted on the biological, physicochemical, and serologic properties of the Washington isolate. An in vitro assay system, utilizing a cultured primary of white-tailed deer fetal cells from an entire fetus, was employed for isolation and propagation of the virus. Cytopathic effect was characterized by focal development of rounded and clumped cells. Propagation was unsuccessful in suckling mice, BHK-21, and Vero cell cultures. The virus was resistant to treatment with ether, sodium deoxycholate, trypsin, oxytetracycline hydrochloride, and was sensitive to chloroform. Virus yield was not affected when infected cultures were treated with 5-iodo-2'-deoxyuridine, but dactinomycin (actinomycin D) treatment of infected cultures reduced virus yield. The virus was inactivated when heated at 70 C for 5 minutes or when exposed to pH 5 for 18 hours at 4 C. The virus was completely excluded from the filtrate by a 0.10- micronm (APD) membrane filter. Staining of infected cells with acridine orange indicated the presence of double-standard nucleic acid in the cytoplasm. Serum-neutralization tests with antiserums against the homologous virus and the New Jersey and Alberta strains of epizootic hemorrhagic disease virus resulted in neutralization of the Washington isolate. The Washington virus was not neutralized by bluetongue virus antiserum. Cells infected with the Washington isolate exhibited intracytoplasmic fluorescence by the indirect fluorescent antibody method with New Jersey and Alberta epizootic hemorrhagic disease antiserums but not with bluetongue antiserum.     

蓝舌病病毒17型VP2蛋白单克隆抗体的制备及其抗原表位的鉴定

为制备蓝舌病病毒(BTV)血清17型VP2蛋白的单克隆抗体(MAb)及鉴定其抗原表位,本研究用原核表达系统部分重叠表达的两段VP2蛋白共同免疫BALB/c小鼠,采用细胞融合技术获得杂交瘤细胞,通过以重组VP2蛋为白包被抗原的间接ELISA筛选获得2株稳定分泌抗BTV17 VP2蛋白的MAbs杂交瘤细胞株,分别命名为3F4和4H10.Ig亚类鉴定2株MAbs均为IgG1/k链.Western blot证明,2株MAbs均能识别重组VP2蛋白.间接免疫荧光试验表明:2株MAbs均与BTV17呈阳性反应,其中MAb 3F4与BTV1、BTV2、BTV3、BTV5、BTV8、BTV11、BTV13、BTV16、BTV23、茨城病病毒(IBAV)、牛轮状病毒(BRV)、牛呼肠孤病毒(RV)均呈阴性反应,但与BTV10和BTV24呈弱阳性反应.利用合成多肽对VP2抗原表位鉴定结果表明,MAb 3F4识别的抗原表位为540DPWNNR545,MAb 4H10识别的抗原表位为540DPWNNRA546.本研究结果为建立BTV17型特异性检测方法及VP2功能研究奠定了基础.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.