Interferon induction in bovine and feline monolayer cultures by four bluetongue virus serotypes.

The interferon inducing ability of bluetongue viruses was studied in bovine and feline monolayer cultures inoculated with each of four bluetongue virus serotypes. Interferon was assayed by a plaque reduction method in monolayer cultures with vesicular stomatitis virus as challenge virus. Interferon was produced by bovine turbinate, Georgia bovine kidney, and Crandell feline kidney monolayer cultures in response to bluetongue virus serotypes 10, 11, 13 and 17. The antiviral substances produced by the bluetongue virus infected cultures had properties of interferon.     

Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21

Genetic reassortment studies of bluetongue virus (BTV) Types 20 and 21 have revealed a reassortant genotype that was not neutralized serotype-specifically. In reciprocal neutralization tests, BTV 20 and 21 were neutralized specifically by homologous antiserum. Similarly, reassortants that possessed both outer capsid proteins (i.e., VP2 and VP5) from the same parent virus reacted with that antiserum specifically. However, two reassortants, 16(9) and 19(1), with VP2 of BTV 20 and VP5 of BTV 21 had intermediate neutralization characteristics. These reassortants were neutralized to high titres by antiserum to BTV 20 and to lower, but significant titres by antiserum to BTV 21. In addition, antiserum to BTV 20 induced 10-16-fold higher titres in plaque reduction neutralization (PRN) tests with these two reassortants compared with BTV 20 itself. Evidence of the serological cross-reactivity of Reassortants 16(9) and 19(1) was also found with respect to reductions in plaque sizes observed in the PRN tests. The average plaque sizes of these reassortants were reduced to differing extents by antiserum to BTV 20 and 21, while those formed by the parent viruses were reduced in size by homologous antiserum only. Immunoblotting analysis of the structural proteins of BTV 20 and 21 demonstrated that VP2 alone was antigenically distinct, therefore confirming its role in determining serotype specificity in virus-neutralization tests. Electrophoretic analysis revealed considerable migrational differences between VP2 and VP5 of the parent viruses, suggesting that there was some divergence in their molecular weights, intrinsic charges or structural compositions. Taken together, the data suggest that the intermediate neutralization characteristics of the reassortants that contain VP2 and VP5 from different parent viruses are due to conformational alterations in their outer capsid structure which allow antibody recognition of common neutralizing epitopes that are not exposed on BTV 20 or BTV 21.     

Haemophilus parahaemolyticus serotypes. Serological response

Serotypes 1,2,4 and 5 of Haemophilus parahaemolyticus were inoculated into, respectively, 4,4,2 and 5 pigs. Serum samples were tested for circulating antibodies by the modified complement fixation test (CF test). When individual serotypes were used as antigen, titers were found only to the serotype which had been used for inoculation. Using antigen in which the serotypes were pooled, antibodies were demonstrated in sera from all the pigs. The CF titers obtained with the pooled antigen were equivalent to those found with each serotype separately. When the CF test was used for serological examination of field sera there was full agreement between the results obtained with the pooled antigen and those obtained with serotype 2 antigen alone. No cross reactions were found with the pooled antigen in herds that were sero-positive to Haemophilus parasuis, strain 4800. The experiment has shown that there is no serological cross reaction between serotypes 1, 2, 4 and 5 when they are used as antigen in the CF test. Also, the results imply that with a pool of the different serotypes of Haemophilus paralyticus as antigen similar results may be obtained as with the single serotype 2 antigen.     

用间接ELISA检测鸭疫里默氏菌不同血清型之间的交叉反应

鸭疫里默氏菌（Riemerella anatipestifer，RA）感染即鸭传染性浆膜炎，是目前危害我国肉鸭养殖业的主要疾病，因该病可导致较高的死亡率和淘汰率，其发生常给广大养鸭户造成巨大的经济损失。迄今为止，国际上已报道RA存在21个血清型一，我国许多养鸭地区也已流行10多种血清型，这种流行现状进一步增加了控制鸭传染性浆膜炎的难度，研究表明，l、2、6、10型是我国一些地区主要流行的血清型。     

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.     

14型蓝舌病病毒VP7蛋白原核表达及间接ELISA抗体检测方法的建立

为建立蓝舌病(bluetongue,BT)的血清学诊断方法,本研究通过得到14型蓝舌病病毒(bluetongue virus,BTV)的s7基因,并在大肠杆菌表达系统中进行表达,原核表达得到VP7蛋白,并对表达的包涵体VP7蛋白进行纯化,Western blot分析表明,纯化的VP7蛋白具有良好的抗原性。以纯化的VP7蛋白作为包被抗原,建立间接ELISA检测方法。该方法与羊痘阳性血清,羊口蹄疫阳性血清和羊的小反刍兽疫阳性血清均无交叉反应,与中和试验比较,两者符合率为90%,ELISA批内和批间重复性试验显示,D值的变异系数小于10%。说明本方法具有良好的特异性和重复性。本研究在国内首次建立了14型BTV抗体间接ELISA方法,可用于BTV感染的流行病学调查以及疫苗接种动物的抗体水平监测,为以后相关BTV ELISA检测试剂盒研发提供技术平台。     

Identification of seven serotypes of bluetongue virus from the People's Republic of China

Seven serotypes (1, 2, 3, 4, 12, 15 and 16) of bluetongue virus were isolated from the blood of sheep and cattle in the People's Republic of China between 1986 and 1996. Six of these viruses were isolated in Yunnan province. The sheep from which serotypes 1 and 16 were isolated showed obvious signs of bluetongue disease, whereas the cattle from which serotypes 2, 3, 4, 12 and 15 were isolated were clinically normal. Phylogenetic analyses of these viruses indicate that they are more closely related to one another, and to an Australian strain of serotype 1, than they are to prototype strains of bluetongue virus serotypes 2, 10, 11, 13 and 17 from the USA.     

Serological evidence of the occurrence of bluetongue in Iraq.

Precipitating antibodies against bluetongue were detected in sheep and goat serum samples collected from animals slaughtered in Baghdad abattoir. Out of 294 sheep serum samples and 110 goat serum samples examined, 28 and 18 samples respectively showed precipitating activity. In addition, examination of sheep serum samples collected from localities where clinical cases similar to bluetongue were previously reported revealed the presence of bluetongue precipitating antibodies in 101 sera out of 198 samples examined. This is the first report confirming the occurrence of bluetongue in Iraq.     

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.     

用抗原捕获ELISA方法及过氧化物酶染色法检测蓝舌病病毒

蓝舌病 (Bluetongue,BLU)是由呼肠弧病毒科环状病毒属蓝舌病病毒引起的羊等反刍动物的严重传染病。该病传播快 ,发病率和死亡率都较高 ,因而对畜牧业的发展有着极大的影响 ,进出境动物检疫中将其列为一类传染病。蓝舌病血清抗体的检验方法通常较简便 ,如琼脂扩散 (AGID)、补体结合试验(CFT)及竟争性酶联免疫吸附试验 (C- ELISA)等 ,然而对其病毒的直接检测 ,通常比较复杂 ,并要较长时间 ,一般很难在 45天规定的隔离检疫期内完成任务 ,不符合口岸动物检疫工作快速、准确、简便的基本要求。为了改变这种状况 ,我们采用了一种新程序 ,引…     

A comparison of an australian bluetongue virus isolate (CSIRO 19) with other bluetongue virus serotypes by cross-hybridization and cross-immune precipitation

No major differences in size were observed when both the double-stranded RNA and the polypeptides of the Australian bluetongue virus (BTV) isolate CSIRO 19 (BTV-20) were compared with those of other BTV serotypes such as BTV-10 and BTV-4. Minor capsid polypeptide P6 of both BTV-20 and BTV-4, which electrophoreses as a single band on continuous phosphate buffered gels, in separated into 2 distinct bands on discontinuous glycine-buffered gels. This was not the case with BTV-10. Cross-immune precipitation of BTV-20 with BTV-10, BTV-17, BTV-4 and BTV-3 indicated strong immunological cross-reaction of the group-specific antigen P7 of the different serotypes. There was also some cross-immune precipitation of the serotype-specific polypeptide P2 of BTV-20 and BTV-4. This result is in agreement with the observed cross neutralization of these 2 viruses. The main distinction between BTV-20 and the other BTV serotypes was observed in cross-hybridization experiments. The homology between the nucleic acid of BTV-20 and other BTV serotypes was less than 30%, whereas homology normally found between BTV serotypes is at least 70%. The hybridization products of the different BTV serotypes were analysed by electrophoresis and fluorography. Two main hybrid segments were observed in all heterologous hybridizations with BTV-20 as a compared with 7 hybrid segments in hybridizations between BTV-4 and BTV-10. In order to determine from which genome segment of BTV-20 these 2 hybrid segments were derived, the hybridizations were carried out with individually purified double-stranded RNA segments. These results indicate that the 2 segments of BTV-20 that show the largest homology to corresponding segments of a heterologous BTV serotype are No. 7 and 10.     

Neutralizing antibody responses to bluetongue and epizootic hemorrhagic disease virus serotypes in beef cattle

Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (BTV) immunodiffusion test (IDT) and virus-neutralization test (VNT), for 5 BTV serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (EHDV) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the BTV IDT at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconverted as assessed by results of the BTV IDT. In the 16 seropositive cattle in 1984, neutralizing antibodies were detected to BTV serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to BTV-2 or BTV-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the IDT. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to BTV serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1985, no cattle seroconverted to BTV-2. Clinical diseases attributable to BTV or EHDV was not detected in these cattle in 1984 or 1985.     

A competitive ELISA for detection of antibodies to the group antigen of bluetongue virus.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect antibodies to the group antigen of bluetongue virus (BTV). The epitope recognized by the BTV-specific monoclonal antibody was confirmed, by immunofluorescence staining of monolayers of virus-infected Vero cells, to be present on BTV serotypes 2, 10, 11, 13, and 17 but not on epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2. Sera from BTV-inoculated ruminants and rabbits were used to evaluate the cELISA and to compare its specificity and sensitivity with that of the conventional BTV-specific agar gel immunodiffusion (AGID) and serum neutralization (SN) tests. Rabbit antisera to the 5 serotypes of BTV present in the United States had cELISA titers (inverse of the final dilution of serum that gave greater than 20% inhibition) that ranged from 32 to greater than 1.024. Seroconversion of the 8 calves and lambs inoculated with BTV was detected by all 3 serologic tests (SN, AGID, cELISA) by 6 weeks after inoculation. Specificity of the cELISA test was confirmed with bovine sera that contained neutralizing antibodies to EHDV but not to the 5 serotypes of BTV present in the United States; these sera gave positive results by AGID test but were negative by cELISA. The sensitivity and specificity of the cELISA test was further confirmed by analysis of a panel of bovine test sera supplied by the National Veterinary Services Laboratories, indicating that the cELISA is a superior test for detection of BTV group-specific antibodies in sera from ruminants in the United States.     

Temporal appearance, geographic distribution, and species of origin of bluetongue virus serotypes in the United States.

Beginning in 1973, all available laboratory and field strains of bluetongue virus (BTV) from the United States were serotyped. Of the viral strains serotyped, 27 were collected from 1953 through 1972; 173 were collected from 1973 through 1977. Although 20 BTV serotypes have been found worldwide, only BTV serotypes 10, 11, 13, and 17 have been found in the United States. Since 1973, serotypes 11 and 17 have been the prevalent serotypes. Samples were collected over a 24-year period in the United States and represent a wide geographic area and diverse host sources (sheep, cattle, wild ruminants, and insect vectors). The collection was not a statistical sampling.     

Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection of bluetongue virus antibodies in cattle.

A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (BTV) is described for the detection of BTV antibodies to BTV in cattle sera. Dots of BTV antigens were adsorbed to nitrocellulose (NC) strips and/or NC mounted in the windows of dipsticks. After blocking the remaining sites of the NC paper with milk powder solution and immersion in the test sample, the NC strips and dipsticks were exposed to mAb. Bound mAb was detected with peroxidase conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in the test sample, BTV antigen sites were reactive with mAb as indicated by a brown colored dot in the presence of the enzyme substrate, hydrogen peroxide and diaminobenzidine. In the presence of sufficient anti-BTV antibodies no color reaction was observed. The performance of these assays in detecting anti-BTV antibody in field blood eluate samples, prepared from whole blood dried on filter paper, from 395 bluetongue-free cattle in Canada and 635 sentinel cattle in Florida, USA, was evaluated and compared with the standard competitive (C) ELISA. The specificity of the dipstick B-dot ELISA was identical to that of the C-ELISA in testing of BT-free Canadian cattle but not in the testing of samples from the sentinel cattle in Florida, resulting in values of 100% diagnostic and 88.9% relative specificity, respectively. Based on the C-ELISA, the specificity of the NC strip B-dot ELISA was low and in the same order as that of the dipstick assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus

The aim of this study was to estimate the diagnostic value of different commercially available ELISA kits for the detection of bluetongue virus (BTV) antibodies in infected and vaccinated animals. The relative specificity of ELISA kits was evaluated using a panel of sera originating from healthy cattle, never vaccinated nor exposed to BTV. All ELISA kits applied had a high relative specificity (99.3 - 100%). The relative sensitivity of ELISA kits assessed using a panel of sera collected from BTV infected cattle was also high and similar for all the kits (97.3 - 100%). However, the relative sensitivity evaluated on the basis of testing vaccinated animals was different: the highest sensitivity was found for Ingenasa, PrioCHECK and ID VET ELISAs (96.5 - 98.3%). Slightly lower sensitivity was calculated for Pourquier and LSI kits (82.8% and 85.4%, respectively) and much lower sensitivity was found for VMRD ELISA kit (69.5%). The repeatability of BTV ELISA kits was expressed as a coefficient of variation (CV) of results of sera tested 5 times in the same day and in different days by the period of 2 months, by the same person, in the same conditions, and by using the same equipment. The CVs of sera tested in all ELISA kits ranged from 6.1 to 9.8% and were below 10% threshold adopted as a maximum for the acceptable repeatability of the method. In conclusion, it can be stated that the applied ELISA kits can be a valuable diagnostic tool for the serological monitoring studies in the BTV contaminated premises. All the methods are very specific and sensitive when testing BTV infected animals. Nevertheless, the Ingenasa and PrioCHECK can be the most useful in sero-surveillance of livestock following vaccination.