A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.

Section: Discussionmentioning

confidence: 99%

A Duplex Fluorescent Microsphere Immunoassay for Detection of Bluetongue and Epizootic Hemorrhagic Disease Virus Antibodies in Cattle Sera

Drolet

Reister-Hendricks

2021

“…A multiplex fluorescence microsphere immunoassay (FMIA) was developed to detect IgM and IgG antibodies in ruminant sera to RVFV structural and non-structural proteins. Preliminary results demonstrate the potential of the FMIA diagnostic platform for the development of diagnostic tests that can be used to differentiate vaccinated from infected animals and for simultaneous differential diagnosis of several abortive and zoonotic pathogens [ 48 , 49 ].…”

Section: Introductionmentioning

confidence: 99%

“…The inhibition ELISA based on a whole tissue culture-derived, inactivated antigen [ 40 ] and competitive ELISA based on recombinant NP antigen [ 50 , 51 , 52 ] allow multi-species RVFV antibody detection using the same diagnostic procedure without requirements for species-specific conjugates. While in the last 15 years, several groups have developed and evaluated various ELISA formats based on RVFV recombinant NP antigen [ 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 ], more extensive evaluation of their diagnostic performance was only achieved in humans [ 58 ], buffalo [ 59 ], and cattle [ 60 ] to date.…”

Section: Introductionmentioning

confidence: 99%

Large-Scale International Validation of an Indirect ELISA Based on Recombinant Nucleocapsid Protein of Rift Valley Fever Virus for the Detection of IgG Antibody in Domestic Ruminants

Pawęska

Vuren

Msimang

et al. 2021

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.

“…Luminex technology, which uses colored magnetic polystyrene beads to capture antigens, is well known and widely applied for the detection of analytes [23]. Because of its multiplexing capability, this method has been used for differentiating infected from vaccinated animals for viral pathogens such as foot and mouth disease virus [24], West Nile virus [25], avian influenza virus [26], and Rift Valley fever virus [27]. ELISA is a traditional diagnostic method for detecting a viral antigen or antibody [28].…”

Section: Introductionmentioning

confidence: 99%

Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies

Wei

Wang

et al. 2020

Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni–nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.