European Inter-Laboratory Proficiency Test for Dourine Antibody Detection Using the Complement Fixation Test.

Dourine is a sexually transmitted parasitic disease affecting equids. Its causative agent is referred to as Trypanosoma equiperdum and the prescribed serodiagnosis method is the complement fixation test (CFT). In the context of our European Reference Laboratory mandate for equine diseases (excluding African horse sickness), we organised dourine CFT inter-laboratory proficiency tests (ILPTs) in 2015, 2018 and 2022 to evaluate the performance of the European Union network of National Reference Laboratories (NRLs) for dourine. ILPT panels were composed of horse sera with or without antibodies against Trypanosoma spp. originating from non-infected, immunised or experimentally infected horses. Twenty-two NRLs participated in at least one of the three sessions. In 2015, 2018 and 2022, the percentage of laboratories obtaining 100% of the expected results was 57, 90 and 80, respectively. These dourine CFT ILPTs showed the benefits of standardising the method's detection limit and underlined the constant need to evaluate NRLs to improve the network's performance. These results also argue in favour of the need for a representative bio-bank to improve the representativeness of ILPT samples and to allow the adoption of alternative serological methods for international surveillance of dourine.

Development of a microsphere-based immunoassay for the serological diagnosis of equine trypanosomosis.

Trypanozoon infections in equids are caused by three parasite species in the Trypanozoon subgenus: Trypanosoma equiperdum, T. brucei and T. evansi. They are respectively responsible for infectious diseases dourine, nagana and surra. Due to the threat that Trypanozoon infection represents for international horse trading, accurate diagnostic tests are crucial. Current tests suffer from poor sensitivity and specificity, due in the first case to the transient presence of parasites in the blood and in the second, to antigenic cross-reactivity among Trypanozoon subspecies. This study was designed to develop a microsphere-based immunoassay for diagnosing equine trypanosomosis. We tested beads coated with eight Trypanosoma spp. recombinant antigens: enolase, GM6, PFR1, PFR2, ISG65, VSGat, RoTat1.2 and JN2118HU. Of these, GM6 was identified as the best candidate for the serological diagnosis of Trypanozoon infections in equids. Using a receiver operating characteristic (ROC) analysis on 349 equine sera, anti-GM6 antibodies were detected with an AUC value of 0.994 offering a sensitivity of 97.9% and a specificity of 96.0%. Our findings show that the GM6 antigen is a good target for diagnosing equine trypanosomosis using a microsphere-based immunoassay. This promising assay could be a useful alternative to the official diagnostic tool for equine trypanosomosis.

Molecular detection of 7SL-derived small RNA is a promising alternative for trypanosomosis diagnosis.

Equine trypanosomosis comprises different parasitic diseases caused by protozoa of the subgenus Trypanozoon: Trypanosoma equiperdum (causative agent of dourine), Trypanosoma brucei (nagana) and Trypanosoma evansi (surra). Due to the absence of a vaccine and the lack of efficacy of the few available drugs, these diseases represent a major health and economic problem for international equine trade. Development of affordable, sensitive and specific diagnostic tests is therefore crucial to ensure the control of these diseases. Recently, it has been shown that a small RNA derived from the 7SL gene (7SL-sRNA) is produced in high concentrations in sera of cattle infected with Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei. Our objective was to determine whether 7SL-sRNA could serve as a marker of active infection in equids experimentally infected with Trypanosoma equiperdum by analysing the sensitivity, specificity and stability of the 7SL-sRNA. Using a two-step RT-qPCR, we were able to detect the presence of 7SL-sRNA between 2 and 7 days post-infection, whereas seroconversion was detected by complement fixation test between 5 and 14 days post-infection. There was a rapid loss of 7SL-sRNA signal from the blood of infected animals one day post-trypanocide treatment. The 7SL-sRNA RT-qPCR allowed an early detection of a treatment failure revealed by glucocorticoid-induced immunosuppression. In addition, the 7SL-sRNA remains detectable in positive sera after 7 days of storage at either 4°C, room temperature or 30°C, suggesting that there is no need to refrigerate serum samples before analysis. Our findings demonstrate continual detection of 7SL-sRNA over an extended period of experimental infection, with signals detected more than six weeks after inoculation. The detection of a strong and consistent 7SL-sRNA signal even during subpatent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.