Journal of Life Science and Biomedicine  
J Life Sci Biomed, 10 (3): 29-43, 2020  
ISSN 2251-9939  
Molecular detection of Trypanosoma theileri and a new  
Trypanocidal drug, a review  
Tewodros FENTAHUN  
Unit of Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, P.O.Box 196, Gondar, Ethiopia  
Corresponding author’s Email:;  
Original Article  
Introduction. Trypanosoma theileri (T.theileri) is apathogenic, cosmopolitan and commensal  
protozoa of cattle. Despite apathogenic in healthy, but not in stressed cattle; it’s getting  
recent attention as a tool to tackle pathogenic ones. These days, researchers are giving due  
attention to study the biology and feasibility of T.theilerito use it as a model candidate for  
novel drug discovery. In addition, in-silico analysis using common antitrypanosome drug  
targets couldn’t show significant similarity both at DNA and protein level. Nevertheless,  
homologous sequences have been identified among drug targets for Ornithine  
decarboxylase. This indicated the possibility to consider T.theileri as a model to search novel  
drugs once having whole genome sequences. The SDM 79 is an appropriate medium to  
PII: S225199392000005-10  
Rec. 11 March 2019  
Rev. 15 May 2019  
Pub. 25 May 2020  
cultivate at 26 C, without CO2. Gradient PCR amplification has been used to detect using T.  
Novel Trypanocidal drug,  
SDM 79,  
theileri. The specific primer (Tth625) which reveals 465 bp amplification product and also the  
full length 18S ribosomal DNA sequence of T.theileri DNA detectable at 730 base pairs are  
commonly used. Whole genome and transcriptome analysis can show the phylogenetic  
relationship between T.theileri and other pathogenic Trypanosomes which can be the basis  
for novel drugs development. Aim. The purpose of this paper is toreview the nature of  
Trypanosoma theileri, its molecular identification and also using its apathogenic nature as  
an opportunity to discover anew Trypanocidal drug.  
Trypanosoma theileri  
The Trypanosomatid parasites cause one of the most notorious human and animal Trypanosomiasis  
throughout Africa and South America.Even if trypanosomes are core causes of human (Sleeping sickness) and  
animal (Nagana) diseases, many other species are non-pathogenic [1]. Such apathogenic trypanosomatids are  
found worldwide, infecting a wide range of hosts. Among these, Trypanosoma theileri (T.theileri), is the one which  
is a ubiquitous, ‘truly cosmopolitan’ protozoan commensal of cattle found worldwide [1-3]. It was first described  
in cattle by Theiler, Laveran and Bruce in 1902 [4].  
Natural infection can be found throughout all age of cattle though rare for those under a year old. Neither  
its life cycle nor host relationship is fully understood within the mammalian host. The principal vector(s)  
responsible for transmission of the parasite is Tabanidae. However, ticks (Hyalomma anatolicum and Boophilus  
microplus) have been also reported as a vector later [5]. It is typically characterized by stercorarian type of  
transmission [5]. Following ingestion of infected blood meal, trypanosomes develop in the hindgut of the  
vector, infection is then transmitted to new hosts through fecal contamination on mucus membrane or through  
skin abrasions [6]. In newly infected host, the epimastigotes multiplies in the bloodstream by binary fission.  
Besides, epimastigotes and large trypomastigotes in peripheral blood, flagellates have been also found in extra-  
vascular sites of lymph nodes, kidney, spleen and brain [6].  
As a result of low parasitemia, the parasite is rarely detected during microscopic examination of the stained  
smears of peripheral blood; rather it lives for many years without being detected by such routine diagnostic  
technique [7]. Nevertheless, it can be detected by culturing peripheral blood using in vitro techniques. In order  
to isolate pathogenic African Trypanosomes, a kit called KIVI (Kit for In Vitro Isolation of trypanosome) was  
designed [8]. Similarly, Verloo et al. [2] proved that this kit can be used as an excellent tool to  
isolateT.theileriwith a much highersensitivity than the Roswell Park Memorial Institute medium (RPMI). On the  
other hand, there was evidence which endorsed the growth in RPMI medium easily [3]. However, the easier  
growth of T.theileri might reduce its efficiency to isolate pathogenic African Trypanosomes [2]. Furthermore,  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Hoare [4] reported that epimastigote form was grown 60 times more in cell culture media at 25°C than in blood  
agar media. A similar study in baby hamster kidney (BHK) cell culture inoculated with non-thrown blood buffy  
coat showed a typical morphological characteristics of T.theileri [3].  
The current drugs available to treat Trypanosomiasis are not satisfactorybecause they cause significant  
public health problem with toxic side effects and have poor efficacy [9]. Among these drugs, Pentamidine,  
Diminazeneaceturate (Berenil), Isometamidium chloride (Samorin), and Ethidium bromide, are important  
antitrypanosomal drugs [10] Thus, there is an urgent need for new, improved antitrypanosomal drugs by  
relaying on the non-pathogenic T.theileri as a tool.  
High-throughput screening (HTS) and virtual screening are used as a standard tool in drug discovery to  
identify novel lead compounds that target a biomolecule of interest. However, the latter is considered as a cost-  
effective tool [11, 12]. Trypanosomatid RNA editing can be used to identify drug target for protozoan parasites  
which are causing diseases like Trypanosomiasis. Amaro et al. [13] reported that RNA-editing ligase-1 (REL-1)  
can be used as drug-like inhibitors of a key enzyme in the editing Machinery. Identification of inhibitors was  
done through a strategy employing molecular dynamics to account for protein flexibility [13]. Currently, new  
parasitic inhibitors have been identified due to the availability of automated high content microscopy approach  
For a better pharmacology hypothesis and tests, the development of computational (In Silico) methods is  
also playing a significant role. This method comprises pharmacophores, databases, quantitative structure-  
activity relationships, homology models and other molecular modeling approaches, machine learning, network  
analysis tools and data analysis tools that use a computer. These are primarily used alongside the generation of  
in vitro data both to create the model and to test it. Such models have seen frequently used in the discovery and  
optimization of novel molecules with affinity to a target, the clarification of absorption, distribution,  
metabolism, excretion and toxicity properties as well as physicochemical characterization [11].  
Genome sequence analysis is used to find out molecules which are responsible for controlling parasite’s  
pathogenicity, virulence, immunity and chemotherapeutics responses. Genetic analysis is vital to characterize  
targets for defining the degree of variation within a gene encoding an identified molecule as a potential drug  
target [15] At present, for organisms with known genome sequence, the search for these molecules are  
undergone through the application of bioinformatics, which enhances the speed with which genes, can be  
identified. Further challenges remain in bioinformatics-based screening of compounds from predicted protein  
structure and the computerized docking of small molecules onto these structures as a means for high  
throughput screening of lead targets. Although the algorithms underlying these programs have improved  
significantly, the predictions are imperfect [15]. There are plenty of challenges for the development of tools for a  
trypanosome vaccine, new therapeutics or specific diagnostics. Genome sequence data, differential gene  
expression analysis and comparative genomics are playing significant role for identification of remarkable  
candidate of targets [15].  
Though T.theileri is nonpathogenic naturally, but it can cause illness to stressed cattle. Moreover, little is  
known about the parasite so far. However, since recently, it has become area of interest by considering it as a  
tool and vector to treat the pathogenic microorganisms; particularly protozoan parasites. For instance,  
according to a report by Mott et al. [1], T. theileri can offer significant potential to target multiple infections by  
using as a novel vehicle to transport targeted vaccine antigens and other protein of interest, and also has a  
potential to deliver therapeutics including the lytic factor to protect cattle from trypanosomiasis [1].  
Furthermore, mixed infection they cause with pathogenic trypanosome on same host (cattle), and the presence  
of homologous sequences with specific sequences of anti-trypanosome drug targets from pathogenic  
Trypanosomes may guide us to use this parasite as a model candidate for the development of novel drugs to  
treat the pathogenic Trypanosomes. Though T.theileri is nonpathogenic naturally, but it can cause illness to  
stressed cattle. Moreover, little is known about the parasite so far. However, since recently, it has become area  
of interest by considering it as a tool and vector to treat the pathogenic microorganisms; particularly protozoan  
parasites. Therefore, the main objective of this paper is to review the possible detection ways and the role of  
T.theileri in new Trypanocidal drug discovery.  
Trypanosomes are members of the Order Kinetoplastida; Family Trypanosomatidae and Genus  
Trypanosoma. Based on the site of the parasite’s growth in the vector, they are further classified as Stercoraria  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
and Salivaria type. Stercorarian trypanosomes develop in the hindgut of the vector, following ingestion of  
infected blood meal, and infection is transmitted to new host through fecal contamination [6]. Trypanosomes  
from bovids and cervids formed the clade T. theileri in phylogenetic trees, which corresponds to the type-species  
of the subgenus Megatrypanum, a taxon comprised solely of trypanosomes from ruminants [16, 17].  
Today, molecular testing has shown the phylogenetically useful genes at the clade, lineage, and genotype  
levels, supporting at least six genotypes within the clade T. theileri [17]. Moreover, Hoar [4] reclassified all  
trypanosomes from cattle and buffalo as T. theileri. Nevertheless, , only cattle isolates should be considered  
synonyms of T. theileri, whereas all Megatrypanum isolates from species of Artiodactyla other than cattle,  
including those of other Bovidae spp. with the exception of isolates from goats and sheep, should be classified as  
T.theileri-like. Trypanosomes, morphologically similar to T. theileri have been identified at least 24 different  
species of ruminants. Besides T. theileri of cattle, only T. melophagium (sheep) and T. theodori (goat) are  
considered to be separate species occurring in Bovidae because experimental cross-infections have established  
their host restriction to vertebrate and invertebrate hosts. Similarly, the inability to infect bovids supports the  
separateness of the Cervidae trypanosomes T. cervi and T. mazamarum from T. theileri. The knowledge of most  
Megatrypanum spp. of any other order other than Artiodactyla is restricted to morphology of blood forms [18].  
The sequencing of polymorphic internal transcribed spacer of ribosomal DNA (ITS rDNA), spliced leader  
(SL) and CATL genes disclosed two phylogenetic lineages, TthI and TthII, and five genotypes of T. theileri.  
Phylogenetic analysis clustered together all T. theileri isolates from cattle so far examined, from North and  
South America, Europe and Asia [16, 17, 19].  
Table 1. Taxonomy of Trypanosomes in general.  
Classical classification [4]  
Clade classification [86]  
Species and subspecies  
T. bruceibrucei  
T. bruceigambiense  
T. bruceirhodesiense  
T. evansi  
T. equiperdum  
T. congolense  
T. simiae  
T. brucei (T. brucei and related species  
mostly transmitted by tsetse flies)  
T. godfreyi  
T. vivax  
T. uniforme  
T. suis  
T. cruzi  
T. cruzimarinkellei  
T. vespertilionis  
T. dionissi  
T. rangeli  
T. lewisi  
T. musculi  
T. nabiasi  
T. cruzi (T. cruzi and related species  
transmitted by triatomine bugs)  
Rodent(presumably transmitted by  
T. microti  
T. theileri  
T. melophagium  
T. conorhini  
T. minasense  
Other Trypanosoma clades  
T. pestanai  
T. cyclops  
T. theodori  
T. carassi, T. cobitis, T. granulosum, T.  
sinipercea, T. ophiocephali, T. chelodinae, T.  
nudigobii, T. blennidini, T. haploblephari, T.  
boissoni, T. capigobii, T. semegalense, T.  
murmanense, T. pleuronectidium, T. triglae, T.  
rotatorium, T. ranarum, T. chattoni, T. binneyi,  
T. therezieni, T. mega, T. neveulemairei, T. fallisi  
Aquatic(transmitted by insects and  
leeches, also includes trypanosomes  
of platypuses and aquatic tortoises)  
T. avium, T. bennetti, T. corvi, T. culicavium, T.  
gallinarum, T. polygranularis, T. anguiformis  
Avian(transmitted primarily by black  
flies and hippoboscid flies)  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Morphology is the traditional taxonomic criterion for the classification of T.theileri and allied species, which  
trypanosomes that share large blood trypomastigotes, restricted mammalian hosts, worldwide distribution,  
lack of pathogenicity and contaminative transmission by tabanid or hippoboscid flies [4]. It has been used as the  
exclusive taxonomic criterion to classify trypanosomes in the subgenus Megatrypanum, whose members have  
the largest mammalian blood trypanosomes with a small kinetoplast situated very close to the nucleus. Species  
within this subgenus are defined based on their host restriction. According to morphology and to the  
assumption that Megatrypanum spp. are mammalian host-specific, or at least infective to very closely related  
species, all isolates from cattle have been classified as T. theileri. It has been considered as an independent  
trypanosome occurring in distinct species and or races of Bovidae or in different countries, resulting in  
considerable synonymy [17].  
In general, T. Theileriis large in size (25-120 µm) long with long-pointed posterior end (Figure 1). The  
kinetoplast is large in size and has well developed undulating membrane and free flagellum. Both  
trypomastigote and epimastigote may occur in blood. Body is somewhat curved and is drawn to fine point at  
posterior end. Important character in this trypanosome is the possession of well-marked myonemes [17].  
Figure 1. Photomicrograph of trypomastigotes found in blood-derived mononuclear cell cultures from different  
naturally infected cows, from San José del Nus, Antioquia, morphologically characterized as T. theileri [21].  
This species is a cosmopolitan parasite of cattle with high incidence on every continent except Antarctica  
[4,16]. There are numerous records of T. theileri in cattle. For example in Western Europe, T. theileri is the only  
trypanosome species occurring in cattle [2]. Hence, it has been reported in European countries (France,  
Germany, England, Scotland, Belgium, Italy and Spain), North America (USA, Canada), South America (Brazil)  
and Asia (Korea and Bangladesh) and Africa [2]. Besides cattle, morphologically indistinguishable trypanosomes  
related to T. theileri have been described in otherdomestic and wild artiodactyls, including buffalo, sheep, goat,  
antelopes, and cervids [4, 16].  
A principal vector for T. theileri is Tabanidae (Diptera) flies, commonly known as horse flies, deer flies, clegs,  
or blind flies. Different species such as Haematopota pluvialis, Tabanus glaucopis and T. striatus were found as  
intermediate host experimentally [4]. It is among the nuisance pests for people and livestock because of their  
painful and irritating bite, persistent biting behavior, and blood ingestion. They are seasonally present in all  
kinds of landscapes, latitudes, and altitudes [22]. In addition, ticks can also maintain the development of  
T.theileri. A high infection rate of the tick with T. theileri was reported by Latif et al. [5]. Morzaria et al. [23] in the  
Sudan reported the first biological transmission of T. theileri to cattle by the tick, H. a. anatolicum. Asimilar study  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
was done in Brazil showed that epimastigote forms were found in the hemolymph of the cattle tick Boophilus  
microplus in the state of Rio Grande do Sul, southern Brazil [24].  
Life cycle  
So far, the life cycle of T.theileriwithin mammalian host has rarely been reported [3, 22]. It has an indirect life  
cycle and transmitted cyclically by tabanid. Trypanosomes undergo development in the hind gut of the tabanid  
flies [4, 25]. Cattle are infected commonly though contamination of the mucous membrane with flies faeces  
containing the small metacyclic forms (Stercoraria) [25]. In vitro infections showed that transmission occurs by  
excretion of metacyclic form in tabanid feces, which gain entrance into a new host either by the bite wound of  
the vector or by skin abrasions. Furthermore, ingestion of feces or the vector itself by the host can cause  
infection [26, 25].  
The parasite has several development stages (Figure 2). Epimastigotes undergo multiplication asexually by  
binary fissionin the lymph nodes and in various internal organs and in the bloodstream which is hardly  
detectable [19]. As epimastigotes and large trypomastigotes have been found in peripheral blood, flagellates are  
also found in extra-vascular sites of lymph nodes, kidney, spleen, and brain [27]. Based on the report by Bose et  
al. [26]; the infective stages were found in the hind hut and faeces of Tabanids. Although “amastigote-like” forms  
were described within bovine lymphocyte cultures, the existence of intracellular stages of T. theileri in tissues of  
vertebrates has not been reported [28].  
Figure 2. Morphological analysis of T.theileri. Images of Giemsa-stained T. theileri. The trypomastigote form  
(upper panel) and epimastigote and intermediate forms (lower panel) were observed under a microscope [29].  
Megatrypanum spp. are cyclically transmitted by Tabanid and transmission is contaminative [26, 25].  
Furthermore, infection of T. theileri established in the calves with the Trypanosomatid flagellates from the tick  
H. a. anatolicum [30]. The transmission was contaminative by excrements containing metacyclictrypo-  
mastigotes. In the vertebrate host, the stercorarian trypanosomes multiply as amastigotes or epimastigotes.  
Trypomastigote stages found in the peripheral blood, do not divide and their sole function is to spread the  
infection into other organs and enable the transmission into the vector. T.theileri and T.melophagium are  
stercorarian species transmitted by tabanid and hipoboscid flies to cows and sheep, respectively [31].  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Despite its nonpathogenicity, detectable parasitemia is usually associated with concomitant disease,  
suggesting that host immunity is involved in parasite control [32]. The parasiteinduces chronic and cryptic  
infections which is potentially pathogenic for those under severe physical and nutritional stress, for newborn  
and pregnant cows, and when associated with concurrent infectious diseases.Prolonged infection persists  
without any clinical signs. The depression of the immune system is thought to allow for increased parasitemia  
and dispersion of T. theileri through several organs including central nervous system [27, 33-35].  
Diagnosis and confirmation of T.theileri  
So far, the diagnosis of this species requires isolation by blood culture, which is a laborious, time consuming  
and expensive method. Although no serological diagnostic method has been developed specifically for T. theileri,  
a lack of cross-reactivity has been demonstrated for methods used to diagnose T. evansi, T. congolense, T. vivax  
and T. b. brucei [36]. Furthermore, T. theileri can be distinguished from other trypanosomes by PCR-  
amplification of ITS rDNA, SL and CATL sequences [16, 17, 37] Nevertheless, such molecular studies are limited  
by the low levels of parasitemia; by rare parasites in blood smears despite positive hemocultures [16, 17].  
a) Cell Culture  
Isolation of T.theileri from whole or defibrinated blood of infected cattle can be done in a variety of culture  
media. Though it is easier to isolate the organism, its continuous maintenance in culture is much more difficult.  
The presence of blood cells, the temperature of cultivation and the type of culture vessel used appear to be  
important [38]. Cultivation at room temperature results in multiplication in the crithidial stage, and only in a  
few instances has the organism been cultivated at 37°C in the trypanosome stage [39]. Attempts by Herbert [38]  
to obtain continuous culture of T. theileri at 37°C were unsuccessful. Later, T.theileri in the trypanosome form  
has been reported as a contaminant in cultures, at 37oC, of normal bovine fetal kidney cells or blood  
lymphocytes and lymph node cells of cattle with lymphatic leukemia or viral diarrhea. Tissue culture fluids,  
supplemented with newborn calf serum and blood or blood products, are suitable for the isolation and  
continuous cultivation of T.theileri at 37°C [39]. Cultivation of the trypanosomes at 37oC has been reported in  
non-defined or partially defined monophasic liquid media containing erythrocytes or erythrocyte products, in  
embryonal chicken eggs and in various types of cell cultures [40]. Furthermore, the KIVI culture systemhas  
been designed for the isolation of pathogenic African trypanosomes, particularly, T. Brucei gambiense which is  
found applicable for T.theileri too [8]. Similarly, Rosewell Park Memorial Institute (RPMI) based culture systems  
have been described for culturing bloodstream forms of T. b. brucei [41]. Hence, in an attempt to isolate a stock  
of T.theileri, blood samples can be inoculated into both RPMI 10%+feeder and KIVI culture medium [2].  
b) Polymerase Chain Reaction (PCR)  
A large variety of molecular diagnostic testshave been developed for human and animal African  
trypanosomiasis based on detection of trypanosome DNA or RNA [42]. It can be applied on all biological  
specimens that may contain the parasite’s nucleic acids such as blood, CSF, lymph node and chancre aspirate,  
biopsy and histological material. Even from stained microscopic slides, DNA can be extracted for subsequent  
PCR [42, 43]. PCR allow discrimination of the trypanosome species in one single run and even in the same  
specimen in case of mixed infection. For instance, the 18S-PCR-RFLP where cleaving of the amplicon with two  
restriction enzymes generating fragment profiles that are characteristic for T. congolense, T. vivax, Trypanozoon  
and T. theileri (Table 6) [43]. Similarly, differential diagnosis can be obtained with the less complex ITS1 PCR  
which generates amplicons of taxon specific lengths [37, 44]. Species specific PCRs can be also hampered by  
genomic variation within the target taxon. According to Njiru [45], a Loop mediated isothermal amplification  
(LAMP) of DNA is a less demanding technology that may prove a real breakthrough in molecular diagnosis. In  
LAMP, DNA is amplified without prior heat denaturation and the amplicons are visualised by turbidimetry,  
colori metric reactions or fluorescence [46].  
c) Two-Dimensional Gel Electrophoresis (2-DE)  
After having sequenced whole genomes, better understanding of cellular mechanisms relies on studying  
the functional units of gene expression-proteins. Among these approaches in proteomicsis the use of two-  
dimensional gel electrophoresis as a means of separation and visualization of complex protein mixtures  
extracted from biological samples. Despite several limitations of the method, its ability to separate large  
numbers of proteins, including their post-translationally modified forms, ensures that it will continue to be  
popular in several well-defined areas of proteomics [47, 48]. Gene expression profiling is the survey of a large  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
number of genes and/or their expression products, typically in an effort to identify differentially expressed  
genes, or broad patterns of gene expression under different experimental conditions. The utility of gene  
expression profiling for understanding molecular processes, elucidation of drug target interactions, clinical  
diagnosis, etc., cannot be overstated. Several DNA based methods for profiling of gene expression have been  
previously described [48]. Two-dimensional electrophoresis (2-D electrophoresis) is a powerful and popular  
technique to sort proteins based on two independent properties and discrete steps: the first-dimension step,  
isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-dimension  
step, SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), separates proteins according to their molecular  
weights. Each spot on the resulting two-dimensional array corresponds to a single protein species in the  
sample. Many different proteins can be separated, and information like pI, apparent molecular weight, and the  
amount of each protein is obtained [49, 50]. It has also a distinct capability to detect post- and co-translational  
modifications, which cannot be predicted from the genome sequence. Its applications include proteome  
analysis, cell differentiation, and detection of disease markers, monitoring therapies, drug discovery, cancer  
research, purity checks, and micro-scale protein purification. Furthermore, precast IPG strips are used  
(Immobiline Dry Strip gels) which is available from Amersham Biosciences [51].  
d) High Throughput Microscopy  
High-throughput microscopy (HTM) shows images which are generated automatically upon many different  
treatments for a certain period. This technology has opened the way to conduct large-scale, image-based  
screens to discover novel genes and their functions [52]. Now days, there are several commercially available  
platforms of high-content high-throughput microscopy. They consist a fully automated wide-field or confocal  
microscope with specialized hardware and software to manage large sets of information. They are equipped  
with state-of-the-art optics enabling a broad range of fluorescence excitation and multi-channel which enables  
to detect several spectrally distinct fluorophores parallelly. Such imaging platforms are capable of imaging at  
different magnifications, which assists in imaging of both large populations of cells or sub-cellular details in a  
group of selected cells [53]. For systematic analyses of protein function, quantitative fluorescence microscopy is  
now becoming one of the tools of choice in large-scale [54]. Compared to a non-microscopic screening  
approaches, fluorescence microscopy provides information at the single-cell or even sub-cellular level. It is  
enables an enhanced statistical data analyses for the identification of distinct phenotypic populations in one  
culture treated in similar way. On the other hand, Microscopic analyses can also be performed in living cells for  
extended periods of time to return vital data on the dynamics of fluorescent molecules [53, 55], or the order of  
occurrence of phenotypes during the observation period [56]. Overall, to apply this technology, there are several  
steps and optimization involved in an experiment. These are sample preparation, image acquisition, image  
analysis, data storage and handling, data mining and modelling (Figure 3). Hence, it enables unsupervised data  
collection with high information content on the temporal and spatial distributions and states of fluorescent  
markers [54, 53].  
Figure 3. The principle: Steps in high-content high-throughput microscopy; adapted from Starkuviene and  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Current state of therapy and recent developments  
The idea of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and  
arsenical compounds that displayed selectivity against trypanosomes [57].  
Commonly used drugs to treat sleeping sickness  
Since half a century ago, we have been using only certain drugs to treat Human African Trypanosomiasis  
(HAT). There is a very limited arsenical drug with shortcomings such as high toxicity with emerging-resistance.  
Early stages of HAT are treated with pentamidine, an aromatic diamidine, and suramin, a naphtaline derivative.  
Adverse effects for these drugs are significant and the failure rate is high. Late stages of HAT are treated with  
Melarsoprol, a melaminophenyl arsenical compound that is able to cross the blood brain barrier [58].  
Melarsoprol (Arsenicals) have been the most effective drugs universally to treat sleeping sickness despite  
resistance of trypanosomes and toxicity to patients. Side effects are severe and up to 5% of those treated die of  
drug-induced reactive encephalopathy [58]. On the other hand, Pentamidine, Suramin and Berenil® are active  
only in the first stage of the disease when the parasite is restricted to the blood and lymphatic circulatory  
system [36, 59]. However, neither of these compounds crosses the blood-brain barrier to treat central nervous  
system infections. On the contrary, Nifurtimox and alpha-difluoro-methylornithine provide interesting  
alternatives totreat the central nervous systeminfection. Nevertheless, DFMO is known to be less active against  
T.rhodesiense [60].  
Recently, eflornithine-nifurtimox combined therapy islaunchedand found the safest treatment for late-  
stage trypanosomiasis [59]. This mixed drug allows significant reduction of eflornithine dose and treatment  
duration. However, like eflornithine alone, this combined therapy is only effective in the second stage of T. b.  
gambiense infections. This leaves melarsoprol which causes reactive encephalopathy in about 10% of treated  
patients, as the only effective drug against both T. b. rhodesiense and T. b. gambiense late-stage infections.  
Because of this, the increasing rate of melarsoprol treatment failures is alarming [61].  
Table 2. Problems associated with current drugs for human African trypanosomiasis  
Effective against early stage of T. b.  
Ineffective against early stage of T. b. gambiense and late  
stage of both HATs  
Effective against early stage of T. b.  
Ineffective against early stage of T. b. rhodesienseand late  
stage of both HATs  
Effective against late stage of both HATs Toxic (kills up to 5% of patients) Resistance observed in field  
Effective against late stage of T. b.  
Ineffective against late stage of T. b. rhodesiense Difficult  
dosing scheme, and cost of hospitalisation  
Commonly used drugs to treat animal trypanosomiasis  
These days, several chemotherapeutic agents have been used to treat animal trypanosomiasis. However,  
most of these have a narrow therapeutic index which makes administration of the correct dose essential. Drug  
resistance occurs and should be considered in refractory cases [63].  
Table 3. Drugs commonly used for Trypanosomiasis in domestic animals  
Main Action  
Diminazene Aceturate  
Homidium Bromide  
Vivax, congolense,brucei  
Vivax, congolense,brucei  
Cattle, Equids  
Curative, some prophylactic activity  
Isometamidium Chloride  
Quinapyramine Sulfate  
Vivax, congolense  
Curative and prophylactic  
Horses, camels,  
pigs, dogs  
Vivax, congolense,brucei, evansi,equiperdum,si  
Horses, camels,  
pigs, dogs  
Vivax, congolense,brucei, evansi,equiperdum,si  
Horses, camels,  
Brucei, evansi  
Curative, some prophylactic activity  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Mechanism of actions  
Arsenical drugs that are used to treat late-stage African trypanosomiasis kill cells sooner in lowdrugdosage.  
It results in oxidative stress via nonspecific covalent binding to thiol groups [64]. Whereas,  
Difluoromethylornithine (DFMO/Eflornithine) is a suicide inhibitor of ornithine decarboxylase [65].  
Furthermore, Isometamidium is an ethidium bromide derivative which intercalates selectively in the kDNA. As  
consequence, it affects the kDNA integrity by preventing networkdivision [66]. Pentamidine,  
Diminazeneaceturate (Berenil), Isometamidium chloride (Samorin), and Ethidium bromide, which are important  
antitrypanosomal drugs, promote linearization of Trypanosoma equiperdumminicircle DNA. This effect occurs at  
therapeutically relevant concentrations [10]. Having know-how on the mechanism of action of anti-  
trypanosomal drug efficacy and resistance, will help us to develop a new therapeutic principle to combat drug  
resistance and contributes to have the idea on the mode of actions of anti-trypanosomal resistance at molecular  
level [14].  
Now a day, there is a breakthrough of T. theileri which has been used as a novel vehicle to deliver vaccine  
antigens and other proteins to control pathogenic trypanosomatids [1]. Favorable conditions for the growth and  
transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or  
specific localisation at the cell interior or surface using trafficking signals. Engineered vehicle could establish in  
the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific  
immune responses to an expressed Babesia antigen at protective levels. It also has the potential to deliver  
therapeutics to cattle, including the lytic factor which protects humans from cattle trypanosomiasis [1].  
Drug resistance  
Drug resistance is a major impediment in the treatment of the diseases given that existing drugs are old  
with severe side effects. Most resistance mechanisms developed by these parasites are related with a decreased  
uptake or increased efflux of the drug due to mutations or altered expression of membrane transporters.  
Different new approaches have been elaborated that can overcome these mechanisms of resistance including  
the use of inhibitors of efflux pumps and drug carriers for both active and passive targeting [61].  
The two principal drugs, Melarsoprol and Pentamidine have been used to treat both sleeping sickness in  
humans and Nagana in livestock. However, since half a century ago, cross-resistance to these drugs was found  
and remains the only example of cross-resistance among sleeping sickness therapies. Adenosine transporter in  
T.brucei, is well-known for its role in the uptake of these drugs. Furthermore, aquaglyceroporin-2 (AQP2) loss-  
of-functions was linked to melarsoprol-pentamidine cross-resistance. AQP2, a channel that appears to facilitate  
drug accumulation, may also be linked to clinical cases of resistance [67].  
Target sequences screening  
At the molecular level, the main targets for drugs are proteins (mainly enzymes, receptors and transport  
proteins) and nucleic acids [68]. Drugs bind to their corresponding targets and perform the desirable  
therapeutic effects. These days, the identified drug targets have been stored in databases such as DrugBank [69]  
Therapeutic Target Database (TTD), Potential Drug Target Database (PDTD) and TDR Targets Database [70, 71].  
Searching for novel genes required for survival or virulence of any organism was based on several genetic  
methods involving genetic crosses, gene knockouts and induced mutations, followed by screening for the  
relevant phenotype. In trypanosomes, these approaches have been hampered by the inability to generate  
sufficient numbers of progeny clones from genetic crosses and the lack of a fine genetic map. These problems  
have affected the ability to determine precisely the statistical significance of segregating phenotypes in relation  
to the number of loci or alleles determining the trait of interest [72].  
Current methods for target-based drug discovery usually involve screening diverse or focused libraries  
against the molecular target (Table 4). The hits are then characterized and optimized through rounds of design,  
synthesis and testing against protein and cell. The testing usually includes assays for potency, selectivity and  
pharmacokinetic properties. In terms of target validation, it is important to prove that the compounds are  
acting on-target for inhibition or modulation of the target that is causing the death of the parasite. The  
compounds are then assayed in animal models of infection. It is only at this stage that it becomes clear if the  
proposed drug target has a reasonable degree of validation. It shows that it takes both time and resource before  
getting evidences which shows the target chosen, is truly valid drug target. Therefore, it is important to choose  
drug targets very carefully at the outset of a drug discovery programme [9].  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Elucidation of molecular targets is very important for lead optimization during the drug development  
process. A direct method to find targets of antitrypanosomal compounds against T.brucei using a trypanosome  
overexpression library was done by Begolo et al. [73]. As proof of concept, the library was treated with  
Difluoromethylornithine and DDD85646 and identified their respective targets, ornithine decarboxylase and N-  
myristoyltransferase. The overexpression library could be a useful tool to study the modes of action of novel  
antitrypanosomal drug candidates [73].  
RNA interference (RNAi) is a phenomenon through which double stranded RNA induces potent sequence-  
specific degradation of homologous transcripts. Besides its function in cellular defense and developmental  
regulation, it has emerged as an invaluable tool for elucidation of gene function and drug target validation. This  
is particularly useful when substantial genome sequence data are available. Gene silencing using RNAi can aid  
translation of raw genomic sequence data into biologically relevant information toward the development of new  
and/or improved control strategies [74].  
Table 4. Common antitrypanosomal drug targets.  
Accession No  
Difluoromethylornithine Ornithine decarboxylase  
Diminazene and  
Cytosolic serine Oligopeptidase  
Trypanosomal proteasome  
exhibiting high trypsin-like but  
low chymotrypsin-like  
Proteasome inhibitors  
T. bruceibrucei  
Cysteine protease  
inhibitors, Peptidyl  
inhibitors and Non-  
peptidyl inhibitors)  
Cathepsin L-Like Cysteine  
Protease (Brucipain and  
T. brucei  
T. grayi  
T. grayi  
T. brucei  
NCBI database  
Pyruvate kinase  
T. bruceibrucei  
T. bruceibrucei  
DNA topoisomerase  
mitoxantrone and  
Tubulin directed drugs  
and Inhibition of lipid  
DNA topoisomerases  
T. bruceibrucei  
High-throughput screening of trypanosome RNA interference (RNAi) libraries is playing a significant role  
in understanding drug mechanism of action and up take [78]. However, it’s becoming challenging to use such  
reduced-function approach to find out specific drug targets. Usually, drugs mode of action of antitrypanosomal  
drug is by inhibiting essential enzymes. Even without using drug, depleting the target can be life threatening.  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Theoretically, weak RNAi result in slowly growing cells with enhanced drug susceptibility. In normal  
circumstance, there are coincidences of the cells to die without adding drug for treatment. It implies that as a  
result of loss of function of one of cellular path way inside, there is an opportunity of the cell to be more  
susceptible to drug than the targeted pathway. Hence, gain-of-function approaches are the best choice for  
direct determination of the targets of drug of choice [79].  
An experiment done by Alsford et al. [80], on five current HAT drugs for genome-scale RNA interference  
(RNAi) target sequencing (RIT-seq) screens in T. brucei were used , revealing the transporters, organelles,  
enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling  
identifies both known drug importers and the only known pro-drug activator, and links more than fifty  
additional genes to drug action [80].  
A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while  
the AP-1, lysosomal proteases and major lysosomal transmembrane protein and alsospermidine and N-  
acetylglucosamine biosynthesis all contribute to suramin action [80]. If an antitrypanosomal drug inhibits the  
function of a single protein, then over expression of that protein or a fragment containing the drug binding site  
could result in reduced drug sensitivity. Due to loss of function of a single protein by antitrypanosomal drug,  
over expression of specific protein of interest or part of it with drug binding site is happened; consequently it  
leads to loss of drug sensitivity [81]. Glycolysis is perceived as a promising target for new drugs against  
parasitic trypanosomatid protozoa, because this pathway plays an essential role in their ATP supply. However,  
Bloodstream form T.theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely  
rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism.  
Potential criteria to aid target selection are described below [9].  
Table 5. Criteria for molecular targets developed [82, 83].  
Genetic or chemical  
No or weak genetic or  
chemical validation that the  
target is essential for  
survival of the organism  
No drug-like, small  
molecule inhibitors are  
known and the active site is  
not druggable  
Genetic and chemical validation  
that the target is essential for  
survival of the organism  
validation that the target is  
essential for survival of the  
Drug-like, small molecule  
inhibitors are known or the  
Drug-like, small molecule  
inhibitors are known and there  
is a druggable active site (clinical active site is potentially  
activity within the target family) druggable  
Robust assay in plate format  
amenable to high-throughput  
development into robust,  
screening developed and active  
protein supply assured within  
appropriate time-lines  
In vitro assay exists,  
No in vitro assay developed  
and/ or significant  
plate format feasible, but not problems with protein  
yet achieved  
Target has no known isoforms  
within the same species and is  
not subject to escape from  
Target has isoforms within  
same species or may be  
subject to escape from  
Target has multiple gene  
copies or isoforms within  
same species and is subject  
to escape from inhibition  
Resistance potential  
Toxicity potential  
No human homologue of the  
target present, or the human  
homologue is known to be non-  
essential and inhibition of this  
shows no effect on the human  
Human homologue of the  
target is present and little  
or no evidence (structural  
or chemical) that selective  
inhibition is possible  
Human homologue of the  
target is present, but  
evidence (structural or  
chemical) that selective  
inhibition is possible  
Structure without ligand  
available and/or poor  
resolution (>2·3 Å) or  
opportunity to build a good  
homology model (high  
sequence homology to  
Ligand-bound structure of  
targetor ligand in closely related  
homologue available at high  
resolution (<2·3 Å)  
No structure of target or  
closely related homologu  
Adopted from Gilbert [9].  
Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI:  
Table 6. Primers for T.theileri.  
Primer (forward and reverse)  
Tth625a (5’-CCG CTG GAG CTA AGA ATA GA-3’) and  
Tth625b (5’-AAT TGC ATA AAC ACA GCT CCC-3’)  
For species-specific PCR amplification  
Forward primer 18STnF2 (5’-CAA CGA TGA CAC CCA TGA ATT GGG GA-3’) and  
Reverse primer 18STnR3 (5’-TGC GCG ACC AAT AAT TGC AAT AC-3’)  
The full length 18S ribosomal DNA  
sequence Analysis  
For single PCR amplification to anneal  
internal transcribed spacer of ribosomal  
genes (ITS) sequence  
Kin1 reverse (5’- GCG TTC AAA GAT TGG GCA AT-3’) and  
Kin2 forward (5’-CGC CCG AAA GTT CAC C-3’)  
Trypanosome genomics  
Two complementary approaches are being followed insequencing the trypanosome genome: genomic  
DNAsequencing and expressed sequence tag (EST) sequencing.Initial data emerging from the T. brucei shotgun  
genomesequence indicate that the overall sequence organizationof chromosomes is distinct from that of other  
highe reukaryotic organisms. For example, trypanosome genesare very tightly packed on chromosomes, which  
mightexplain in part the high incidence of alternative splicing ofpre-mRNAs and the generation of polycistronic  
RNAs. The transcripts appear to be unidirectional over long stretchesof the chromosomes [84].  
The availability of genome sequence data has facilitated the development of high-throughput genetic  
screening approaches in microbial pathogens. In the African trypanosome, T.brucei, genome-scale RNA  
interference screens have proven particularly effective in this regard. These allow for identification of the genes  
that contribute to a pathway or mechanisms of interest. The approach has been used to assess loss-of-fitness,  
revealing the genes and proteins required for parasite viability and growth. Consequently, the results from  
these screens predict essential and dispensable genes which facilitate drug target prioritization efforts. The  
approach has also been used to assess resistance to anti-trypanosomal drugs, revealing the genes and proteins  
that facilitate drug uptake and action. These outputs also highlight likely mechanisms underlying clinically  
relevant drug resistance [85].  
Though T.theileri is nonpathogenic naturally, but it can cause illness to stressed cattle. Moreover, little is known  
about the parasite so far. However, since recently, it has become area of interest by considering it as a tool and  
vector to treat the pathogenic microorganisms; particularly protozoan parasites. T.theiler grows successfully in  
vitro in SDM 79 at 26 oC. The growth pattern, viability and its response for pentamidine can be checked through  
resazurin assay. The parasite (T.theileri), is confirmed using PCR amplification by species specific primers.  
Furthermore, the BLAST and MSA with common antitrypanosome target sequences. Subsequently, there is no  
significant similarity both at DNA and protein level with T. theileri unlike the antitrypanosome targets from  
pathogenic Trypanosomes. There is a chance to not to isolate the parasite immediately as a result of low  
parasitemia naturally. Hence, most of the isolation of T.theileri from natural infection was not confirmed  
purposively. Rather, it has been found during unexpectedly time. It can be isolated while researchers were  
doing for other objectives. For example, during macrophage culture, Total leukocyte and differential counts,  
PCV, while studying Bovine leukemia virus (BLV) from lymphocyte cultures of cows infected with it.  
Competing interests  
The author declares that there are no competing interests.  
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Citation: Fentahun T. Molecular detection of Trypanosoma theileri and a new Trypanocidal drug, a review. J Life Sci Biomed, 2020; 10(3): 29-43; DOI: