Trypanosoma evansi in Asia

Trypanosoma evansi has the widest geographical range of all the pathogenic trypanosome species and infects domesticated livestock in many countries of South America, Africa and Asia. In spite of this wide distribution, T. evansi has been less intensively investigated than the African tsetse-transmitted trypanosomes and there is correspondingly less information available on the incidence and economic importance of the disease that it causes. Many of the new techniques in immunology and molecular biology, which have provided much fundamental information on the tsetse-transmitted trypanosomes, have yet to be applied to T. evansi. Interest in T. evansi is increasing and a Working Group has now been established to coordinate and promote future research (Box 1). T. evansi is an important aetiological agent of disease in the livestock of Asia; this article evaluates both the historical perspective and our current knowledge of the epidemiology and pathology of T. evansi in this region.     

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.     

Trypanosoma evansi: cloning and expression in Spodoptera frugiperda [correction of fugiperda] insect cells of the diagnostic antigen RoTat1.2

A complementary DNA encoding the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigenic type (RoTat)1.2, currently used for experimental serological diagnosis of T. evansi infection in livestock, was cloned as a recombinant plasmid and sequenced. A recombinant baculovirus containing the coding region of RoTat1.2 VSG was constructed to express the protein in Spodoptera frugiperda [corrected] insect cells. From this, sufficient quantities of the recombinant protein are being produced for empirical and wide-scale objective assessment of the diagnostic potential of this antigen. The gene encoding the RoTat1.2 VSG was shown by PCR to be present in the genomes of many different cloned isolates of T. evansi, but not T. brucei, from geographically separate regions of Africa, Asia, and South America. With the recombinant RoTat1.2 at hand, it is now possible to investigate the extent to which epitopes on this VSG are conserved among different T. evansi isolates.     

The detection of non-RoTat 1.2 Trypanosoma evansi

The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR amplification for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from different trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR amplification of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp amplification product was present in all five non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is specific for non-RoTat 1.2 T. evansi.     

Trypanosomiasis of domestic animals in China

Trypanosomiasis caused by Trypanosoma evansi (surra) is a problem of great economic importance in livestock in China because it affects important working animals: buffaloes in the south and east, and horses and cattle in the north and west. In addition, buffaloes are an important source of meat and leather for the Chinese population. In the north and west, T. evansi is found mainly in camels, whereas in the east, the south and the southwest, it is primarily buffaloes, cattle and horses that are affected by this parasitic flagellate. Although trypanosomiasis is one of the most important parasitic diseases of domestic animals in China, little information on this disease has been published. In this article Zhao-Rong Lun, Yuang Fang, Chang-Ji Wang and Reto Brun evaluate trypanosomiasis of domestic animals in China and review epidemiological studies of the past 40 years.     

A possible role for rusa deer (Cervus timorensis russa) and wild pigs in spread of Trypanosoma evansi from Indonesia to Papua New Guinea

Movement of transmigrants and livestock from western Indonesia to southeastern areas of Irian Jaya near the border with Papua New Guinea may pose a risk of introducing Trypanosoma evansi into Papua New Guinea via feral Rusa deer (Cervus timorensis russa) and wild pigs which inhabit these areas in large numbers. Pilot experimental studies were conducted to observe infection in pigs and Rusa deer with a strain of T. evansi isolated in Indonesia. Parasitaemia and signs of clinical disease were monitored each second day for 120 days. Trypanosomes were observed in haematocrit tubes at the plasma-buffy coat interface of jugular blood of deer and pigs on 86% and 37% of sampling occasions respectively. Parasitaemia was at a high level in deer for 35% of the time but for only 11.5% of the time in pigs. Results indicate that both Rusa deer and pigs have a high tolerance for infection with T. evansi. The deer suffered mild anaemia evidenced by a 25% reduction in packed cell volume (PCV) 14 days after infection which coincided with the initial peak in parasitaemia. However, PCV had returned to pre infection values by the end of the experiment. The pigs showed no change in PCV. There were no visual indications of disease in either species and appetite was not noticeably affected. It was concluded that both Rusa deer and pigs were capable reservoir hosts for T. evansi but that Rusa deer, with their more persistent higher levels of parasitaemia, have more potential to spread T. evansi into Papua New Guinea from West Irian than pigs.     

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.     

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.     

Trypanosoma evansi: molecular homogeneity as inferred by phenetical analysis of ribosomal internal transcribed spacers DNA of an eclectic parasite

The protozoan Trypanosoma evansi is described as presenting high morphological and genetic similarities among the isolates despite its biological heterogeneity and wide geographical distribution. PCR amplification of the internal transcribed spacers of the ribosomal gene in combination with the coding region of the 5.8S ribosomal subunit further submitted to restriction enzymes digestion were carried out in DNAs extracted from 41 T. evansi strains isolated from horses, dogs, coatis and capybaras from two distinct regions of the Brazilian Pantanal. We also used one T. evansi isolate from Africa, one from Asia and one isolate of T. b. brucei from Africa. Analysis of the RFLP profiles yielded a unique "riboprinting" that does not vary intraspecifically. These results provide insights on the ribosomal gene organization of T. evansi and showed that ITS analysis by RFLP show high genetic similarity of this locus among isolates of this protozoan parasite.     

VSG 117 gene is conservatively present and early expressed in Trypanosma evansi YNB stock

African trypanosomes, including Trypanosoma brucei and the closely related species Trypanosoma evansi, are flagellated unicellular parasites that proliferate extracellularly in the mammalian bloodstream and tissue spaces. They evade host immune system by periodically switching their variant surface glycoprotein (VSG) coat. Each trypanosome possesses a vast archive of VSGs with distinct sequence identity and different strains contain different archive of VSGs. VSG 117 was reported as a widespread VSG detected in the genomes of all the T. brucei strains. In this study, the presence and expression of VSG 117 gene was observed in T. evansi YNB stock by RT-PCR with VSG-specific primers. We further confirmed that this VSG tends to be expressed in the early stage of T. evansi infections (on day 12-15) by immuno-screening the previously isolated infected blood samples. It is possible that the VSG 117 gene evolved and spread through the African trypanosome population via genetic exchange, before T. evansi lost its ability to infect tsetse fly. Our finding provided an evidence of the close evolutionary relationship between T. evansi and T. brucei, in the terms of VSG genes.     

Trypanosoma evansi: demonstration of a transferrin receptor derived from expression site-associated genes 6 and 7.

In Trypanosoma brucei, uptake of host transferrin is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor derived from the 2 expression site-associated genes 6 and 7 (ESAG6 and ESAG7). By using specific antibodies, it is shown here that T. evansi, a trypanosome species transmitted mechanically by biting flies, also expresses a transferrin receptor composed of ESAG6 and ESAG7. The cellular uptake of transferrin in T. evansi is completely inhibited with anti-T. brucei (ESAG6/7 heterodimer) antibodies. The demonstration of a functional ESAG6/7 transferrin receptor in T. evansi supports further its close relationship to T. brucei.     

Trypanosoma equiperdum: master of disguise or historical mistake?

After 100 years of research, only a small number of laboratory strains of Trypanosoma equiperdum exists, and the history of most of the strains is unknown. No definitive diagnosis of dourine can be made at the serological or molecular level. Only clinical signs are pathognomonic and international screening relies on an outdated cross-reactive serological test (the complement-fixation test) from 1915, resulting in serious consequences at the practical level. Despite many characterization attempts, no clear picture has emerged of the position of T. equiperdum within the Trypanozoon group. In this article, we highlight the controversies that exist regarding T. equiperdum, and the overlap that occurs with Trypanosoma evansi and Trypanosoma brucei brucei. By revisiting the published data, from the early decades of discovery to the recent serological- and molecular-characterization studies, a new hypothesis arises in which T. equiperdum no longer exists as a separate species and in which current strains can be divided into T. evansi (the historical mistake) and Trypanosoma brucei equiperdum (the master of disguise). Hence, dourine is a disease caused by specific host immune responses to a T. b. equiperdum or T. evansi infection.     

Mechanical transmission of Trypanosoma evansi and T. congolense by Stomoxys niger and S. taeniatus in a laboratory mouse model

Abstract .Mechanical transmission of Trypanosoma evansi (South American origin) and T. congolense of Kilifi DNA type (Kenyan origin) was studied in laboratory mice using the African stable flies Stomoxys niger niger and S. taeniatus . Altogether, 355 flies were interrupted after feeding on infected blood and then transferred immediately to an uninfected mouse to complete feeding. Microscopy and subinoculation of triturated flies into uninfected mice demonstrated the survival of T. congolense in Stomoxys for up to 210 min and T. evansi for up to 480 min. Parasites survived for much longer periods in the digestive tract than inside or on the mouthparts. Trypanosoma congolense was transmitted only by S. n. niger , and only at low rates of 3, 8 and 10% using flies of different feeding histories: fed on blood the previous day, freshly caught, and teneral. Trypanosoma evansi was transmitted by both Stomoxys species at higher rates: S. taeniatus range 13–18%; S. n. niger range 17–35%. The highest transmission rate occurred with the combination of teneral S. n. niger and T. evansi.     

Therapeutic effect of bleomycin on experimental infection of mice with Trypanosoma gambiense and Trypanosoma evansi

Intraperitoneal injection of 10 mg/Kg bleomycin into mice 24 h after inoculation with Trypanosoma gambiense or Trypanosoma evansi, reduced the incidence of infection 62.1%, and 95.2%, respectively. No parasitemia was not found in these mice. Treatment of mice with parasitemia with 30 mg/Kg of bleomycin decreased the number of parasites within about 5 h and caused complete cure without relapse in 45% and 75% of mice infected with T. gambiense and T. evansi, respectively. Treatment of mice infected with T. gambiense with bleomycin in combination with ethidium bromide was highly effective and resulted in a high incidence of complete cure, even in heavily infected mice. The mode of action of bleomycin and ethidium bromide on trypanosomes in relation to p-rosaniline resistance is discussed.     

RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate

Drug resistance of trypanosomes is now a problem, but its underlying mechanisms are not fully understood. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. The adenosine transporter-1 gene, TbAT1, encoding a P2-like nucleoside transporter has previously been cloned from Trypanosoma brucei brucei, and when expressed in yeast, it showed very similar substrate specificity to the P2-nucleoside transporter, but could not transport diamidines (pentamidine and diminazene). We have cloned and sequenced a similar gene (TevAT1) from Trypanosoma evansi and found it to have 99.7% identity to the TbAT1 gene. To elucidate the role of the TevAT1 gene on diamidine trypanocidal effect, we genetically engineered T. evansi for conditional knock-out of the TevAT1 gene by RNA interference (RNAi). Induction of the RNAi resulted in 10-fold depletion of TevAT1 mRNA, with concomitantly significant resistance to diminazene aceturate (berenil). The induced parasites propagated normally and attained peak cell density at an in vitro concentration of berenil, 5.5-fold higher than the IC(100) of the wild-type. TevAT1 knock-out had no effect on the trypanocidal activity of suramin and antrycide, but conferred some resistance to samorin. Our findings validate the significance of the TevAT1 adenosine transporter-1 gene in mediating the trypanocidal effect of diamidines in T. evansi. Further, we show for the first time that RNAi gene silencing in T. evansi can be induced using plasmids designed for T. brucei. We also demonstrate the usefulness of real-time PCR in rapidly quantifying mRNA levels in trypanosomes.     

Immunohistochemical demonstration of Trypanosoma evansi in tissues of experimentally infected rats and a naturally infected water buffalo (Bubalus bubalis)

Trypanosoma evansi was demonstrated by an immunohistochemical technique in formalin-fixed paraffin-embedded tissues of experimentally infected rats. Trypanosoma evansi was visible readily, nuclei were stained darkly, the cytoplasm was stained moderately, and the cell membranes were delineated clearly. The parasites were present in small- to large-sized blood vessels of all organs, in extravascular spaces of ventricles and neuropil of the brain, and in interstitial tissues of the lung and testes. This method also stained nuclei but not cytoplasm or cell membranes of Trypanosoma congolense, and did not stain Trypanosoma theileri. In a water buffalo (Bubalus bubalis) with nonsuppurative meningoencephalitis, the presence of T. evansi could not be demonstrated by conventional histological stains. However, the trypanosomes were recognized readily in the Virchow-Robin spaces and neuropil of the brain by the immunohistochemical method.     

High Trypanosoma vivax infection rates in water buffalo and cattle in the Brazilian Lower Amazon

Highly sensitive and accurate molecular diagnostic methods have not yet been employed for livestock trypanosomosis in the Brazilian Lower Amazon although the first reports of Trypanosoma vivax and Trypanosoma evansi in Brazil were in water buffalo (Bubalus bubalis) in this region. The present study assessed trypanosomosis in buffalo and cattle raised in communal and seasonally flooding pastures in the state of Pará using the fluorescent fragment length barcoding (FFLB) method. T. evansi was not detected, but high infection rates of T. vivax and T. theileri were revealed by a simplified FFLB standardized in the present study that discriminates all trypanosome species infective to livestock in South America. T. vivax infection rates detected by TviCATL-PCR were 24.6% for cattle (n = 61) and 28.1% for buffalo (n = 89). Using the FFLB method, overall T. vivax infection rates increased to 59.6% and 44.3% for buffalo and cattle, respectively. Furthermore, the predominance of a single microsatellite-based genotype of T. vivax was reinforced in the Lower Amazon. Relevant T. vivax infection rates detected in clinically healthy buffalo and cattle through the sampled years (2008–2017) highlight the need for systematic studies to demonstrate the endemic steady state of T. vivax in this region. Our findings provide baseline information for livestock management, including control of T. vivax dispersal, and the introduction of naïve animals. The growing international trade of live livestock from this very important livestock breeding region represents a serious risk for T. vivax spreading outside Amazonia and Brazil.