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SummaryEquine 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 analyzing 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 sub-patent parasitemia and the early detection of treatment failure highlight the very promising nature of this new diagnostic method.

Trypanosoma brucei evansi and T. brucei equiperdum are animal infective trypanosomes conventionally classified by their clinical disease presentation, mode of transmission, host range, kinetoplast DNA (kDNA) composition and geographical distribution. Unlike other members of the subgenus Trypanozoon, they are non-tsetse transmitted and predominantly morphologically uniform (monomorphic) in their mammalian host. Their classification as independent species or subspecies has been long debated and genomic studies have found that isolates within T. brucei evansi and T. brucei equiperdum have polyphyletic origins. Since current taxonomy does not fully acknowledge these polyphyletic relationships, we re-analysed publicly available genomic data to carefully define each clade of monomorphic trypanosome. This allowed us to identify, and account for, lineage-specific variation. We included a recently published isolate, IVM-t1, which was originally isolated from the genital mucosa of a horse with dourine and typed as T. equiperdum. Our analyses corroborate previous studies in identifying at least four distinct monomorphic T. brucei clades. We also found clear lineage-specific variation in the selection efficacy and heterozygosity of the monomorphic lineages, supporting their distinct evolutionary histories. The inferred evolutionary position of IVM-t1 suggests its reassignment to the T. brucei evansi type B clade, challenging the relationship between the Trypanozoon species, the infected host, mode of transmission and the associated pathological phenotype. The analysis of IVM-t1 also provides, to our knowledge, the first evidence of the expansion of T. brucei evansi type B, or a fifth monomorphic lineage represented by IVM-t1, outside of Africa, with important possible implications for disease diagnosis.

Trypanosoma brucei evansi and T. brucei equiperdum are animal infective trypanosomes conventionally classified by their clinical disease presentation, mode of transmission, host range, kinetoplast DNA (kDNA) composition and geographical distribution. Unlike other members of the subgenus Trypanozoon, they are non-tsetse transmitted and predominantly morphologically uniform (monomorphic) in their mammalian host. Their classification as independent species or subspecies has been long debated and genomic studies have found that isolates within T. brucei evansi and T. brucei equiperdum have polyphyletic origins. Since current taxonomy does not fully acknowledge these polyphyletic relationships, we re-analysed publicly available genomic data to carefully define each clade of monomorphic trypanosome. This allowed us to identify, and account for, lineage-specific variation. We included a recently published isolate, IVM-t1, which was originally isolated from the genital mucosa of a horse with dourine and typed as T. equiperdum. Our analyses corroborate previous studies in identifying at least four distinct monomorphic T. brucei clades. We also found clear lineage-specific variation in the selection efficacy and heterozygosity of the monomorphic lineages, supporting their distinct evolutionary histories. The inferred evolutionary position of IVM-t1 suggests its reassignment to the T. brucei evansi type B clade, challenging the relationship between the Trypanozoon species, the infected host, mode of transmission and the associated pathological phenotype. The analysis of IVM-t1 also provides, to our knowledge, the first evidence of the expansion of T. brucei evansi type B, or a fifth monomorphic lineage represented by IVM-t1, outside of Africa, with important possible implications for disease diagnosis.

2.AbstractTrypanosoma brucei evansi and Trypanosoma brucei equiperdum are animal infective trypanosomes conventionally classified by their clinical disease presentation, mode of transmission, host range, kDNA composition and geographic distribution. Unlike other members of the subgenus Trypanozoon, they are non-tsetse transmitted and predominantly morphologically uniform (monomorphic) in their mammalian host. Their classification as independent species or subspecies has been long debated and genomic studies have found that isolates within T. b. evansi and T. b. equiperdum have polyphyletic origins. Since current taxonomy does not fully acknowledge these polyphyletic relationships, we re-analysed publicly available genomic data to carefully define each clade of monomorphic trypanosome. This allowed us to identify, and account for, lineage specific variation. We included a recently published isolate, IVM-t1, which was originally isolated from the genital mucosa of a horse with dourine and typed as T. equiperdum. Our analyses corroborate previous studies in identifying at least four distinct monomorphic T. brucei clades. We also found clear lineage specific variation in the selection efficacy and heterozygosity of the monomorphic lineages, supporting their distinct evolutionary histories. The inferred evolutionary position of IVM-t1 suggests its reassignment to the T. b. evansi type B clade, challenging the relationship between the Trypanozoon species, the infected host, mode of transmission and the associated pathological phenotype. The analysis of IVM-t1 also provides the first evidence of the expansion of T. b. evansi type B, or a 5th monomorphic lineage represented by IVM-t1, outside of Africa, with important possible implications for disease diagnosis.3.Impact statementTrypanosoma brucei are unicellular parasites typically transmitted by tsetse flies. Subspecies of T. brucei cause human African trypanosomiasis and the animal diseases, nagana, surra and dourine. T. b. evansi and T. b. equiperdum have branched from T. brucei and, by foregoing tsetse transmission, expanded their geographic range beyond the sub-Saharan tsetse belt. These species can only reproduce asexually and exhibit morphological uniformity in their host (‘monomorphism’). T. b. evansi and T. b. equiperdum have historically been classified based on fragmentary information on the parasites’ transmission routes, geographic distribution, kDNA composition and disease phenotypes. Our analysis of genome sequencing data from monomorphic T. brucei supports at least four independent origins with distinct evolutionary histories. One isolate, IVM-t1, typed as T. equiperdum, is a closer relative to T. b. evansi, highlighting the risk of using pathognomonic descriptors for subspecies assignment. We show clear lineage specific variation in the selection efficacy in monomorphic T. brucei. Using the evolutionary relationships between lineages, we suggest it would be beneficial to reconcile phylogeny and pathology in monomorphic trypanosomes.4.Data summaryThe data used in this study is available from the Sequence Read Archive or the Wellcome Sanger Institute. The accessions can be found in Supplementary file 1.