2020 SCIENCELINE

Journal of Life Science and Biomedicine

J Life Sci Biomed, 10 (3): 29-43, 2020

ISSN 2251-9939

DOI: https://dx.doi.org/10.51145/jlsb.2020.5

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: tedyvet@gmail.com;

: 0000-0002-2955-5638

ABSTRACT

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

Keywords

Model,

o

cultivate at 26 C, without CO₂. Gradient PCR amplification has been used to detect using T.

Novel Trypanocidal drug,

PCR,

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

INTRODUCTION

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:

https://dx.doi.org/10.51145/jlsb.2020.5

29

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

[14].

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.

DISCUSSION

Taxonomy

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:

https://dx.doi.org/10.51145/jlsb.2020.5

30

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]

Section

Subgenus

Species and subspecies

T. bruceibrucei

T. bruceigambiense

Trypanozoon

T. bruceirhodesiense

T. evansi

T. equiperdum

T. congolense

T. simiae

T. brucei (T. brucei and related species

mostly transmitted by tsetse flies)

Salivaria

Nannomonas

T. godfreyi

T. vivax

T. uniforme

Duttonella

Pycnomonas

T. suis

T. cruzi

T. cruzimarinkellei

T. vespertilionis

T. dionissi

T. rangeli

T. lewisi

T. musculi

T. nabiasi

Schizotrypanum

Herpetosoma

T. cruzi (T. cruzi and related species

transmitted by triatomine bugs)

Rodent(presumably transmitted by

fleas)

Stercoraria

T. microti

T. theileri

T. melophagium

T. conorhini

Megatrypanum

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)

Adapted from Fraga et al . [20]

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:

https://dx.doi.org/10.51145/jlsb.2020.5

31

Morphology

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].

Epidemiology

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].

Vector

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:

https://dx.doi.org/10.51145/jlsb.2020.5

32

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].

Transmission

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:

https://dx.doi.org/10.51145/jlsb.2020.5

33

Pathogenesis

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 37^oC, 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 37^oC 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:

https://dx.doi.org/10.51145/jlsb.2020.5

34

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

Pepperkok [53].

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:

https://dx.doi.org/10.51145/jlsb.2020.5

35

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

S.No

Drug

Use

Problem

Effective against early stage of T. b.

rhodesiense

Ineffective against early stage of T. b. gambiense and late

stage of both HATs

1.

Suramin

Effective against early stage of T. b.

gambiense

Ineffective against early stage of T. b. rhodesienseand late

stage of both HATs

2.

3.

4.

Pentamidine

Melarsoprol

Eflornithine

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.

gambiense

Ineffective against late stage of T. b. rhodesiense Difficult

dosing scheme, and cost of hospitalisation

Adapted from Wilkinson and Kelly [62].

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

Drug

Animal

Trypanosoma

Main Action

Diminazene Aceturate

Homidium Bromide

Cattle

Vivax, congolense,brucei

Curative

Cattle, Equids

Curative, some prophylactic activity

Isometamidium Chloride

Quinapyramine Sulfate

Cattle

Vivax, congolense

Curative and prophylactic

Curative

Horses, camels,

pigs, dogs

Vivax, congolense,brucei, evansi,equiperdum,si

miae

Quinapyramine

Dimethylsulfate

Horses, camels,

pigs, dogs

Vivax, congolense,brucei, evansi,equiperdum,si

miae

Prophylactic

Horses, camels,

dogs

Suramin

Brucei, evansi

Curative, some prophylactic activity

Curative

Melarsomine

Dichlorhydrate

Camels

Evansi

Adapted from Cynthia et al. [63].

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:

https://dx.doi.org/10.51145/jlsb.2020.5

36

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

a

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:

https://dx.doi.org/10.51145/jlsb.2020.5

37

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.

Drugs

Target

Accession No

Organism

Sources

XM_824336

T.bruceibrucei

1.

Difluoromethylornithine Ornithine decarboxylase

Begolo et al. [73]

AF042286

T.bruceigambiense

J02771

T.brucei

XM_822811

XM_009314999

HQ587038

T. bruceibrucei

T.grayi

T.cruzi

2.

3.

DDD85646

N-myristoyltransferase

Begolo et al. [73]

Rory et al. [75]

Pentamidine,

Diminazene and

Suramin

Cytosolic serine Oligopeptidase

(OP-Tb)

XP_009306351

T.grayi

Trypanosomal proteasome

exhibiting high trypsin-like but

low chymotrypsin-like

activities

Proteasome inhibitors

Dietmar and

Kevin [76]

4.

5.

-

T. bruceibrucei

XM_009318006

GU299414

GU299413

GU299412

GU299411

T.grayi

T.theileri

T.grayi

Cysteine protease

inhibitors, Peptidyl

inhibitors and Non-

peptidyl inhibitors)

Dietmar and

Kevin [76]

Cathepsin L-Like Cysteine

Protease (Brucipain and

Cruzipain)

GU299410

XP_009316281.

CAA44870.1

XP_009309990

KEG11772.1

T. brucei

T. grayi

ORC92589.1

CAA41018.1

3QV9_B

T.theileri

T. brucei

NCBI database

6.

Pyruvate kinase

T.cruzi

CCC52316.1

CCC94421.1

XP_827894.1

ESL09806.1

EAN78782.1

T,vivax

T.congolense

T. bruceibrucei

T.rangeli

T. bruceibrucei

DNA topoisomerase

inhibitors

Aline et al. [77],

Dietmar and

Kevin [76]

(Camptothecins,

Anthracyclines,

mitoxantrone and

Fluoroquinolones

Tubulin directed drugs

and Inhibition of lipid

synthesis

DQ887563

7.

8.

DNA topoisomerases

Tubulin

T.cruzi

Dietmar and

Kevin [76]

-

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:

https://dx.doi.org/10.51145/jlsb.2020.5

38

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].

Items

Green

Amber

Red

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

organism

Drug-like, small molecule

inhibitors are known or the

Essentiality

Drug-like, small molecule

inhibitors are known and there

Druggability

Assayability

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

supply

Target has no known isoforms

within the same species and is

not subject to escape from

inhibition

Target has isoforms within

same species or may be

subject to escape from

inhibition.

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

host

Human homologue of the

target is present and little

or no evidence (structural

or chemical) that selective

inhibition is possible

Structure

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

homologue)

Ligand-bound structure of

targetor ligand in closely related

homologue available at high

resolution (<2·3 Å)

Structural

information

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:

https://dx.doi.org/10.51145/jlsb.2020.5

39

Table 6. Primers for T.theileri.

Primer (forward and reverse)

Purpose

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

(Tth625-PCR)

1

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

2

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’)

3

Source: Lee et al. [3], Rodrigues et al. [16] and Geysen et al. [43]

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].

CONCLUSION AND RECOMMENDATIONS

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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