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This review focuses on the most reliable and up-to-date methods for diagnosing trypanosomoses, a group of diseases of wild and domestic mammals, caused by trypanosomes, parasitic zooflagellate protozoans mainly transmitted by insects. In Africa, the Americas and Asia, these diseases, which in some cases affect humans, result in significant illness in animals and cause major economic losses in livestock. A number of pathogens are described in this review, including several Salivarian trypanosomes, such as Trypanosoma brucei sspp. (among which are the agents of sleeping sickness, the human African trypanosomiasis [HAT]), Trypanosoma congolense and Trypanosoma vivax (causing “Nagana” or animal African trypanosomosis [AAT]), Trypanosoma evansi (“Surra”) and Trypanosoma equiperdum (“Dourine”), and Trypanosoma cruzi , a Stercorarian trypanosome, etiological agent of the American trypanosomiasis (Chagas disease). Diagnostic methods for detecting zoonotic trypanosomes causing Chagas disease and HAT in animals, as well as a diagnostic method for detecting animal trypanosomes in humans (the so-called “atypical human infections by animal trypanosomes” [a-HT]), including T. evansi and Trypanosoma lewisi (a rat parasite), are also reviewed. Our goal is to present an integrated view of the various diagnostic methods and techniques, including those for: (i) parasite detection; (ii) DNA detection; and (iii) antibody detection. The discussion covers various other factors that need to be considered, such as the sensitivity and specificity of the various diagnostic methods, critical cross-reactions that may be expected among Trypanosomatidae, additional complementary information, such as clinical observations and epizootiological context, scale of study and logistic and cost constraints. The suitability of examining multiple specimens and samples using several techniques is discussed, as well as risks to technicians, in the context of specific geographical regions and settings. This overview also addresses the challenge of diagnosing mixed infections with different Trypanosoma species and/or kinetoplastid parasites. Improving and strengthening procedures for diagnosing animal trypanosomoses throughout the world will result in a better control of infections and will significantly impact on “One Health,” by advancing and preserving animal, human and environmental health. Graphical Abstract

The stable fly, Stomoxys calcitrans, is a cosmopolite pest causing direct and indirect nuisances on livestock, due to painful bite, harassment, blood despoliation and biological or mechanical transmission of viruses, bacteria and parasites. Its control is mainly based on direct applications of pyrethroid insecticides on livestock, although evidences show limited efficiency due to short term effects and high levels of phenotypic and genotypic resistance. Diagnosis of genetic resistance is currently based on a series of point mutation PCRs showing limitations. Based on fly specimens trapped in southern France, this study aims to establish a new diagnosis method to investigate kdr allele frequencies in livestock farms. A total of 144 S. calcitrans specimens were collected from seven farms (1 with insecticide usage, and 6 without) and processed individually through a single PCR using newly designed primers amplifying a 340 bp fragment including the mutation site of the voltage-sensitive sodium channel domain II; PCR products were then sequenced. Among the 104 individuals successfully sequenced, the methodology allowed to detect the wild-type , kdr-his and kdr genotypes encoding for leucine, histidine and phenylalanine, respectively. Although the wild-type was the most prevalent, resistance alleles were detected in all farms, especially at the veterinary school, where insecticides are commonly used. These results highlight that genotypic insecticide resistance in S. calcitrans populations is widespread. This single PCR-sequencing method, simple, cost-effective and reliable, will allow determining prevalence, distribution and resilience of genotypic pyrethroid insecticide resistance, a primary data to support a conversion from chemical vector control toward environmental-friendly strategies.

Reliable diagnostic tools are needed to choose the appropriate treatment and proper control measures for animal trypanosomoses, some of which are pathogenic. Trypanosoma cruzi , for example, is responsible for Chagas disease in Latin America. Similarly, pathogenic animal trypanosomoses of African origin (ATAO), including a variety of Trypanosoma species and subspecies, are currently found in Africa, Latin America and Asia. ATAO limit global livestock productivity and impact food security and the welfare of domestic animals. This review focusses on implementing previously reviewed diagnostic methods, in a complex epizootiological scenario, by critically assessing diagnostic results at the individual or herd level. In most cases, a single diagnostic method applied at a given time does not unequivocally identify the various parasitological and disease statuses of a host. These include “non-infected”, “asymptomatic carrier”, “sick infected”, “cured/not cured” and/or “multi-infected”. The diversity of hosts affected by these animal trypanosomoses and their vectors (or other routes of transmission) is such that integrative, diachronic approaches are needed that combine: (i) parasite detection, (ii) DNA, RNA or antigen detection and (iii) antibody detection, along with epizootiological information. The specificity of antibody detection tests is restricted to the genus or subgenus due to cross-reactivity with other Trypanosoma spp. and Trypanosomatidae, but sensitivity is high. The DNA-based methods implemented over the last three decades have yielded higher specificity and sensitivity for active infection detection in hosts and vectors. However, no single diagnostic method can detect all active infections and/or trypanosome species or subspecies. The proposed integrative approach will improve the prevention, surveillance and monitoring of animal trypanosomoses with the available diagnostic tools. However, further developments are required to address specific gaps in diagnostic methods and the sustainable control or elimination of these diseases. Graphical Abstract

Background Tsetse flies (Diptera: Glossinidae) are vectors of human and animal trypanosomes. The Glossina palpalis gambiensis Burkina Faso (BKF) colony, established in 1972 and rejuvenated once in 1981, is a long-standing closed colony used extensively for research and vector control. While its performance and competitiveness for sterile insect technique (SIT) programs are regularly monitored, its vector competence (VC) data are outdated. This study aimed to update the VC data of this 47-year-old colony (from the onset of experiment in 2019) against Trypanosoma congolense and T. brucei brucei in laboratory conditions using trypanosome clone and tsetse fly individuals from the BKF colony. Methods Vector competence was studied by infecting rats with T. congolense IL1180 and T. b. brucei BE8P2P2, on which tsetse flies received their first blood meal. Dissections were subsequently performed at different time intervals. Results Following experimental infections with T. congolense IL1180, 10.58% (20/189) of G. p. gambiensis developed mature infections (trypanosomes in the proboscis), resulting in an average VC index of 0.106. For T. b. brucei BE8P2P2, 4.21% (11/261) of flies developed mature infections (trypanosomes in the salivary glands), yielding an average VC index of 0.042. Conclusions The VC for T. b. brucei aligned with previous findings from 21 years ago, though a different trypanosome isolate was used at that time. However, using the same trypanosome strain, the observed competence for T. congolense was 4.8 times higher than previously reported. These results raise questions about the long-term effects of insectary rearing on VC, particularly in the absence of prolonged parasite exposure. Graphical abstract

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

Background Surra, a vector borne disease caused by Trypanosoma (T.) evansi, affects the health, productivity and working capacity of camels. Since clinical signs are not pathognomonic, diagnosis must be confirmed by laboratory methods. This is a first study on the prevalence of surra in Cholistan Desert, Pakistan using a broad variety of diagnostic tests thereby emphasizing it as a risk for the dromedaries of Pakistan. Methods In a cross sectional study, 1005 dromedary camels from three districts in the Cholistan Desert were sampled to assess the prevalence of trypanosomosis due to T. evansi by means of parasitological (Giemsa stained thin smear), serological (formol gel test, CATT/ T. evansi, ELISA/VSG RoTat 1.2 , immune trypanolysis) and molecular tests (TBR1/2 PCR and RoTat 1.2 PCR). Kappa was calculated to assess the degree of agreement between different tests whereas chi-square test along with odds ratios and their 95 % confidence intervals were used to study influence of breed, gender, age and locality on disease prevalence. Results Overall prevalence was 0.7 % with Giemsa stained thin smears (GST), 40.1 % with formol gel test (FGT), 47.7 % with CATT/ T. evansi , 44.2 % with ELISA/VSG RoTat 1.2, 39.9 % with immune trypanolysis (TL), 31.9 % with TBR1/2 PCR and 30.5 % with RoTat1.2 PCR. Based on these results, the Cholistan Desert appears to be a high risk area for surra. According to TL and TBR1/2 PCR, camels at Bahawalpur are approximately two times more likely to be infected than those in Bahawalnagar (OR = 1.8; 95 % CI: 1.38-2.42) and Rahim Yar Khan (OR = 1.9; 95 % CI: 1.30-2.75). Test agreement of TL was moderate with CATT/ T. evansi (k = 0.43; 95 % CI: 0.378-0.489) and ELISA/VSG RoTat 1.2 (k = 0.54; 95 % CI: 0.489-0.594) and poor with the other tests. Test agreement between TBR1/2 PCR and RoTat1.2 PCR was almost perfect (k = 0.96; 95 % CI: 0.950-0.984). We didn't find evidence for the presence of T. evansi type B in the studied population. Conclusion Our study supports using antibody detection tests, rather than parasitological and molecular examination, to assess surra prevalence in camels. It also calls for implementation of measures to control surra in the Cholistan Desert.

Animal African trypanosomosis is an important vector-borne disease of livestock in sub-Saharan Africa. Pigs seem relatively tolerant to trypanosome infection and could act as a reservoir of trypanosomes affecting animals and humans. Our ability to reliably detect trypanosome infection in pigs depends on the performance of diagnostic tools, which is not well known. In pigs experimentally infected with Trypanosoma brucei brucei , we evaluated the performance of parasitological Buffy Coat Technique (BCT), two molecular (TBR and 5.8S PCR) and four serological tests (CATT, HAT Sero- K -Set rapid diagnostic test–RDT, indirect ELISA, immune trypanolysis). Most diagnostic tests showed high specificity, estimated at 100% (95% CI = 74–100%) with the exception of CATT and RDT whose specificity varied between 100% (95% CI = 74–100%) to 50% (95% CI = 7–93%) during the experiment. The sensitivity of each test fluctuated over the course of the infection. The percentage of positive BCT over the infection (30%) was lower than of positive PCR (56% and 62%, depending on primers). Among the serological tests, the percentage of positive tests was 97%, 96%, 86% and 84% for RDT, ELISA, immune trypanolysis and CATT, respectively. Fair agreement was observed between both molecular tests (κ = 0.36). Among the serological tests, the agreement between the ELISA and the RDT was substantial (κ = 0.65). Our results on the T . b . brucei infection model suggest that serological techniques are efficient in detecting the chronic phase of infection, PCR is able to detect positive samples several months after parasites inoculation while BCT becomes negative. BCT examination and RDT are useful to get a quick information in the field, and BCT can be used for treatment decision. ELISA appears most suited for epidemiological studies. The selection of diagnostic tests for trypanosomosis in pigs depends on the context, the objectives and the available resources.

Surra is a vector-borne disease, caused by a flagellate protozoan, Trypanosoma evansi , infecting all domestic mammals, including herbivores and dogs, and, very rarely, humans. In Tunisia, it affects mainly dromedaries ( Camelus dromedarius ) in the southern part of the country, causing heavy economic losses due to high morbidity, abortions and mortality. Trypanosoma evansi is mainly transmitted by mechanical vectors (Stomoxyine flies and tabanids), but also vertically, orally (to carnivores) and iatrogenically. In the present paper, we review and discuss the studies published on surra in Tunisia and show that the antibody seroprevalence in Tunisian dromedaries varies between 22.2% and 37%. The review also highlights the absence of a comprehensive database containing the most relevant information on the occurrence of T. evansi in Tunisia. We also underscore the urgent need for data collection and analyses. These data should be related to different aspects: epidemiological data (spatial and temporal distribution) and entomological data (main vectors involved in the transmission and their activity dynamics). Le surra est une maladie vectorielle, causée par un protozoaire flagellé, Trypanosoma evansi , qui infecte tous les mammifères domestiques, y compris les herbivores et les chiens, et, très rarement, l’homme. En Tunisie, il touche principalement les dromadaires ( Camelus dromedarius ) dans le sud du pays, entraînant de lourdes pertes économiques dues à une morbidité, des avortements et une mortalité élevés. Trypanosoma evansi est principalement transmis par des vecteurs mécaniques (Stomoxes et Tabanidae), mais aussi verticalement, oralement (aux carnivores) et par voie iatrogène. Cet article passe en revue et discute les études publiées sur le surra en Tunisie et montre que la séroprévalence des anticorps chez les dromadaires tunisiens varie entre 22,2 % et 37 %. L’étude souligne également l’absence d’une base de données exhaustive contenant les informations les plus pertinentes sur la présence de T. evansi en Tunisie, et souligne le besoin urgent de collecte et d’analyse de données. Ces données devraient être liées à différents aspects : données épidémiologiques (distribution spatiale et temporelle) et données entomologiques (principaux vecteurs impliqués dans la transmission et leur dynamique d’activité).

Background and Aim: Trypanosoma evansi infection has been reported in Thai livestock such as beef and dairy cattle. However, there is little information on T. evansi infection in bullfighting cattle in Southern Thailand. The aim of this study was to investigate the infection of T. evansi in bullfighting cattle presented for health checks at the Animal Hospital, Faculty of Veterinary Science, Prince of Songkla University, Thailand. Materials and Methods: Blood and serum samples were collected from 177 bullfighting cattle from April 2016 to February 2017 after bullfighting matches. Animal inspected showed signs of fever, weight loss, or exercise intolerance. Investigation of T. evansi infection was tested using polymerase chain reaction (PCR) with TBR primers and using indirect enzyme-linked immunosorbent assay with T. evansi crude antigen. Results: The seroprevalence of T. evansi in bullfighting cattle was 22.60% (40/177). The PCR results detected no parasite DNA in this study. However, bullfighting cattle may serve as T. evansi reservoirs. Conclusion: Health checking procedures for T. evansi should be promoted for bullfighting events so that infected animals can be quarantined in the preparatory stages of such events.