Detection Of Bluetongue Virus Antibodies Research Articles

Detection of bluetongue virus antibodies in sheep from Paraná, Brazil

Bluetongue (BT) is an infectious and non-contagious disease caused by bluetongue virus (BTV) belonging to the genus Orbivirus. It is transmitted by a hematophagous vector, Culicoides sp., to ruminants, particularly to sheep, which are most susceptible to this disease. The main serological tests are agar gel immunodiffusion (AGID), which is recommended by the World Organization for Animal Health (OIE), and the competitive enzyme-linked immunosorbent assay (cELISA), which has the advantage of no cross-reaction with other orbiviruses. The aim was to compare the results of these two tests by conducting them on sera collected from sheep in the state of Paraná, Brazil. From March to October 2017, serum samples were collected from 270 sheep from 10 farms in six mesoregions of Paraná. The samples were subjected to AGID and cELISA to detect antibodies against BTV. Based on the test results, we classified the sheep as low, moderate, and high occurrence. The results demonstrated that 64.81% (175/270) of the sheep were seropositive through the cELISA test, showing a high occurrence, and 41.11% (111/270) were seropositive through the AGID test, indicating a moderate occurrence. The concordance between the tests was moderate (0.51) as determined by the Kappa coefficient. Among the studied farms, 90% (9/10) presented at least one seropositive sheep, and the number of animals tested positive by the cELISA test was higher than those by the AGID test. Favorable climate, which favors the presence and multiplication of the culicoid vector and the occurrence of infection, was the biggest predominant factor responsible for the obtained results. The low occurrence in farms with milder climate suggest that the presence of antibodies also occurs due to the low pathogenicity of circulating serotypes in the different mesoregions studied. It is concluded that BTV infection is present in the sheep herds in Paraná, and the occurrence was moderate detected by AGID test and high detected by cELISA test.

Immuno-reactivity of recombinant non-structural protein 3 N-terminus (rNS3Nt) in indirect-ELISA for detection of bluetongue viral antibodies in serum samples

Bluetongue, an arthropod borne non-contagious disease of ruminants especially sheep, is caused by bluetongue virus (BTV). Detection of BTV antibodies in susceptible hosts is considered to be of significance in disease diagnosis and differentiation. In the present study, a partial NS3 gene encoding for non-structural protein-3 N-terminus (1MT117 aa) of BTV-23, produced as purified recombinant NS3Nt fusion protein (~32 kDa) using prokaryotic expression system (Escherichia coli), was evaluated as a candidate antigen in an indirect-ELISA (rNS3Nt-ELISA) to measure the serologic response to NS3 protein in small ruminants. The rNS3Nt fusion protein obtained in sufficient quantity and quality has good reactivity in detecting NS3 specific antibodies in field serum samples by indirect-ELISA. As NS3 protein is highly conserved, rNS3Nt-ELISA has potential for NS3 specific detection of antibodies in BTV affected animals irrespective of different viral serotypes. In comparison to structural protein (VP7) based c-ELISA kit and i-ELISA kit, the diagnostic sensitivity (85.1%, 86.2%) and specificity (92.5%, 93.2%) of rNS3Nt-ELISA were found to be relatively lower, respectively. Nevertheless, the study indicated the potential utility of rNS3Nt-ELISA as an alternate assay in routine sero-diagnosis of BTV infection and possible sero-surveillance of ruminants under DIVA strategy.

Detection of bluetongue virus antibodies in goats of Delhi

Seropositivity for bluetongue virus antibodies in nondescript adult goats of Delhi, India was evaluated by indigenously developed Agar Gel Immunodiffusion test (AGID). Soluble bluetongue virus antigen was prepared from Baby Hamster Kidney-21 (BHK-21) adapted bluetongue The Indian Journal of Small Ruminants 2015, 21(1): 135–137 Bluetongue virus antibodies in goats virus serotype 1 using ammonium sulphate precipitations. Out of 469 serum samples of non-descript adult goats (371 males and 98 females) 62 serum samples (53 males and 9 females) were found positive with overall seropositivity of 13.21%. Serum samples collected in winter showed greater seropositivity (18.60%) than those collected in monsoon (3.57%). Seropositivity in male and female goats was 14.28 and 9.28%, respectively.

Occurrence of Dual Infection of Peste-Des-Petits-Ruminants and Goatpox in Indigenous Goats of Central India

Peste-des-petits-ruminants (PPR), bluetongue (BT) and goatpox (GP) have been well recognized as causes of significant economic losses in the small ruminant population of Asia and Africa. We describe here the occurrence of these three in an outbreak noticed in non-descript goats from a subtropical region of central India. An investigation was carried out to confirm the aetiology of the heavy mortality in goats (74.6%, 112/150), with testing of samples from 12 surviving animals exhibiting mixed clinical signs indicative of PPR, BT and GP. Sandwich ELISA was used to detect PPR virus antigen and competition ELISA to detect PPR virus and BT virus antibodies. GP was confirmed on the basis of nodular lesions and an immunodiffusion assay. Eight of the 12 affected animals (66.7%) were positive for PPR virus and BT virus antibodies, and two goats (16.7%, 2/12) exhibiting clinical lesions of pox were also found positive for PPR virus/antibodies and BT virus antibodies, respectively. Although BT virus could not be identified in any sample, detection of BT virus antibodies indicated previous or possibly concurrent infection with BT virus in these goats. The N-gene-based RT-PCR was used to confirm the PPR infection in these goats, and one of the amplicons was sequenced. The sequence and phylogenetic analysis revealed close proximity to PPR virus isolates from Tibet and China, with sequence homology of up to 96.9%. The sequence homology was relatively low with the majority of other Indian isolates (72.7-93.5%). The detection of this new PPR virus sequence indicates the circulation of cross-border strains in this region of India. It is presumed that the heavy mortality observed in goats is possibly attributable to the occurrence of mixed infection of PPR and GP, or PPR, BT and GP.

Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs

Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein's upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.

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 absorbed 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 fron 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 loq and in the same order as that of the dipstick assay. The sensitivity of dipstick and NC strips assays in testing of 265 samples relative to C-ELISA results were 83.2% and 91.3%, respectively. The B-dot ELISA which is rapid, simple and inexpensive may be an alternative field test for screening and monitoring cattle, and possibly other ruminants, exposed to BTV for control and surveillance programs.

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 days 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.

Dot immunobinding assay in comparison with enzyme-linked immunosorbent assay for the detection of bluetongue virus antibodies in sheep

A total of 384 sheep serum samples collected from two organised sheep farms was tested by dot immunobinding assay (DIA) and indirect enzyme-linked immunosorbent assay (I-ELISA) for the presence of bluetongue virus (BTV) antibodies. The results of both these assays were compared to find a sensitive, specific, rapid, easily performed and economical test for the diagnosis of bluetongue disease. DIA detected BTV antibodies in 210 samples (54.94%) and I-ELISA detected 157 positive samples (40.88%). Competitive ELISA (C-ELISA) was performed to check the discrepancies in I-ELISA and DIA. On the basis of these tests the overall agreement, relative specificity and sensitivity between ELISA and DIA were 75%, 87.6% and 100%, respectively. DIA was found to be a rapid, sensitive, easily performed and economical test as compared to ELISA.

Expression of bluetongue virus group-specific antigen VP3 in insect cells by a baculovirus vector: its use for the detection of bluetongue virus antibodies.

DNA representing RNA segment 3 of bluetongue virus (BTV) serotype 17, corresponding to the gene that codes for a group-specific antigen VP3, has been inserted into a baculovirus transfer vector in lieu of the 5' coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After cotransfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived recombinant transfer vector DNA, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with one of these recombinant viruses, a protein that was similar in size and antigenic properties to the BTV VP3 protein was synthesized. Antibodies raised in mice or rabbits to the baculovirus-expressed VP3 protein immunoprecipitated the VP3 protein of BTV-17 as well as that of BTV-10. The expressed antigen reacted with antisera representing four U.S.A. BTV serotypes in an indirect ELISA test.

Studies on the fluorescent and neutralizing antibodies to bluetongue virus in sheep.

The sensitivity of the indirect fluorescent antibody technique (FAT) was compared with the serum-virus neutralization test (S-VNT) in the detection of bluetongue virus antibodies.