WOLIATA SODO UNIVERSITY

SCHOOL OF VETERINARY MEDICINE

TRYPANOCIDAL DRUG AND DEVELOPMENT OF DRUG RESISTANCE IN

AFRICAN ANIMAL TRYPANOSOMOSIS

A paper submitted for the course Senior Seminar on Animal Health (EMPH 504)

BY: Elias Kalel

Advisor: Dr.Asefa Asmare (PhD)

April, 2015

Woliata Sodo, Ethiopia

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TABLE OFCONTENTS PAGE

LIST OF TABLES III

LIST OF FIGURES IV

LIST OF ABBREVATIONS V

ACKNOWLEDGEMENT VI

SUMMARY VII

1. INTRODUCTION 1

2. AFRICAN TRYPANOSOMES 3

2.1. Morphology and characterization ............................................................................ 3

2.2. Life cycle ..................................................................................................................... 4

2.3. The vector of African trypanosomosis ..................................................................... 5

2.4. Host ............................................................................................................................. 6

2.5. Risk factors of trypanosomosis ................................................................................. 7

2.5.1. Animal factor 7

2.5.2. Environmental factor 8

2.6. Trypanosomosis control methods ............................................................................. 8

2.6.1. Sequential aerosol technique 8

2.6.2. Stationary attractive devices (traps and targets) 9

2.6.3. Live bait technique 9

2.6.4. Sterile insect technique 10

3. TRYPANOCIDAL DRUGS 11

3.1.Prophylactic treatments ........................................................................................... 11

3.2.Curative treatments .................................................................................................. 12

3.3.Mechanism of action of current used drugs ........................................................... 12

3.3.1. Diminazene aceturate (Berenil®) 12

3.3.2. Isometamidium and homidium 12

3.3.3.Suramin (Polysulphonated naphtyl urea) 13

3.3.4. Melarsomine hydrochloride (Cymelarsan®

) 13

4. TRYPANOCIDAL DRUG RESISTANCE 14

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4.1. Definition .................................................................................................................. 14

4.2. Causes of trypanocidal drug resistance ................................................................. 14

4.3. Types of trypanocidal drug resistance ................................................................... 15

4.4. Cross and multiple drug resistance ........................................................................ 16

4.5. Mechanism of drug resistance ................................................................................ 17

4.6. Current situation of resistance ............................................................................... 18

4.7. Impact of drug resistance ........................................................................................ 19

4.7.1. Factor influencing development of resistance to Trypanocidal drugs 19

4.8. Detection of drug resistance .................................................................................... 20

4.8.1.Field methods 20

4.8.2. Drug sensitivity studies in experimental animals 21

4.8.3. In vitro assays 23

4.8.4. Xenodiagnosis 23

4.8.5. Serological techniques 24

4.8.6. Molecular techniques 24

4.9. Measures to combat drug resistance in the field................................................... 25

4.9.1. Use of the correct dose 25

4.9.2. Changes of drugs 26

4.9.3. Sanative treatment 26

4.9.4. High dose and repeat treatment regimen 26

4.9.5. Use of combined drugs 27

4.9.6. Beware of fake drugs 27

4.10. Quality assurance of trypanocidal drugs ............................................................. 27

5. FUTURE PROSPECT IN TREATMENT FOR TRYPANOSOMOSIS 28

6. CONCLUSSION AND RECOMMENDATIONS 29

7. REFERENCES 30

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LIST OF TABLES

Page

Table 1: Drug resistant trypanosomes in African countries ------------------------------------- 15

Table 2: Cross-resistance between trypanocidal drugs ------------------------------------------ 17

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LIST OF FIGURES

Page

Figure 1. Schematic representation of the digenetic life cycle of Trypanosoma brucei in the

mammalian host and in the tsetse fly vector --------------------------------------------- 5

Figure 2. The structures of the four most commonly used drug in the chemotherapy and

chemoprophylaxis of animal trypanosomosis Africa ---------------------------------- 11

Figure 3. African countries with reported resistance to trypanocidal drugs ---------------- 15

Figure 4. Some factors influencing the development of resistance trypanocidal drugs ------20

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LIST OF ABBREVATIONS

AT African Trypanosomosis

DIM Diminazene aceturate

GPI Glycosyl-Phosphatidyl-Inositol

HAT Human African Trypanosomosis

Ig M Immunoglobulin M

ILRAD International Laboratory for Research on Animal Diseases

ISMM Isometamidium Chloride

Square kilometres

PCR Polymerase Chain Reaction

RFLP Restriction Fragment Length Polymorphism

USD US Dollars

VSG Variant Surface Glycoprotein

SSA Sub-Saharan Africa

HOM Homidium Salts

BCT Phase Contrast Buffy Coat' Technique

i.m Intra-muscular

PCV Packed cell volume

ED Effective dose

DIIT Drug incubation infectivity test

DIGIT Drug incubation Glossina Infectivity Test

bp Base pairs

SMT Standard Mouse Test

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ACKNOWLEDGEMENT

I would like to express my heartfelt thanks to Dr. Asefa Asmare for his valuable advice, over

all guidance and unreserved effort he made to supply with essential materials as well as to

correct this paper. In fact, this is a good opportunity to express my warm appreciation for his

exemplary professional quality.

Furthermore, I sincerely address my appreciation to Dr, Eyob Eshetu, Dr Taju Nugusse and

My father Kalel Mohammed, My mother Ahado Badhaso, My uncle Addus Mohammed and

My Brother Mohammed Xaha for their direct and indirect contribution in preparation of this

seminar paper.

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SUMMARY

The general features of trypanosomosis, drugs for the treatment and drug resistance in African

trypanosomoses are briefly reviewed in this paper and measures to combat drug resistance

especially at field level are also suggested. Trypanosomosis is the most serious animal health

problem in Sub-Saharan Africa and prevents the keeping of animals over 10 millions square

kilometers of potentially productive land making 50 to 70 million cattle at risk. To prevent

and treat trypanosomosis trypanocidal drugs belonging to different chemical families are used

quite intensively in veterinary medicine. Prophylactic and curative treatment of animal

trypanosomosis is carried out using three main drugs known as homidium salts, diminazene

aciturate and isometamidium chloride. About thirty five million doses of trypanocidal drugs

are used annually in the treatment of animal trypanosomosis in Africa. Most of these drugs are

very old and have been utilized for a long period of time. Hence, treatment of trypanosomosis

is complicated by development of drug resistance. Trypanocidal drug resistance has been

widely distributed in Africa and was reported in 17 countries. Like bacterial resistances to

antibiotics, resistance to trypanocides is associated with genetic modification that transforms a

susceptible population of trypanosome into resistant ones. The role of pressure selection on

the development of drug resistant trypanosomes cannot also be undermined. Uncontrolled

infections of drug-resistant trypanosomes will have a severe impact on both survival and

productivity of animals. It is very unlikely that new trypanocidal drugs will be released into

the market in the near future because these diseases are found in marginalized communities of

the developing countries. Therefore, it is essential to maintain the efficacy of the currently

available drugs through proper utilization of trypanocidal drugs such as use of correct dose,

change of drugs, sanative treatment, high dose, use of combined drugs and quality assurance

of trypanocidal drugs.

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1. INTRODUCTION

African animal trypanosomosis (nagana) is a group of diseases of ruminants, camels, equines,

swine and carnivores caused by different trypanosome species. The major pathogenic species in

African cattle are T. congolense, T. vivax, and, to a lesser extent, T. brucei (Taylor and Authie,

2004). Although hosts acquire infection principally via the bite of infected tsetse flies, other

haematophagous insects like Tabanids and Stomoxys species also transmit trypanosomosis

mechanically (Eisler et al., 2004).

Trypanosomosis in humans, also known as sleeping sickness, is caused by two sub-species of

Trypanosoma brucei:T. b. gambiense, which is the agent of a late on set, chronic form that is

endemic in Western and Central Africa, and T. b. rhodesiense which is responsible for an early-

onset, acute disease found in Eastern and Southern Africa (Solano et al., 2003). Human African

trypanosomosis represents a major public health threat in Africa and together with nagana, the

animal form of African trypanosomosis, is considered a main obstacle for development of rural

regions of the continent (Simarro et al., 2008). Estimates put the number of cattle at risk from

trypanosomosis between 50-70 million animals (Geerts and Holmes, 1998).The impact of the

tsetse-associated disease extends in sub-Saharan Africa over 10 million (a third of the

continent). Trypanosomosis in Africa costs livestock producers an estimated 1340 million USD

each year (Radostits et al., 2007). If lost potential in livestock and crop production are considered,

then trypanosomosis is costing Africa an estimated five billion USD per year (ILRAD,1994).

To prevent and treat trypanosomosis different trypanocidal drugs are used. The frequency with

which treatment has to be applied is often to economically unacceptable levels. About 35 million

doses of drugs are used in Africa each year, with about 50-70 million animals at risk from

trypanosomosis (Geerts et al., 2001). Since most of trypanocidal drugs have been in use for more

than half a century, they can cause the appearance of the drug resistant strain of trypanosomes.

Until new trypanocidal drugs are available, it is of outmost importance that measures are taken to

avoid or delay the development of resistance so that the efficacy of the currently available drugs is

maintained.

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Shared characteristics of the different species of African trypanosomes include the ability to

produce almost unlimited antigenic variation of their variant surface glycoprotein (VSG) and to

induce a predominantly T cell-independent antibody response to the VSG, profound

immunosuppression, polyclonal B cell activation and persistent hypo-complementemia in infected

mammalian hosts (Pan et al., 2006). Infection of the mammal host leads to cycle of parasitemia

associated with new VSGs. Each new VSG initially elicits a strong immunoglobulin M (IgM)

anti-VSG response which leads to phagocytosis of the trypanosomes, predominantly by

macrophages of the liver (Naessens, 2006). The present day methods for the control of African

trypanosomosis, namely, systemic case detection and treatment, tsetse control, do not more than

limit the disease although both these approaches have been shown to be effective where they have

been vigorously applied (Delespaux et al., 2008).

Therefore, the general objective of this seminar paper is:

►To review the drug and drug resistance of African animal trypanosomosis

Specific objectives:

►To illustrate causes of trypanocidal drug resistance

►To discuss types of trypanocidal drug resistance

►To highlight mechanism of development of trypanocidal drug resistance

► To show current situation of trypanocidal drug resistance

► To indicate impact of trypanocidal drug resistance

► To elaborate methods of detection of trypanocidal drug resistance

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2. AFRICAN TRYPANOSOMES

Trypanosomes belong to the phylum Sarcomastigophora, order Kinetoplastida, family

Trypanosomatidae, and genus Trypanosoma. They are unicellular flagellated protozoan

haemoparasites characterized by one nucleus and one flagellum, either free or attached to the

parasites body by means of an undulating membrane (Bourn et al., 2005). Trypanosomes also

usually contain a small, compact kinetoplast, a disc-shaped DNA-containing organelle, situated

within a large mitochondrion (Brun et al., 2009). Within the subgenus, Nannomonas there are

three major trypanosome species; T. brucei, T. equiperdum, and T. evansi. T. brucei can further be

divided into three subspecies, of which T. brucei rhodesiense and T. brucei gambiense are the

causative agents of the debilitating sleeping sickness disease. T. b. brucei and T.b. congolense

(belonging to the subgenus, Nannomonas) and T. vivax (belonging to the subgenus Duttonella),

are associated with trypanosomosis of domestic (Hoare, 1972).

2.1. Morphology and characterization

The trypanosome species differ in morphological characteristics as described by (Maudlin et al.,

2004). All these trypanosome species have a size range of 15-55 μm and typically live in the

blood, lymph, and tissues of their hosts. Bloodstream forms of trypanosomes are covered by a

protective surface coat consisting of variant surface glycoproteins (VSG) linked in turn to the

plasma membrane surface by means of glycosyl-phosphatidyl-inositol (GPI) anchors (Vickerman,

1985).

African trypanosomes are characterized on the basis of their size, shape, position of the nucleus,

size, and location of the kinetoplast, host range, and geographical distribution. Generally they are

elongated, spindle-shape organisms with a single flagellum (Morrison et al., 2009). The flagellum

originates from the basal body near the kinetoplast and runs the length of the trypanosome. The

pellicle, the layer bordering the cytoplasm, while maintaining a definite shape, is flexible enough

to permit a certain degree of body movement. The pellicle and the cytoplasm are pinched up into a

thin sheet of tissue along the length of the body forming the undulating membrane (Soltys and

Woop, 1997).

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2.2. Life cycle

Trypanosomes are pleomorphic, single-celled parasites with a two-host life cycle: mammalian and

arthropod (Brun et al., 2009). With the exception of T. equiperdum and T. evansi, the majority of

trypanosome species undergo a developmental phase in insect vectors, the tsetse fly (Vickerman,

1985). The cycle (Figure 1) starts when blood from a trypanosome infected animal is ingested by

the tsetse fly. It is within the insect vector that trypanosomes undergo a chain of events involving

differentiation, multiplication and biochemical alterations, such as swapping their energy

metabolism from glucose (in bloodstream forms) to proline (in procyclic forms), before migrating

to the salivary glands, where they progress into infective metacyclic forms by re-gaining their

VSG coat and are then ready to be inoculated into a new host during the next blood meal

(Vickerman, 1985). Trypanosoma brucei group of trypanosomes migrate from the gut to the

proventriculus, to the pharynx and eventually to the salivary glands; the cycle for T. congolense

stops at the hypopharynx, and the salivary glands are not invaded; the entire cycle for T.vivax

occurs in the proboscis. The animal-infective form in the tsetse salivary gland is referred to as the

metacyclic form (Delespaux et al., 2008).

Once inoculated in a new host, trypanosomes quickly lose their surface coat, transform into the

long slender trypomastigotes and proliferate by binary fission at the site of the bite for a few days,

leading to an inflammatory chancre. The parasites, then, spread to the draining lymph nodes and

the bloodstream (first or early haemolymphatic stage of infection), through which they reach other

organs such as the spleen, liver, heart and endocrine system (Delespaux et al., 2008). The T.

brucei group of trypanosomes (T.b.brucei, T. b. gambiense, T. b. rhodesiense) and T. evansi

mostly invade tissues (humoral) whereas, T. congolense and to a lesser extent T. vivax and T. cruzi

predominantly restrict themselves to the blood circulation (haemic) (Morrison et al., 2009).

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Figure 1. Schematic representation of the digenetic life cycle of Trypanosoma brucei in the

mammalian host and in the tsetse fly vector

Source: Blum et al., 2008

2.3. The vector of African trypanosomosis

Tsetse flies, the vectors for African trypanosomosis (AT), belong to the family Glossinidae, order

Diptera the two winged flies. There are 31 recognized Glossina species and sub-species, divided

into three groups (morsitans, palpalis and fusca) which have been given sub-generic status

(Solano et al., 2010). Recently, comparative gene sequence analysis and geometric wing

morphometry have been proposed to help in the Glossina group identification (Patterson and

Schofield, 2005). The morsitans group that includes G.morsitans morsitans, G. m. submorsitans,

G. pallidipes, G. longipalis and G. austeni is found mainly in the savannah ecosystems. They are

the most important vectors of bovine trypanosomosis (Leak, 1999). The palpalis group is found

mainly in the riverine galleries of West and Central Africa but sometimes extends into savannah

regions between the river systems (Hendrickx et al., 2004). The palpalis fly species are less

mobile than the morsitans group, often relying on sight rather than smell to locate their hosts

(Leak, 1999). In West Africa, important bovine trypanosomosis vectors among the palpalis group

include G. palpalis palpalis, G. p. gambiensis and G. tachinoides (Solano et al., 2010). The fusca

group flies settle mainly in forests and are therefore less important vectors of bovine

trypanosomosis. Glossina longipennis and G. brevipalpis found in the drier areas of Kenya are

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exceptions among the fusca group, as they have been demonstrated to transmit trypanosomes

(Makumi et al., 2000).

Adult Glossina species are dull in appearance, varying in colour from light yellowish brown to

dark blackish brown (Leak, 1999). In some species the abdomen may have alternate darker and

lighter bands. The smallest species is 6-8 mm long and the largest 10-14 mm (Jordan, 1986). The

adult female produces a single egg, which hatches to first stage larva in the uterus. After a period

of development and moulting, a third stage larva is deposited on the ground. Females produce one

full grown larva every 8-10 days which pupates in light clay or sandy soil. The adult fly will

emerge after a puparial period that varies according to temperature but may be around 30 days at

240C. Consequently, tsetse flies have a very low rate of reproduction, closer to that of a small

mammal than to most insects. This reproductive method of tsetse flies is known as adenotropic

viviparity (Leak, 1999). Females are receptive to males as soon as they start seeking food and are

often mated soon after taking their first blood meal (Leak, 1999). They are usually mated once

with viable sperms remaining stored in the spermathecae throughout the life of the female from

where the sperms get nourished by secretions from alayer of cells surrounding the cuticular lining

of the lumen of each spermathecae. In certain instances, mating may happen more than once. Male

flies may not mate soon after emergence from the pupa and they are not fully fertile until they are

a few days old. Other than tsetse flies, other haematophagous insects like tabanids and stomoxys

species also transmit trypanosomosis mechanically as has been demonstrated by (Desquesnes and

Dia (2003).

2.4. Host

Trypanosomosis is known to affect a number of mammalian vertebrates, either as African animal

trypanosomosis (AAT) and or as human African trypanosomosis (HAT) (Leak, 1999).

Trypanosoma vivax, T. congolense and T. brucei, for example, affect various ungulates including

cattle, sheep, goats, horses, pigs and camels (Maudlin et al., 2004). Other animals like dogs, cats

and the wild carnidae are also affected (Hoare, 1972). Trypanosoma evansi, principally a parasite

of camels and equines, also infects other animals like water buffaloes, sheep, goats, cattle and

deers. Trypanosoma vivax and T. evansi by virtue of their transmission by haematophagous biting

flies occur in sub-Saharan Africa, Asia, Central and North and Southern America (Hoare, 1972).

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The silvatic cycle that involves wild animals is known to greatly influence the epidemiology of

trypanosomosis since wild animals serve as reservoirs for both human and animal trypanosomosis

(Taylor and Authie, 2004). Trypanosomosis maintains large areas of Africa (so-called ʻfly beltsʼ)

free of livestock, and it is presumed that wildlife have developed an evolutionary immuno

tolerance to these parasites with which they have cohabitated for millennia (Reichard, 2002).

2.5. Risk factors of trypanosomosis

2.5.1. Animal factor

A number of factors contribute to the severity of disease in its various host. The animal and

environmental factors,play important roles in modulating the severity of the disease (Taylor and

Authie, 2004). For animal factor exotic animals (dairy cattle) are more severely affected by

trypanosomosis than the local genotypes, which exhibit a range of breed and individual animal

susceptibility. The West African taurine breeds, like the N'Dama, Baoule and their crosses with

zebu (Dieteren et al., 1998), and certain zebu cattle in East Africa (Njogu et al., 1985) can survive

and remain productive under trypanosomosis risk. This phenomenon is called trypanotolerance

and involves the ability of the animals to control parasitaemia, maintain weight and resist anaemia

(Murray et al., 1982). Established through experimental work that animals within the

trypanotolerant breeds with previous exposure to trypanosomosis suffer less severe

trypanosomosis effects as compared to those without previous exposure (naive animals). Suckling

calves are also known not to suffer from serious attacks of trypanosomosis, possibly because of

the influence of maternal antibodies in their systems (Dwinger et al., 1992). There is also

evidence from studies that tsetse get attracted mostly to larger cattle from which they feed rather

than smaller ones (Torr and Mangwiro, 2000).

According to Torr and Mangwiro (2000) that a large ox was bitten ~10 times more often by tsetse

than a calf. Within herd differences have also been registeredwhere ~75% of the tsetse feed from

~25% of the herd (Torr et al., 2001). It would be expected that animals with concomitant

infections with other parasites like haemonchus species would develop serious disease when

infected by trypanosomes, particularly T. congolense, as was reported in the Gambia (Kaufmann

et al., 1992).

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2.5.2. Environmental factor

The environment allows for the interaction between the Glossina species, vertebrate hosts and the

trypanosomes in order for trypanososmosis to be produced. In West Africa, tsetse habitats have

been sub-divided along distinct North-South climatic gradients, with predominantly riverine tsetse

species in the North and a mixture in the South (Hendrickx et al., 2004). In the North, arid

conditions prevent fly spread and riparian vegetation constitutes suitable niches for the localized,

well-demarcated pockets of tsetse populations. Outside these favourable microclimates, tsetse

hardly survives and it would appear that no links exist between pockets, except occasionally and

in spatially limited neighbouring areas during the rainy seasons. In the intermediary band, climatic

conditions and vegetation become gradually more suitable. Distinct fly pockets tend to merge and

tsetse distribution patterns become more linear along main streams. Tsetse populations still remain

concentrated in pockets during the dry season, but disperse during the rainy season over large

parts of the river systems, including important tributaries and savannah buffers (Bouyer et al.,

2006). In the humid South, there are no climatic limitations to fly distribution and flies are present

along river sytems and even the surrounding humid woodlands and forests. Due to increasing

human population and consequently the opening up of more land for crops, the morsitans group is

disappearing in most places of West Africa (Djiteye et al., 1997).

2.6. Trypanosomosis control methods

Controlling the vector remains theoretically the most desirable way of containing trypanosomosis

(Leak, 1999). It is a strategy that has worked well in many areas where multiple drug resistance

has been reported before (Fox et al., 1993). Vector control methods available include:

2.6.1. Sequential aerosol technique

The sequential aerosol technique involves the ultra-low volume spraying of non-residual

insecticides 10-15 metres above tree canopy by fixed wing aircraft or helicopter (in more difficult

terrain) in 5-6 subsequent spraying cycles, separated by 16-18 days depending on temperature

(Allsopp and Hursey, 2004). The goal is to kill all adult tsetse flies in the first spraying cycle and

then kill all emerging flies in the subsequent cycles before they start reproducing. It remains a

perfect method if done under global positioning system navigation, especially for effective area-

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wide tsetse suppression (the efficacy of this technique still needs to be assessed in dense humid

forest ecosystems) or even eradication (in open savannah-type ecosystems) (Allsopp and Hursey,

2004). The disadvantage with the method is that insecticides sprayed may also kill non-target

insects.

2.6.2. Stationary attractive devices (traps and targets)

Stationary attractive devices attract and either kill the flies through tarsal contact with insecticides

embedded in the fabric or the flies are guided and trapped in a non-return cage (Reichard, 2002).

This technique is suitable for deployment by local farmer communities to treat small areas, but the

high target densities required against certain species and in certain dense habitats make the use of

these devices over large areas uneconomical (Kappmeier et al., 2007). The major disadvantage of

Stationary attractive devices is that the active ingredient gets washed off by rain water, hence

compromising its efficacy. Increasing the concentration of insecticide to 0.6-0.8% allows the

Stationary attractive devices to remain deployed even during the wet season, retaining tsetse

mortality rates of > 90% for about 300 days (Torr et al., 1992). Blue fabrics lose colour and

become inefficient after a short time, depending on the dye used and method of fixation. Theft of

the targets, bush fires and maintenance problems are some further problems associated with use of

Stationary attractive devices (Leak, 1999).

2.6.3. Live bait technique

This method involves the application of insecticide onto cattle as pour-ons, sprays or dips so that

tsetse flies attempting to feed on the treated cattle get killed on picking up a lethal deposit of the

insecticide through their tarsi and pre-tarsi. Unlike Stationary attractive devices, the live bait

technique is less prone to theft and does not suffer from maintenance problems. Because of its

added advantage of also controlling ticks, the use of live baits is appreciated as a private good and

can easily be adopted by the rural farming community (Torr et al., 2002). Other disadvantages

with the live bait treatment schemes are the high treatment frequency, the high cost of the

insecticides, insecticide residues in cattle dung, motivation and participation of farmers and the

potential development of resistance to the insecticides in ticks and insects with a high

reproductive rate (Vale and Grant, 2002). It is also thought that the use of insecticides on live

10

animals has the profound effect of interfering with the enzootic stability that is manifested in

young indigenous cattle when exposed to tick challenge (Bourn et al., 2005). Since the attachment

sites of ticks and the feeding sites of tsetse differ, treating only legs and bellies of older cattle will

help, as >95% of tsetse feed come from on adult cattle (Torr et al., 2001). This approach will

more effectively control tsetse without threatening the enzootic stability in calves.

2.6.4. Sterile insect technique

The Sterile insect technique is used if the objective is tsetse eradication. The introduction of the

Sterile insect technique helped eradicate the fly from this island in 1996 in a campaign that had

been commenced two years earlier (Reichard, 2002). As a prerequisite, tsetse density has to be

suppressed through the widespread application of insecticide treated stationary attractive devices,

live baits or fly trapping to a point where the sterile insect technique is considered feasible. In

Zanzibar, a sterile insect plant producing 70,000 irradiated pupae weekly was constructed that

made the release of over 7.8 million sterile male flies possible. Dispersal of the irradiated males

over time was done to achieve an estimated ratio of 50 sterile males for every 1 wild male in order

to overwhelm the residual wild tsetse population (Reichard, 2002). The released sterile males in

the target area do out-compete the wild male population for wild females (Vreysen, 2005). Mating

of the sterile males with virgin, native females results in no offspring. With each generation, the

ratio of sterile to wild insects will increase, making this technique more and more efficient with

lower wild female population densities (inversely-density dependent). The Sterile insect technique

is non-intrusive to the environment, has no adverse effects on non-target organisms, is species-

specific and can easily be integrated with biological control methods such as parasitoids, predators

and pathogens (Leak, 1999). There is no threat of resistance development to the effects of sterile

males, provided that adequate quality assurance is assured during the production process and that

the sterile insects cannot get established in released areas as is the case with other biological

control programmes (Vreysen, 2001). In addition, the sterile insect technique necessitates efficient

release and monitoring methods, which have to be applied on an area-wide basis (Vreysen, 2005).

Since the irradiated tsetse are fully capable of developing and transmitting mature trypanosomes

of all three main species pathogenic to cattle (Moloo and Kutuza, 1984), the sterilized tsetse are

fed on either uninfected blood meals or blood-meals are medicated with trypanocidal drugs before

11

the sterilized insects are fed. Sterilized tsetse are less likely to become infected (at least with

Nannomonas and Trypanozoon parasites) after they have taken an uninfected blood meal and

trypanocidal drugs in medicated meals helps reduce the establishment of infections in subsequent

meals.

3. TRYPANOCIDAL DRUGS

In the 37 African countries where animal trypanosomosis is endemic, trypanocides are used for

the control of the disease (Geerts and Holmes, 1998). Drugs have proven sustainably and

sufficiently attractive to the livestock keepers. Three compounds - isometamidium

chloride(ISMM), homidium salts (homidium bromide (Ethidium®) and homidium chloride

(Novidium®)) and diminazene aceturate (DIM) have been and are still in use more than 50 years

since they were released in the market (Holmes et al., 2004).

Figure 2. The structures of the four most commonly used drug in the chemotherapy and

chemoprophylaxis of animal trypanosomosis Africa

Source: Anene et al., 2001

3.1.Prophylactic treatments

Prophylactic treatments target all animals in a herd or a particular group of valuable or ʻat-riskʼ

animals (Holmes et al., 2004). Isometamidium administered intramuscularly at a dose rate of 0.5-

1mg/kg b.w. provides up to 3 months (a range between 2‒22 weeks) protection against pathogenic

trypanosomes of cattle and against T. vivax especially in small stock, T. brucei in equidae and T.

evansi in camels (Geerts et al., 2001). Isometamidium is given either as routine block

treatment(pre-determined intervals) or as strategic block treatment (when challenge reaches a

predetermined threshold). It is recommended that once a year, additional to ISMM, the animals

12

are separately treated with DIM in order to delay the development of resistance, following the

concept of `sanative pair' (Whiteside, 1962).

3.2.Curative treatments

Diminazene aceturate and HOM salts are the main therapeutic drugs used in the management of

clinical trypanosomosis in animals (Holmes et al., 2004). Diminazene aceturate is administered

i.m. at a dose rate of 3.5‒7mg/kg bw the lowest dose being effectiveagainst T. congolense and T.

vivax and the highest dose against T. brucei(Peregrine, 1994). At these dose rates, DIM, in

addition to its curative uses, alsooffers short term protection of up to 2 weeks (Geerts et al., 2001).

Homidium on the otherhand is administered at a dose rate of 1mg/kg b.w. in cattle. In low tsetse

challenge areas, aprophylactic effect of HOM salts has also been observed (Peregrine, 1994).

3.3.Mechanism of action of current used drugs

3.3.1. Diminazene aceturate (Berenil®)

Diamidines are dicationic molecules, which bind to the minor groove of DNA at AT-rich sites.

They exert their biological activity by primarily binding to DNA and then inhibiting one or more

of the DNA dependent enzymes (such as topoisomerases or nucleases) or by directly impeding the

transcription process. Berenil is the only comercial diamidine for treatment of animal

trypanosomosis at this moment, but new diamidines are being investigated as future therapeutic

options. Two diamidine compounds (DB 75 and DB 867) present comparable efficacy at lower

doses than the standard drug diminazene and could be considered as potential clinical candidates

against T. evansi infection (Gilling et al.,2011).

The selectivity of diamidines is primarily due to its selective accumulation in the pathogen rather

than in the host cells. Diminazene aceturate do not cross the blood–brain barrier. The new

diamidine DB75 (furamidine) does not have sufficient penetration capacity into the central

nervous system, but a related compound (DB820, azafuramidine) seems to completely cure a

murine late-stage model of sleeping sickness (Soeiro et al., 2005).

3.3.2. Isometamidium and homidium

13

Isometamidium chloride (Samorin®, Trypamidium®, Veridium®) has been prophylactically or

therapeutically used in the field for livestock suffering from trypanosomosis for several decades. It

was first synthesized by coupling homidium (Ethidium®) with p-aminophenyldiazonium chloride,

this is, by coupling homidium with a part of the diminazene aceturate molecule. It has been

proposed that the main mechanism of action of isometamidium is the cleavage of kDNA-

topoisomerase complexes, causing the desegregation of the minicircle network within the

kinetoplast (Shapiro and Englund, 1990). But Kaminsky et al.(1997) showed that dyskinetoplastic

trypanosomes are at least as sensitive to isometamidium as kinetoplastic lines.

3.3.3.Suramin (Polysulphonated naphtyl urea)

The molecular structures of the dyes provided a starting point for the synthesis of suramin, which

has been used as a trypanocidal drug since 1916 and is still in clinical use although unfortunately

its large scale production for animal use was stopped in the middle of 90’s. Suramin do not cross

the blood brain barrier. Inhibition of several enzymes by Suramin (among others, dihydrofolate

reductase, fumarase, glycerol-3-phosphate dehydrogenase, hexokinase, reverse transcriptase, RNA

polymerase and kinases) has been described (Hagos et al., 2010). Deprivation from cholesterol

and phospholipids by inhibition of the uptake of low density lipoproteins has been proposed as the

main mechanism of action for this drug.

3.3.4. Melarsomine hydrochloride (Cymelarsan®

)

Melarsoprol is an arsenical compound that contains the trivalent arsenic element with a markedly

reactive arsenoxide group. The presence of arsenoxide confers the physiochemical ability of lipid

solubility that allows passage across the blood brain barrier. The veterinary arsenical trypanocide

melarsamine hydrochloride (Cymelarsan®) is a conjugation of melarsen oxide and two equivalents

of cysteamine. It is used mainly to treat T. evansi infections in domestic animals.

14

4. TRYPANOCIDAL DRUG RESISTANCE

4.1. Definition

Drug resistance is the heritable loss of sensitivity of a micro-organism to a drug to which it was

sensitive before. Resistance to the commonly used animal trypanocides has emerged in sub-

Saharan Africa and interfer with effective veterinary management of trypanosomosis.

4.2. Causes of trypanocidal drug resistance

Trypanocidal drug resistance is caused by the exposure of trypanosomes to sub-therapeutic drug

concentrations, resulting from under-dosing and the irrational use of drugs and the lack of proper

diagnosis (Whiteside, 1962). The prolonged and frequent use of trypanocides in high tsetse

challenge areas, even when used at the right doses, is also likely to cause resistance (Geerts and

Holmes, 1998). Furthermore, poor quality drugs have been finding their way on to the market in

some cases, products with no trypanocidal activity have been also identified and in other

situations compound with reduced activity have been marketed. Such products are not only less

effective when used by farmers, but also greatly increase the risk of drug resistance development.

Reduction in drugs accumulation by the target cell or organism and diminished drug activity in

immune-suppressed animals can contribute to the emergence of drug resistance. Thus, drug

resistance can arise either as a consequence of changes in drug concentration at the target site or

alteration in the target, or both. There is experimental evidence that drug-resistant trypanosome

clones accumulate fewer drugs than their sensitive counterparts (Anene et al.,2001).

15

Figure 3. African countries with reported resistance to trypanocidal drugs. *Resistance to

trypanocidal drugs has been reported in animal trypanosomes in that country;

Source: Delespaux et al., 2008

4.3. Types of trypanocidal drug resistance

Two types of resistance against trypanocidal drugs are recognized: single drug resistance and

multiple drug resistance. In single drug resistance, trypanosomosis control still could be achieved

by using one of the drug pairs in which resistance has not developed through the application of the

sanative pair principle (Geerts and Holmes, 1998). However, the second drug should be used with

caution in order to avoid resistance development against it as well. Multiple drug resistance is

resistance concurrently to two or more drugs, making sanative drug pairs ineffective. Multiple

drug resistance can only be counteracted by intervening at the level of the vector (Fox et al.,

1993).

Table 1: Drug resistant trypanosomes in African countries

Country

Trypanosome

species

Animal

Examined

Resistance

% of

Resistance

Resist to

16

D= diminazene; H = homidium bromide (ethidium); I =isometamidium

Source:Geerts and Holmes, 1998

So far, resistance to one or more of the three more commonly used trypanocidal drugs used in

cattle has been reported in at least 17 countries in sub-Saharan Africa (Burkina Faso, Chad, Ivory

coast, Ethiopia, Kenya, Mali, Somalia, Sudan, Tanzania, Uganda, Zimbabwe, Zambia,

Mozambique, Cameroon, Nigeria, Guinea and Central African Republic) (Fig. 3) (Delespaux et

al., 2008). In eight of the 17 countries, multiple resistances have been reported. This is probably

an underestimation of the true situation,because in several countries surveys for resistance have

not yet been carried out or cases of resistance have not been published. Table 1: shows the

Trypanosoma species and type of drugs in which their resistance has been reported (Geerts and

Holmes, 1998).

4.4. Cross and multiple drug resistance

With development mechanisms of drug resistance to one compound, trypanosomes may show of

resistance to compound of the same series and may also to those of other of series thus, in the

phenanthridium series, resistance to pyrithidium bromide leads to resistance to isometamidium

and homidium. There is also cross resistance between quinapyramine and phenanthridium series.

So sometimes, one strain of trypanosome may be resistant to many drugs (multiple drug

resistant), a situation that constitutes a particularly grave threat to livestock production and health

in Africa. In contrast, a number of experimental drug sensitivity studies with suramin and

diminazene in vitro and in rodents have demonstrated that acquisition of resistance to suramin

does not confer resistance to diminazene, suggesting that cross resistance may not exist between

Isolates

Ethiopia T. congolense 12 12 100 D

Burkina faso T. congolense 12 9 75 I

Kenya T. congolense 7 2 29 I

Kenya/Somalia T. vivax 7 6 86 I

Nigeria T. vivax 19 12 63 D, H, I

T. brucei 12 2 17 D, I

Sudan T. congolense,

T.vivax, T.brucei

12 5 42 H

Uganda T. brucei 36 1 12 D, I

Zimbabwe T. congolense 14 6 43 D

17

these drugs (Anene et al.,2001). Table 2 shows the cross-resistance among the five compounds in

use for the treatment of tsetse-transmitted trypanosomosis in livestock.

Table 2: Cross-resistance between trypanocidal drugs

Trypanosome

resistant to

Cross resistance to

At curative dose At increased dose

QP HP PB IM DA HM PB IM DA

QP + + + + + + + - -

HM + + + + - + + - -

PB + + + + - + + - -

IM + + + + - + + - -

DA + - - - + - - - +

QP = Quinapyramine; HM = Homidium; PB = Pyrithidium bromide; IM = Isometamidium, DA =

Diminazene aceturate.+ = resistant; - = not resistant;± = some strains resistant;

Source: Uilenberg, 1998

4.5. Mechanism of drug resistance

An understanding of the mechanisms of drug resistance by trypanosomes, among others, is

important as it can lead to the identification potential and novel drug targets and provide direction

to how new chemotherapeutic strategies can be used to reduce development of resistance. In spite

of the length of time these drug have been available and widespread interest in drug resistance,

relatively little work has been done on how these drug are taken up by trypanosomes and the

processes that are changed when drug resistance emerges. Progress is being made in elucidating

the role of nucleoside transporters in resistance to trypanocidal drugs (Barrett and Fairlamb,

1999). Furthermore, changes in the mitochondrial electrical potential have been demonstrated in

isometamidium resistant trypanosomes. As the mitochondrial electrical potential is closely linked

with rate of isometamidium uptake seems to be a good indicator of the degree of drug resistance.

Measuring mitochondrial electrical potential might be a rapid indication of the degree of drug

resistance. It could be carried out using small number of trypanosomes directly isolated from the

blood of infected animals. Interesting work is also going on to identify genetic markers

forisometamidium resistance which might be developed later on into reagents for the

identification of resistant trypanosomes using polymerase chain reaction (Geerts and Holmes,

1998).

18

In addition, alterations of the transporters can cause development of drug resistance (Carter et al.,

1995). There are numerous reports of resistance to Berenil in different countries and in several

Trypanosoma species. In any case, resistance seems to be limited to highly endemic areas where

the use of this drug is very common. Barret et al. (1995) demonstrated that resistance to

diminazene aceturate in T. equiperdum was due to lack of activity of P2 aminopurine transporter,

required to translocate the drug across the cell membrane. The role of the P2-type purine

transporter in the uptake of arsenical diamidines, pentamidine and DA by T. brucei, T. evansi and

T. equiperdum, and the consequences of inhibition, knocking down or silencing this gene have

been extensively described and reviewed in the literature (De Koning et al., 2004)

These transporters have been cloned and expressed in yeasts to demonstrate their role in resistance

(Matovu et al., 2003). But P2 is not the only significant transporter for melaminophenyl arsenicals

expressed in bloodstream trypanosomes. Some theories of heavy metal resistance in protozoa

proposed the implication of aqua-glyceroporins (Gourbal et al., 2004), thiamine transporters

(Schweingruber, (2004) or high affinity pentamidine transporter (HAPT1) (Matovu et al., 2003) ,

but it seems to be that these molecules do not play an important role in vivo.

4.6. Current situation of resistance

Currently, there are close to 20 African countries in which resistance has already been reported

(Delespaux et al., 2008). In addition to the 13 countries mentioned by Geerts and Holmes, (1998),

resistance has been reported in Mozambique (Jamal, 2005), Mali, Guinea (Diall et al., 2003),

Cameroon (Mamoudou et al., 2008) and recently in Ghana and Benin (Allegye-Cudjoe, 2009). It

is suspected that in several other African countries, resistance is present but is yet to be

demonstrated (Delespaux et al., 2008). Large scale surveys have been conducted in 13 African

countries including Kenya (Mdachi, 1999), Uganda, Tanzania (Eisler et al., 2000), Ethiopia

(Tewelde et al., 2004), Zambia (Shinyangwe et al., 2004), Zimbabwe (Joshua et al., 1995),

Cameroon (Mamodou et al., 2008), Nigeria (Geerts et al, 2001), Burkina Faso(McDermott et al,

2003), Mali, Guinea ( Grace, 2005) Ghana and Benin (Allegye-Cudjoe, 2009) demonstrating area-

wide resistance in at least one regionof these countries.Confirmed reports about resistance in the

cotton zone of West Africa were first made particularly in Burkina Faso in the early 1980s (Pinder

and Authie, 1984).These authors described stocks of T. congolense isolated from cattle in the

19

Samorogouan area in 1982 which were resistant to isometamidium. Later, tests in mice showed

that certain T.congolense strains from the same area were also resistant to diminazene aceturate

indicating existence of multiple drug resistant T. congolense (Authie, 1984). Recent studies on

resistance in the same area of Burkina Faso (McDemott et al., 2003) and in other areas of West

Africa underline that the problem is present and is expanding (Allegye-Cudjoe, 2009).

4.7. Impact of drug resistance

It is essential to assess not only the distribution of drug resistance, but also the constraint it

imposes on effective control. To date, few studies have accurately assessed the impact of drug-

resistant trypanosomes on livestock productivity, although it is generally assumed that

uncontrolled infections will have a severe impact on both survival and productivity of animal. A

useful recent study to assess the impact of drug-resistant trypanosomes on the productivity of the

local cattle was carried out in the Ghibe valley, Ethiopia, where a high prevalence of multiple drug

resistance was reported. In the study, incidence of abortion was increased and the financial and

economic returns were also affected (Anene et al., 2001).

4.7.1. Factor influencing development of resistance to Trypanocidal drugs

The exposure of trypanosomes to sub-therapeutic concentrations of trypanocidal drugs, the

treatment frequency and the degree of drug exposure of the parasite population are important

factors influencing the development of drug resistance (Geerts and Holmes 1998). Furthermore,

some trypanocidal drugs such as ethidium are well-known mutagenic compounds and might

induce mutations, the most resistant of which might be selected under drug pressure (Holmes et

al., 2004). The phenomenon of cross-resistance has now been clearly demonstrated.

Quinapyramine usage has been shown to induce resistance to isometamidium, homidium and

diminazene (Uilenberg, 1998). Research on drug resistance in Plasmodium has shown also that

the genetic structure of a parasite population (clonal or panmictic) is an important parameter

influenced by the transmission intensity, and this in turn might influence the rate of development

of drug resistance (Holmes et al., 2004).

20

Figure 4. Some factors influencing the development of resistance to trypanocidal drugs;

Source:Geerts et al.,2001

4.8. Detection of drug resistance

4.8.1.Field methods

Eisler et al. (2000) proposed a method for the assessment of prevalence of resistance to

isometamidium chloride by monitoring cattle populations under natural challenge in the field.

Briefly, two groups consisting of 30 to 80 cattle each are used. One group is treated with 1 mg/kg

b.w. ISMM and the other is used as untreated control. The two groups then are exposed to natural

challenge and tested for the presence of trypanosomes using the phase contrast buffy coat

technique (BCT) (Murray et al., 1977) every two weeks for two to three months. A comparison

through survival analysis curves is made on the data of new trypanosome infections between the

group of cattle treated with ISMM and the untreated control group (Eisler et al., 2000; Tewelde et

al., 2004). If >25% of the ISMM treated cattle become infected within 8 weeks of exposure, drug

resistance is strongly suspected (Mdachi, 1999; Eisler et al., 2000; Tewelde et al., 2004).

Several epidemiological studies to map field trypanocidal drug resistance, based on the protocol

by Eisler et al. (2000). McDermott et al. (2003) working in the Kenedougou Province of Burkina

Faso, Shinyangwe et al. (2004) working in Eastern Zambia, Tewelde et al. (2004) in Ethiopia,

21

Grace (2005) in Guinea and South-Eastern Mali and Allegye-Cudjoe (2009) in Ghana and in

Benin are some examples. An abbreviated version of the original 8‒12 week-protocol by Eisler et

al. (2000) was validated in the cotton zone of West Africa and found effective and reliable for use

not by researchers but by the national veterinary services. This involves a 4 week long follow-up

(Diall et al., 2003) period in order to reduce costs and still generate data within a very short time.

The abbreviated protocol is effective in areas where trypanomosis risk is high (prevalence is

>10%) as has been demonstrated in the cotton zone of West Africa (Diall et al., 2003; Grace,

2005). Rowlands et al. (1994) developed a model to distinguish new and recurrent infections to

determine if the high infection rates observed in cattle in the Ghibe valley, south-west Ethiopia,

following treatment of T. congolense infections with diminazene aceturate were due to the tsetse

challenge or if they rather were a relapse of infections following treatment. An infection was

defined as new if it was preceded by two previous months in which monthly collected samples

had packed cell volumes (PCV) of ≥ 26% and in which trypanosomes were not detected.

4.8.2. Drug sensitivity studies in experimental animals

Tests in ruminants

Neither the single-dose nor the multiple-dose tests in mice are able to predict accurately the

curative doses of trypanocidal drugs needed to clear trypanosome populations from infected cattle

(Eisler et al., 2001). The test in ruminants should hence be used to just determine whether or not

drugs are principally efficacious at recommended curative doses in cattle infected with a particular

trypanosome populations. The test in calves may further be used for investigations on drug

resistance in T. vivax, which is usually not infective for mice. A group of cattle or small

ruminants, preferably of a breed native to the area and without prior exposure to tsetse or

trypanosomosis are used (Eisler et al., 2001). They should also be negative for anti-trypanosomal

antibodies as determined by the indirect fluorescent antibody test or ELISA (Luckins and Mehlitz,

1978) if these tests are available. Specific detailed protocols on this are as contained in Eisler et

al. (2001). Duereduction in drugs accumulation by the target cell or organism and diminished

drug activity in immune-suppressed animals can contribute to the emergence of drug resistance.

Thus, drug resistance can arise either as a consequence of changes in drug concentration at the

target site or alteration in the target, or both. There is experimental evidence that drug-resistant

trypanosome clones accumulate fewer drugs than their sensitive counterparts (Anene et al.,

22

2001).to individual variation in the response to trypanocidal drug treatment among ruminants

inoculated with the same T. congolense isolate (Peregrine et al., 1991), it is advisable to use a

minimum of three and preferably six animals. However, economic considerations may often

preclude the use of more than a single animal per stabilate for drug-sensitivity testing.The

experimental animals must be kept in a fly-proof stable or in a non-tsetse infested area to

eliminate the risk of reinfection during the study. A breakthrough infection, indicative that one of

the inoculated trypanosome populations was drug-resistant can be inoculated into a group of

calves and mice to determine the level of drug resistance. A variation of this method also exists

whereby blood from a group of infected cattle is pooled and inoculated into a single recipient calf

which is monitored and later, if parasitaemic, treated with trypanocide at the recommended dose.

This technique is appropriate where laboratory facilities are limited but only allows for a

qualitative assessment of resistance. Further constraints of the technique are that not all

trypanosome populations might grow equally well and that sensitive isolates might overgrow

resistant ones when inoculated together (Sones et al., 1989); this however, is not a consistent

observation (Burudi et al., 1994). A useful indication of the level of resistance can be obtained

from studies in ruminants by recording the length of time between treatment and detection of

break through populations of trypanosomes. The shorter the period, the greater the level of

resistance (Ainanshe et al.,1992).

Tests in mice

Either single-dose or multi-dose tests are conducted in mice to provide information on resistant

trypanosome isolates from a given area, as described in the protocol by Eisler et al. (2001). After

expansion of an isolate in a donor mouse, experimental mice are inoculated with the test

trypanosome isolate and treated with a trypanocidal drug. Tail blood wet smears are checked 2‒3

times per week for parasites for a period of up to 60 days. The ED50 and ED95 (effective dose

that gives temporary clearance of the parasite in 50% or 95% of the animals, respectively) can be

calculated as can the CD50 and CD95 (curative dose that gives complete cure in 50 and 95% of

the animals, respectively). Sones et al. (1988) used a group of five mice, which allowed an easy

calculation of ED80 and CD80 values (one out of five mice not cleared or cured). Knoppe et al.

(2006), using the standard mouse test (SMT), screened a number of T. congolense isolates

collected in the Kenedougou Province of Burkina Faso against isometamidium chloride at a dose

23

of 0, 0.25, 1.0, 5.0, 10, 15 or 20 mg/kg b. w. and found the method very sensitive but labour

intensive.

There are however several disadvantages with this method. Firstly, most T. vivax isolates, and also

some T. congolense isolates, do not grow in mice (Holmes et al., 2004). Secondly, although there

is a reasonable correlation between drug sensitivity between mice and cattle, higher doses of drugs

must be used in mice (normally ten times higher) in order to obtain results comparable to those

from cattle, because of the vast different metabolic size (Sones et al., 1988). Thus, the curative

dose for ruminants cannot be extrapolated from the assay results in mice (Sones et al., 1988).

Thirdly, a danger further exists of selecting against particular trypanosome species, particularly in

mixed infections. Fourthly, precise assessment of resistance requires a large number of mice per

isolate. Finally, it takes as long as 60 days to evaluate the drug sensitivity of an isolate.

4.8.3. In vitro assays

In vitro assays use blood stream or metacyclic forms instead of procyclic forms. This technique

has been used to detect resistance in T. brucei and T. congolense (Clausen et al., 2000). It takes up

to 40 to 50 days of in vitro incubation to generate metacyclic trypanosomes (Gray and Peregrine,

1993). The advantage with this technique is that large numbers of isolates can be examined and

tests with metacyclic trypanosomes correlate well with field observations. However, in vitro

cultivation of bloodstream forms is only possible using pre-adapted lines and not isolates directly

from naturally infected animals (Hirumi et al., 1993). A simplified axenic culture system has been

developed by these authors, but further research is still necessary to study the correlation with

field data. A potential problem associated with this lengthy time adaptation is the possible

selection against trypanosomes that possess the phenotype of the original population. Further, in

vitro assays are quite expensive and require good laboratory facilities and well-trained staff.The

drug incubation infectivity test (DIIT) combining both in vivo and in vitro techniques issuitable

for drug sensitivity testing of T. b. brucei, T. evansi and T. vivax (Kaminsky et al., 1990). It was

modified by Sutherland et al. (1991) and it proved suitable for T. congolense.

4.8.4. Xenodiagnosis

24

Xenodiagnosis is the feeding of a clean susceptible vector species on a suspected case of

trypanosomosis, after which it is either dissected and examined for the presence of infection, or

allowed to feed on a clean animal which then is itself examined for the development of infection.

A modification of this approach, the drug incubation glossina infectivity test (DIGIT), in which

trypanosomes are exposed to the trypanocidal drugs in vitro for a short time and thereafter are fed

to tsetse flies to check whether or not they develop into metacyclic forms was successfully

validated and proved sensitive for detecting drug resistance (Clausen et al., 1999). This technique

distinguishes resistant from sensitive isolates and does not require experimental animals.

However, it does require a ready supply of teneral tsetse flies from an artificially reared colony.

4.8.5. Serological techniques

Although not frequently used, enzyme linked immuno-sorbent assay (ELISA) has proved valuable

in diagnosing isometamidium resistance (Eisler et al., 1996). The use of ELISA in the detection of

ISMM in the serum of cattle can becombined with field block treatment studies or for individual

treatment of ruminants to detect resistant trypanosomes (Eisler et al., 1996). The presence of

trypanosomes in animals with an ISMM serum concentration > 0.4ng/ml suggests that parasites

are resistant (Eisler et al., 1997). Similar drug-ELISAs have been developed for the detection of

sub-nanogramme amounts of homidium and diminazene (Holmes et al., 2004). A closely related

technique to drug-ELISAs is the mitochondrial electrical potential (MEP) which determines the

rate of ISMM accumulation in the trypanosome kinetoplast (Wilkes et al., 1997).

4.8.6. Molecular techniques

i. Polymerase chain reaction

Because of the problems associated with the low sensitivity of the parasitological techniques

(Paris et al., 1982) and the long follow-up time of study animals (Eisler et al., 2001), polymerase

chain reaction (PCR) with high sensitivity and specificity is a good solution to these problems.

Gall et al. (2004) used this method in Burkina Faso and found it four times more sensitive

compared to the field parasitological techniques.

ii. PCR-RFLP

25

Molecular methods for the diagnosis of ISMM resistance were recently developed (Afework et

al., 2006). The first method enables discrimination between ISMM-ISMMsensitive and ISMM-

resistant strains of T. congolense by PCR-RFLP (Delespaux et al., 2005). This test is based on the

polymorphism observed in the 381 bp fragment (in sensitive strains) or the 384 bp fragment (in

resistant strains) of a putative gene presenting some homologies with transporter. The second

method has been developed to distinguish ISMM-resistant from ISMM-sensitive strains of T.

brucei (Afework et al., 2006). This PCR-RFLP test is based on the polymorphism of the 677 bp

fragment of the TbAT1 gene. The same set of six point mutations could confer resistance to the

melarsenoxide cysteamine cymelarsan (an arsenical diamidine) and to ISMM (diamidine

compound) and the detection of one of these six mutations could enable reliable identification of

sensitivity or resistance to ISMM (Maser et al., 2003).

4.9. Measures to combat drug resistance in the field

As it was mentioned above, drug resistance in trypanosomes is likely to occur under certain

circumstances such as: i) under large-scale drug use; ii) by using inadequate dosing; and iii) by

using correct dosing with drugs that are slowly eliminated from the body. Furthermore, some

trypanocidal drugs are well-known mutagenic compounds and might induce mutations, the most

resistant of which are certainly selected under drug pressure. Taking into account of these factors

different measures can be proposed in order to reduce the chance of drug resistance. Of these the

most important measures are use of the correct dose, changing of drugs, sanative treatment,

increased dosage, repeative treatment and use of combined drugs. In addition to these, care must

be taken to avoid fake drugs and good quality assurance must be implemented (Geerts and

Holmes, 1998).

4.9.1. Use of the correct dose

Under dosing is one of major causes of resistance development. Sub-therapeutic drug

concentrations exert a strong selective pressure for the emergence of resistant clones that pre-exist

in the trypanosome population. Unfortunately, under dosing occurs very frequently. Farmers have

the tendency to underestimate the weight of their animals when they have to treat them since

26

farmers or unskilled persons in many countries of Africa are administering drugs due to absence

of strict rules about the utilization of veterinary drugs (Geerts and Holmes, 1998).

4.9.2. Changes of drugs

Changing drugs or alterative use of drugs in different time may reduce the chance of drug

resistance. For example one group of chemical can be used for prophylactic purpose and the other

can be applied for curative (Uilenberg, 1998).

4.9.3. Sanative treatment

The concepts of sanative treatment is the use of a pair of trypanocides which are chemically

unrelated and therefore, unlikely to induce cross resistance (Rowlands et al., 1994) Diminazene

and homidium, or diminazene and isometamidium can be used in the field as sanative

combinations. These pairs when strategically employed can be used to maintain herd productivity

in the field without the development of resistance to either of the compounds (Anene et al.,2001).

4.9.4. High dose and repeat treatment regimen

High dose treatment offers the best opportunity for eliminating infections with trypanosomes

which express high degree of resistance to drugs. However, it must be appreciated that the scope

for increased drug dosage is highly dependent on the relationship between the maximal tolerated

dose and the minimal dose required to treat cure (the therapeutic index). This is a major limitation

to high dose treatment with trypanocides as the margin of safety of most of them is usually quite

narrow, trypanocidal drug toxicity being quite common. So this technique is helpful in the

utilization of drugs with wide safety of margin. Studies on the efficacy of repeat treatments of T.

congolense infections with diminazene aceturate indicate that such regimen may be useful

especially if administered at 48 or 96 hour intervals. This tends to support the suggestion that the

efficacy of trypanocides depends not only on the concentration of the drug to which the parasites

are exposed, but also on the length of exposure. But this may not be true for all trypanocidal drugs

(Anene et al.,2001).

27

4.9.5. Use of combined drugs

The rationale for the use of two or more of existing drugs in combinations to increase therapeutic

activity, decrease clinical toxicity and potentially reducing the frequency of the emergence of

drug resistance (Anene et al.,2001).

4.9.6. Beware of fake drugs

Fake trypanocides may be sold in Africa. The drugs are either fake in their composition or are

faked by dilution of the original products or substitutions by an ordinary component apparently a

like (Nere powder for diminazine (berenil), coffee or charcoal for ethidium, potassium

permanganate for isometamidium. For isometamidium, one must pay attention to the information

given on the packages (the examples of a shell found on a fake product labelled “for veterinary

use” and in general carefully check the logo of firms. Use will known products and be regular

customer to trust worthysupply service (Leeflang, 1978).

4.10. Quality assurance of trypanocidal drugs

In recent years, a further issue has arisen associated with the liberalization of veterinary drug

supply and market. The growing problem of poor quality drugs finding their way on to the market

in some cases, products with no trypanocidal activity have been identified and in other situations

compound with reduced activity have been marketed. Such products are not less effective when

used by farmers, but also greatly increase the risk of drug resistance developing (especially when

under dosing also allows the survival for the heterozygote resistant trypanosomes). Unfortunately,

quality control on pharmaceutical products used in the developing world is frequently inadequate

and there is already considerable evidence that the problem is widespread for a variety of

pharmaceutical products (Shakoor et al., 1997).

28

5. FUTURE PROSPECT IN TREATMENT FOR TRYPANOSOMOSIS

Trypanosomosis is controlled either by controlling the vector or by controlling the parasite, or a

combination of both. Over the years, a large arsenal of vector-control tools has been developed.

Nevertheless, the control of animal trypanosomosis in often poor rural communities has and will

continue to rely heavily on the use of trypanocidal drugs. However, trypanocidal drugs resistance

has been reported in 17 African countries. In addition to this, some authors discuss the possible

role of inaappropriate usage of trypanocides in livestock in the development of resistance

observed in human trypanosomosis (Barrett, 2001). Trypanosomes have the capacity for antigenic

variation, which is the basis of their ability to escape the host immune response, and because of

this, prospects for the development of a vaccine against trypanosomosis have been considered

poor. Tonin et al., (2011) assessed the use of diminazene aceturate in association with vitamin E

and sodium selenite in rats, concluding that results in terms of longevity, hematocrit reduction,

leukocytes and lymphocytes number and lipid peroxidation were improved using this combinative

therapy compared to the use of single diminazene aceturate, although sodium selenite showed

better protective action than vitamin E. There is a progressive interest in the utilization of

antioxidants in the prevention and treatment of this disease. T. evansi pathogenic mechanisms

include oxidation of the erythrocytes inducing oxidative stress due to free radical generation

(Habila et al., 2012). Pallavi et al. (2010) investigated the potential of heat shock protein 90

inhibitors as drugs for the treatment of trypanosoma infection in animals. This protein regulates

cell cycle progression and signal transduction.There is an urgent need for new drugs for the

chemotherapy of trypanosomosis. Progress has been made in the identification and

characterization of novel drug targets for rational chemotherapy and inhibitors of trypanosomatid

glycosomal enzymes, trypanothione reductase, ornithine decarboxylase, S-adenosylmethionine

decarboxylase, cysteine proteases and of the purine and sterol biosynthetic pathways

29

6. CONCLUSSION AND RECOMMENDATIONS

Despite limited number of trypanocidal drugs, they are more widely used than means to control

the disease. Drug resistance poses a potential treat to control measures. The exact mechanism how

trypanosomal parasite develop resistant and the factors responsible for the development of drug

resistance are yet to be further established. In addition, it is very unlikely that new trypanocidal

drugs will be released into the market in the near future. Therefore, in order to continue to tackle

the economic lose posed by trypanosomosis by using typanocidal drugs, among others strict drug

quality assurance of trypanocidal drugs coupled with proper utilization of currently available

drugs should be implemented. In addition the latest contol method by Sterile insect technique and

Live bait technique because this two control have advantage over other control such as; Sequential

aerosol technique and Stationary attractive devices (traps and targets).

In line with the above conclusion, the following recommendation are forwarded:

The quality assurance of trypanocidal should be used in overall African country.

The vector control method with Live bite technique shuld be should be used because of its

bivalent advantage.

Using of cattle that trypanotolarent in area with high tsetse fly infestation should be

better.for those people live in that area.

The great attention should be given to applying the latest tsetse control method by Sterile

insect techique.

30

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