Analysis of Trypanosoma equiperdum Recombinant Proteins for the Serological Diagnosis of Dourine.

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.

Production and Characterization of Monoclonal Antibodies Against the VP7 Protein of Epizootic Hemorrhagic Disease Virus.

Monoclonal antibodies (MAbs) against epizootic hemorrhagic disease virus (EHDV) were produced by immunizing BALB/c mice with rec-VP7-EHDV2; 66 clones producing MAbs able to recognize the VP7-EHDV with a strong reaction were obtained and tested in indirect enzyme-linked immunosorbent assay (i-ELISA) against the whole epizootic hemorrhagic disease (EHD) virus serotype 2; potential cross-reactions with related orbiviruses, as Bluetongue virus (BTV) and African horse sickness virus (AHSV), were investigated as well by i-ELISA, Western blot, and immunofluorescence. Fifty-three MAbs were specific for EHDV (VP7 recombinant protein and whole virus) and 13 reacted also with the VP7 of BTV. None of the MAbs reacted with AHSV. MAbs specific for EHDV were further characterized in a competitive ELISA (c-ELISA): 20 among them were found useful to develop a c-ELISA for the detection of antibodies against EHDV in bovine sera. The availability of this extensive set of MAbs provides the opportunity to develop a c-ELISA for the serological diagnosis of EHDV and to tune new methods for the isolation and identification of the virus in biological samples and cell cultures. The experimentation protocol was approved by the Italian Ministry of Health (number 639/2018-PR, Resp. to Prot. BDF16.13#295833199#).

Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels.

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.