Evaluation of a commercial ELISA for detection of epizootic haemorrhagic disease antibodies in domestic and wild ruminant sera.

Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are vector-borne viral diseases affecting domestic and wild ruminants. Both are notifiable under OIE rules. BT and EHD viruses (BTV and EHDV) are closely related Orbiviruses with structural, antigenic and molecular similarities. Both viruses can produce analogous clinical signs in susceptible animals. Serological tests are commonly used for BT and EHD diagnosis and surveillance. Competitive ELISA (c-ELISA) is the most widely used serological test for the specific detection of BTV or EHDV viral protein 7 (VP7) antibodies (Abs). The specificity and sensitivity of the BTV c-ELISA kits available on the market are recognized for the detection of BTV Abs. Concerning EHD, a single commercial EHDV c-ELISA kit (ELISA A kit) commonly used for diagnosis in Europe and Africa was available between 2011 and 2018 but is now no longer on the market. In this study, we evaluated a new commercial c-ELISA to detect ruminant EHDV VP7 Abs in 2,199 serum samples from cattle, sheep, goats, wild deer and zoo animals. The results showed that this ELISA kit is specific and can detect the presence of IgG anti-EHDV VP7 with a very good diagnostic specificity and a satisfactory sensitivity in domestic ruminants, zoo animals and wild deer. Therefore, the evaluated c-ELISA can detect the introduction of EHDV into an area where BTV-seropositive domestic animals are present. The performance of this kit is similar to that of the c-ELISA A kit and can thus be used for diagnosis.

Evaluation of an IGM-specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera.

Competitive-ELISA (c-ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c-ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti-VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM-capture ELISA prototype to detect ruminant anti-BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV-naive, infected, or/and vaccinated with BTV-1, -2, -4, -8, -9, -16, or -27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti-VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV-RNA in IgM-positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV-seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples.

A novel double-antigen sandwich ELISA for the species-independent detection of Crimean-Congo hemorrhagic fever virus-specific antibodies.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease in humans caused by the CCHF virus (CCHFV). The detection of anti-CCHFV antibodies in animals is used to reveal infection risk areas. Therefore a simple, quick and reliable multispecies assay for the detection of CCHFV-specific antibodies is needed. This work presents the development and validation of a novel CCHF double-antigen ELISA for the detection of anti-CCHFV nucleoprotein antibodies. The test requires 30 µl of serum, and results are obtained within 90 min. As the ELISA is based on recombinant N-protein of the IbAr10200 virus, it can be run under standard biosafety conditions. For assay validation, sera from 95 cattle and 176 small ruminants from CCHF-endemic regions (origin: Albania, Cameroon, Kosovo, Former Yugoslav Republic of Macedonia, Mauritania, Pakistan, Turkey) served as a positive reference serum panel. The CCHF antibody status of the positive reference samples had been previously confirmed by two serological assays (species-adapted VectorBest ELISA and Euroimmun IFA). CCHFV strains belonging to three different clades are known to circulate in the countries where the positive samples originated. Sera from 402 cattle and 804 small ruminants from Germany and France served as the negative serum panel, as both countries are considered outside of the CCHFV endemic zone. Sera from monkeys, camels, rats, ferrets, raccoon dogs, raccoons, foxes, hares, pigs and humans were also tested, to determine the suitability of this novel ELISA for these species. All negative reference sera were confirmed by the CCHF double-antigen ELISA, indicating a specificity of 100%. 268 of 271 positive reference sera tested positive for CCHFV-specific antibodies, 8sensitivity of 99%9. Further analysis are needed to ensure a recognition of the IbAr10200 nucleoprotein by antibodies directed against all known CCHFV clades. This is planned to be realized with sera from other regions covering the three missing clades.

Circulation of Crimean-Congo Hemorrhagic Fever Virus in the former Yugoslav Republic of Macedonia revealed by screening of cattle sera using a novel enzyme-linked immunosorbent assay.

BACKGROUND: There are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia. PRINCIPAL FINDINGS: A highly sensitive and specific ELISA was developed to detect CCHFV-specific IgG antibodies in cattle. The assay was validated by using 503 negative serum samples from a country where CCHFV has never been detected until now, and by using 54 positive serum samples. The positive sera were verified by using two commercially available assays (for testing human serum) which we have adapted for use in animals. The sensitivity of the novel ELISA was 98% and its specificity 99%. The presence of Hyalomma ticks was demonstrated in the Former Yugoslav Republic of Macedonia and depending on the region antibody prevalence rates up to 80% were detected in the cattle population. CONCLUSION: This article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.