Diagnosis of animal trypanosomoses: proper use of current tools and future prospects.

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.

A review on the diagnosis of animal trypanosomoses.

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.

Trypanosoma brucei gambiense Group 2: The Unusual Suspect.

Trypanosoma brucei causes human African trypanosomiasis (HAT). Three subspecies were described: T. b. gambiense (Tbg) and T. b. rhodesiense (Tbr) in humans, and T. b. brucei (Tbb) in animals. Molecular markers subdivided Tbg into two groups: Tbg1 and Tbg2, of which the latter is different from Tbg1 and Tbr (absence of the SRA gene), but indistinguishable from Tbb. Tbg2 is considered to be a zoonotic form of HAT in West Africa. Tbg2 was found mainly in Côte d'Ivoire between 1978 and 1992, but the latest description was made in Ghana in 2013. New molecular tools would be welcome to characterize such infections and determine their origins (resistance to human serum or patient immunodeficiency) in the current context of HAT elimination.

Trypanosoma rangeli is phylogenetically closer to Old World trypanosomes than to Trypanosoma cruzi.

Trypanosoma rangeli and Trypanosoma cruzi are generalist trypanosomes sharing a wide range of mammalian hosts; they are transmitted by triatomine bugs, and are the only trypanosomes infecting humans in the Neotropics. Their origins, phylogenetic relationships, and emergence as human parasites have long been subjects of interest. In the present study, taxon-rich analyses (20 trypanosome species from bats and terrestrial mammals) using ssrRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), heat shock protein-70 (HSP70) and Spliced Leader RNA sequences, and multilocus phylogenetic analyses using 11 single copy genes from 15 selected trypanosomes, provide increased resolution of relationships between species and clades, strongly supporting two main sister lineages: lineage Schizotrypanum, comprising T. cruzi and bat-restricted trypanosomes, and Tra[Tve-Tco] formed by T. rangeli, Trypanosoma vespertilionis and Trypanosoma conorhini clades. Tve comprises European T. vespertilionis and African T. vespertilionis-like of bats and bat cimicids characterised in the present study and Trypanosoma sp. Hoch reported in monkeys and herein detected in bats. Tco included the triatomine-transmitted tropicopolitan T. conorhini from rats and the African NanDoum1 trypanosome of civet (carnivore). Consistent with their very close relationships, Tra[Tve-Tco] species shared highly similar Spliced Leader RNA structures that were highly divergent from those of Schizotrypanum. In a plausible evolutionary scenario, a bat trypanosome transmitted by cimicids gave origin to the deeply rooted Tra[Tve-Tco] and Schizotrypanum lineages, and bat trypanosomes of diverse genetic backgrounds jumped to new hosts. A long and independent evolutionary history of T. rangeli more related to Old World trypanosomes from bats, rats, monkeys and civets than to Schizotrypanum spp., and the adaptation of these distantly related trypanosomes to different niches of shared mammals and vectors, is consistent with the marked differences in transmission routes, life-cycles and host-parasite interactions, resulting in T. cruzi (but not T. rangeli) being pathogenic to humans.

Zoonotic trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using human and animal trypanocidal drugs.

Beside typical human trypanosomes responsible of sleeping sickness in Africa and Chagas disease in Latin America, there is a growing number of reported atypical human infections due to Trypanosoma evansi, a livestock parasite, or Trypanosoma lewisi, a rat parasite, especially in Asia. Drugs available for the treatment of T. brucei ssp. in humans are obviously of choice for the control of T. evansi because it is derived from T. brucei. However, concerning T. lewisi, there is an urgent need to determine the efficacy of trypanocidal drugs for the treatment in humans. In a recent study, pentamidine and fexinidazole were shown to have the best efficacy against one stock of T. lewisi in rats. In the present study suramin, pentamidine, eflornitine, nifurtimox, benznidazole and fexinidazole, were evaluated at low and high doses, in single day administration to normal rats experimentally infected with a stock of T. lewisi recently isolated in Thailand. Because none of these treatments was efficient, a trial was made with the most promising trypanocide identified in a previous study, fexinidazole 100mg/kg, in 5 daily administrations. Results observed were unclear. To confirm the efficacy of fexinidazole, a mixed infection protocol was set up in cyclophosphamide immunosuppressed rats. Animals were infected successively by T. lewisi and T. evansi, and received 10 daily PO administrations of 200mg/kg fexinidazole. Drastic effects were observed against T. evansi which was cleared from the rat's blood within 24 to 48h; however, the treatment did not affect T. lewisi which remained in high number in the blood until the end of the experiment. This mixed infection/treatment protocol clearly demonstrated the efficacy of fexinidazole against T. evansi and its inefficacy against T. lewisi. Since animal trypanocides were also recently shown to be inefficient, other protocols as well as other T. lewisi stocks should be investigated in further studies.

Zoonotic trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using veterinary drugs.

A growing number of atypical human infections due to the livestock parasite Trypanosoma evansi, or to the rat parasite Trypanosoma lewisi, are reported in humans in Asia. In some cases, clinical evolutions request treatments, however, so far, there were very few attempts to control T. lewisi using trypanocidal drugs. In a study published elsewhere, the efficacy of human trypanocides is evaluated in laboratory rats, and it concludes that none of them is able to cure rats experimentally infected with T. lewisi. Control of T. lewisi in rat would be a step for identification of drugs against this parasite. In the present study, 4 veterinary drugs: diminazene aceturate, isometamidium chloride, melarsomine hydrochloride and quinapyramine sulfate and chloride, were evaluated at low and high doses, in intra-muscular injections to normal rats experimentally infected with a stock of T. lewisi from Thailand. None of these treatments being efficient, a trial was also made using melarsomine hydrochloride in T. evansi infected rats and in mixed T. lewisi and T. evansi infected rats, in order to demonstrate the efficacy of the drugs under the present protocol. T. evansi was cleared from the rat's blood the day after the treatment, while, T. lewisi remained unaffected until the end of the experiment. These observations clearly demonstrated the efficacy of melarsomine hydrochloride against T. evansi and its inefficacy against T. lewisi. In conclusion none of the veterinary drugs was efficient against this stock of T. lewisi. Other protocols using higher doses or other drugs and T. lewisi stocks should be investigated in further studies. The control of T. lewisi infection in Wistar rats, using veterinary trypanocidal drugs, remains so far unsuccessful.

A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia.

BACKGROUND: Trypanosomais a genus of unicellular parasitic flagellate protozoa.Trypanosoma bruceispecies and Trypanosoma cruziare the major agents of human trypanosomiasis; other Trypanosomaspecies can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma METHODS: Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source. RESULTS: PCR amplification and serological testing identified the infecting species as Trypanosoma evansi.Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive forT. evansi. CONCLUSIONS: We report the first laboratory-confirmed case ofT. evansiin a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden ofT. evansiin local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases.

Chemoprophylaxis and treatment of African canine trypanosomosis in French military working dogs: a retrospective study.

African trypanosomosis is a major threat to livestock production in sub-Saharan Africa. Although the disease mainly concerns cattle, dogs can also be infected by Trypanosoma spp. transmitted by tsetse flies. Between 1997 and 2003, the parasite Trypanosoma congolense was identified in French military dogs sent to Africa. On infected dogs, the diagnosis was made during the mission or just after the return to France, depending on when the symptoms appeared. The high incidence and mortality rate among these dogs led veterinarians of the French Health Service to implement a systematic chemoprophylaxis beginning in 2004. Between 2004 and 2011, the chemoprophylaxis was carried out on more than 400 military dogs. The protocol of chemoprophylaxis relies on the use of isometamidium chloride (Trypamidium(®), Merial). The drug has been used successfully at the dosage of 1mg/kg body weight by deep intramuscular injection, every two or three months. In addition, dogs are given collars impregnated with deltamethrin (Scalibor(®), MSD Animal Health). Isometamidium chloride was also used successfully in the treatment of military dogs infected with T. congolense, with a full recovery and without any relapses.

Multiple infections of Trypanosoma brucei gambiense in blood and cerebrospinal fluid of human African trypanosomosis patients from Angola: consequences on clinical course and treatment outcome.

Human African trypanosomosis, caused by Trypanosoma brucei gambiense, is a chronic disease, although various clinical patterns have been observed, from asymptomatic to acute forms. Since 2001 in Angola, 80% of patients have been found to be in the meningoencephalitic stage of the disease. The existence of an acute form of the disease caused by virulent strains of trypanosomes was suspected. To test this hypothesis, four sensitive and polymorphic microsatellite markers were used to characterize the trypanosome DNA extracted from the blood and cerebrospinal fluid of 100 patients in the meningoencephalitic stage. Twenty-three patients were found with mixed T. b. gambiense genotypes in the blood and/or cerebrospinal fluid. The absence of association between the number of infecting genotypes, the presence of neurological signs and white blood cell counts in the cerebrospinal fluid, seems to indicate, at least in the context of the present study, the absence of virulent strains. However, out of five patients who died from encephalopathy syndrome during treatment with eflornithine, three harbored multiple infections.

Identification of subspecies specific genes differentially expressed in procyclic forms of Trypanosoma brucei subspecies.

Trypanosoma brucei subspecies undergo establishment and maturation in tsetse flies mid-gut and salivary glands, respectively. Successful establishment of trypanosomes in tsetse mid-gut as well as their migration to saliva gland depends on the ability of these parasites to adapt rapidly to new environmental conditions and to negotiate the physical barriers. To identify subspecies specific genes which are differentially regulated during the establishment of T. brucei subspecies in tsetse flies mid-gut, a comparative genomic analysis between different T. brucei subspecies was performed using microarrays containing about 23040 T. brucei shotgun fragments. The whole genome analyses of RNA expression profiles revealed about 274 significantly differentially expressed genes between T. brucei subspecies. About 7% of the differentially regulated clones did not match to any T. brucei predicted genes. Most of the differentially regulated transcripts are involved in transport across cell membrane and also in the purines metabolism. The genes selectively up regulated in T. brucei gambiense and T. brucei rhodesiense (human infective T. brucei) like snoRNA and HSP70 are expressed in response to stress. The high failure rate in the process of establishment and maturation of T. brucei gambiense during cyclical transmission in tsetse flies may result from the incapacity of this parasite to regulate its growth due to the expression of a variety of chaperones or heat shock proteins. Genes selectively up regulated in T. brucei brucei like NT8.1 nucleoside/nucleobase transporters and S-adenosylmethionine synthetase may favour the establishment of this subspecies in tsetse mid-gut. These genes appear as potential targets for investigations on the development of vaccine blocking the transmission of trypanosomes in tsetse flies.

From clonal to sexual: a step in T. congolense evolution?

Although clearly demonstrated in Trypanosoma brucei, genetic exchange remains controversial in other trypanosome species. Recently, Morrison and co-workers applied a population-genetics analysis, and established the existence of mating in Trypanosoma congolense. Starting from this original discovery, we focus here on the important question of how mating is induced during the trypanosome life cycle and discuss the use of statistics to evidence this type of non-obligatory biological process.

First isolation of Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the tsetse fly (Glossina palpalis palpalis) midgut.

This paper reports the first evidence of the presence of bacteria, other than the three previously described as symbionts, Wigglesworthia glossinidia, Wolbachia, and Sodalis glossinidius, in the midgut of Glossina palpalis palpalis, the tsetse fly, a vector of the chronic form of human African trypanosomiasis in sub-Saharan African countries. Based on the morphological, nutritional, physiological, and phylogenetic results, we identified Enterobacter, Enterococcus, and Acinetobacter spp. as inhabitants of the midgut of the tsetse fly from Angola. Enterobacter spp. was the most frequently isolated. The role of these bacteria in the gut, in terms of vector competence of the tsetse fly, is discussed, as is the possibility of using these bacteria to produce in situ trypanolytic molecules.

Development and application of an antibody-ELISA to follow up a Trypanosoma evansi outbreak in a dromedary camel herd in France.

An outbreak of trypanosomosis was observed for the first time in metropolitan France in October 2006, when five camels were proved to be infected by Trypanosoma evansi using parasitological methods. The parasite was isolated and used to produce a soluble antigen for antibody-enzyme linked immunosorbent assay (ELISA) in a protocol derived from a method previously developed for sheep and humans but using protein A conjugate. The animals were treated on three instances, alternatively with melarsomine hydrochloride and quinapyramine and followed up on a monthly basis for 2 years with various diagnostic techniques including parasitological, serological and DNA-based methods. Initially, five animals were detected as being positive using ELISA with 83.3% concordance to parasitological tests. Immediately after the first treatment, parasites and DNA disappeared in all animals; antibody levels decreased regularly until ELISA became negative 3-4 months later. Ten months after the first treatment, parasites and antibodies were detected again in one of the camels previously found to be infected. A retrospective study indicated that the weight of this animal had been underestimated; consequently, it had received underdosages of both trypanocides. However, since hypotheses of re-infection or relapse could not be fully substantiated, it is not known whether the ELISA results for this animal were true- or false-negative over a 7-month period. The study confirmed the value of this ELISA using protein A conjugate to detect antibodies directed against T. evansi in camels and the need to use several diagnostic techniques to optimize detection of infected animals. A warning is raised on surra, a potentially emerging disease in Europe.

Trypanosoma brucei 29-13 strain is inducible in but not permissive for the tsetse fly vector.

Using green fluorescent protein as a reporter, we have shown that the strain 29-13 of Trypanosoma brucei, widely used for inducible down-regulation of mRNA, is inducible in, but not permissive for the tsetse flies Glossina palpalis gambiensis and Glossina morsitans morsitans. Within two weeks post-infection, 42% males and females of teneral and non-teneral tsetse flies harboured intestinal infections, yet not a single infection progressed into the salivary glands.

Genetic characterization of Trypanosoma evansi isolated from a patient in India.

The first human case of trypanosomiasis caused by Trypanosoma evansi was recently discovered in India. We have focused on the parasite to investigate whether this atypical infection was due to a particular genotype of T. evansi. The SRA gene was not detected by PCR in the Indian human T. evansi (TEVH) DNA sample. TEVH appears to be closely related to Vietnam WH, with identical alleles for TRBPA and MT30-33 AC/TC microsatellites. Furthermore, T. evansi has homogeneous kDNA minicircles and the minicircles of isolate TEVH were shown to be of Type A. Thus, the T. evansi isolated from an Indian patient appears to be a typical T. evansi as far as we can judge, suggesting that the explanation for this unusual infection may lie with the patient.

Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report.

We report an Indian farmer who had fluctuating trypanosome parasitemia associated with febrile episodes for five months. Morphologic examination of the parasites indicated the presence of large numbers of trypanosomes belonging to the species Trypanosoma evansi, which is normally a causative agent of animal trypanosomiasis known as surra. Basic clinical and biologic examinations are described, using several assays, including parasitologic, serologic, and molecular biologic tests, all of which confirmed the infecting species as T. evansi. Analysis of cerebrospinal fluid indicated no invasion of the central nervous system (CNS) by trypanosomes. Suramin, a drug used exclusively for treatment of early-stage human African trypanosomiasis with no CNS involvement, effected apparent cure in the patient. This is the first case reported of human infection due to Trypanosoma evansi, which was probably caused by transmission of blood from an infected animal.