RJmJBLIcyJE Dti SENEGAL DIRECTION DES RECHERCHES...
RJmJBLIcyJE Dti SENEGAL
DIRECTION DES RECHERCHES SUR LES
---m-----------
PRODUCTIONS ET LA SANTE ANIMALES
MTNISTERE DE L’,L\\GRICULTURE
--_____--------
--------
LABORATOIRE NATIONAL DE L’ELEV.4GE
INSTITUT SENEGALAIS DE
ET DE RECHERCHES VETERINAIRES
RECHERCHES AGRICOLES (ISRA)
(L. N. E. R.V.) DAKAR-HANN
FIRST RESEARCH COORDINATIONMEETING
OF THE FAOLMm AIBKXV TRYPANOSOMASIS NETWORK
N’AIROBI, KENYA, 7-11 FEBRUARY 1994
(I.A.E.A./I.S.R.A RESEARCH CONTRACT No 7596iR.B)
RAPPORT N.@kPARHO. ANIM.
JANVIER 1994
*: Assistant de Recherches, Laboratoire de Parasitologie.
DETECTION AND CHARACTERISATION
OF SUSPECTED DRUG RESISTANT TRYPANOSOME
STRAINS IN SENEGAL USING B.C.T. ,4ND ,4g-ELISA
DIAGNOSTIC TECHNIQUES
BY
Mamadou SEYE
(ISRA / DRPSA / LNERV, BP 2057,Dakar, Sénégal)
SUMMARY
Animal trypanosomiasis is a major livestock disease in the southern-central region of Senegal.
The major@ of the cattle in this area are of the Diakme breed (cross Zebu-Ndama). Because of their
properties, these animals are widely used by the f%ming community for animal traction, but also for
milk and meat production. Despite their reasonable level of trypanotolerance, many of these animais
are affected by trypanosomiasis in the are& with significant tsetse challenge. For this reason,
trypanocidal drugs such as Berenil and Samorin are being used on a large scale. Their use and
particularly mis-use has lead to the occurence of drug resistant strains. In a previous study (1991-
1992) carried out in this region, results were obtained to indicating this. The proposed study to be
conducted in 1994, is to confüm these fïndings in greater details , using the BCT and Ag-ELISA, and
to identify the most appropriate trypanocidal drug(s) to be used in the Sokone region. The project
activities will involve the screening of approximately 1,000 Diakme cattle in the Sokone area,
followed by the isolation of trypanosomes from infected cattle and their subinoculation into laboratory
animals for subsequent chemotherapeutic trials with Berenil, Samorin and Ethidium. The results
obtained will be provided to the Senegalese Ministry of Agriculture
KEY WORDS
SENEGAL - BOVINE -TRYPANOSOMIASIS - DIAGNOSTIC - ELISA
TRYPANOCID - DRUG RESSISTANCE
DETECTION AND CH/l.RACTERISATION OF SUSPECTED
I>RLJG RESISTANT TRYPA!NOSOME STRAINS IN SENEGAL
LJSING B.C.T AND Ag-ELISA DIAGNOSTIC TECHNIQUES
bY
Mamadou SEYE”
1. INTRODUCTION
The efforts to produce a vaccin against trypanosomiasis bave failed so far, &spite a11
subtantial fùnding and the high level of expertise inputs allocated to this for some decades
throughout the world.
In view of this, the use of trypanocidal drugs remains the only alternative to tr-est
trypanosomiasis afI?ected animais. This bas led to a wide spread use of trypanocidal drugs in
those areas where the disease exists. However, the success of such chemotherapeutic
treatment depends on specific criteria : identification of the causitive trypanosomes, correct
deterrnination of the anima& weight, correct use of effective drug. In field practice however,
it is ofien difficult to meet those requirments. It is known that a frequently used suboptimal
dosage (under-estimated weight, expired drug,etc.) may subsequently lead to the occurence
of a resistance of the trypanosomes to the drug used. As a result, the ill-informed veterinary
technicians Will tend to increase the drug dosage with the risk of producing even more drug
resistance and eventually even reaching the toxic level of the drug. The correct approach
would be to abandon fiuther use of that particular drug and to use an other effective sanative
drug. Ifthis is not done, the occurence and subsequent distribution of such drug resistant
trypanosome strains Will eventually lead to heavy economic losses due to weight loss, loss in
overall animal production and death. The range of trypanocidal drugs available to the
veterinarian is very limited and the occurence of an eventual resistance to these few drugs
Will render a11 possible treatment in future ineffective.
In Senegal, the stock breeders in trypanosomiasis endernic areas regularly request the
local veterinarians for trypanocidal treatrnent of their animais. The conditions under which
these are oRen carried out form a permanent risk in the appearence of chemoresistant
trypanosome s-trains. The major@ of cattle in the Sokone region (southern-Central region)
belong to the Diakore breed (cross Zebu-Ndama). These animais are widely used for bath
milk and meat production and animal traction. The region is almost entirely infested with
Glassina morsitans submorsitans and G. palpalis gambiensis, vectors of Trypanosoma
bru& T. congolense and T. vivax. Since several years, the local veterinarians in the Sokone
region have reported cases of ineffective trypanocidal treatments, in particular with Berenil,
to a lesser extend with Samorin. The widespread use/mis-use of Berenil and Samorin in this
region for many years could have resulted in creating resistant trypanosome strains for one or
bath of these drugs. Results of relevant studies carried out in the region during the period -
199 1-1992 indicated the possible presence of such trypanosome strains. These results Will
bave to be confiirmed by the isolation and identification of these strains following their
subinoculation into laboratory animais and drug triais to determine the effrcacy of the
available trypanocidal drugs.
I.S.R.A/D.R.P.S.AiL.N.E.R.V.,
B.P. 2057, Dakar-Hann, Sénégal (on behalf ofDr. A DIAITE).
The proposed study Will rsquirs the use of both specific and sensitive diagnostic
techniques to enable the detection of possibly a11 infected animals. The combined use of the
BCT and the Ag-ELIS=2 proved to be salisfacto? during ths 199 l-1992 study and will
therefore be utilised again in this proposed study. In addition, suflicient funds shouid be
available to carry out the proposed field visits required to verify the chemotherapeutic trials.
Assistance Will be requested from the FAOIIAEA’s Joint Division of the IAEA to provide
these fùnds for the study.
2. STUDY MATERIALS
- Cattle :
1000 from the Sokone area, from the villages Karang and Keur
Aliou Guèye in particular, where suspected drugresistant strains
were found in 199 l-92.
-Mice:
100 for subinoculation with T. brzwi and T. congolense field strains.
- Goats:
lO- 15 for subinoculation with T. vivux fïeld strains.
- Liquid Nitrogen: 2 X 20 litres for cryopreservation of trypanosome stabilates.
- Diagnostics: FAOUEA Ag-ELISA kit and materials for the serological diagnosis
of T. brucei, T. congolense and T. vivux.
3. STUDY METHODS
The protocol used in 199 l-92 was the following:
- First field visit (= Vi): treatment with Berenil of a11 the study animais;
- Visit 2 (Vl + 15 days): subdivision of the animais into 3 groups:
Group 1: Samorin treatment
Group 2: Samorin treatment
Group 3: No more treatment following VI
- Visit 3 (V2 + 15 days):
Group 1: Ethidium treatment
Group 2: No more treatment following V2
Group 3 : No more treatment following V 1 and V2.
- FoIIow up:
Regular visits at 1 mont-h intervals for 5 months and recording of
the parasitaemic or/a.nd antigenaemic post-therapeutic persisting cases.
Tria1 to obtain natural infection of 2 sheep, but fmaIly cancelled due to
lack of fùnds.
The above described study protocol was highly interesting, however, some problems
remained to be investigated. The effrcacy of Samorin and Ethidium could not be truely
determined for the Berenil sensitive strains which were elirninated by the Berenil treatment
carried out during visit one. In addition, the use and properties of Berenil made it necessary
to plan the second visit two weeks after the first. This period is too short to distinguish
serologically between truiy persisting infections and incompletely cleared parasite
antigenaemia. It is for these reasons that the use of Samorin during the second visit, with a
prophylactic period of approximately 3 months, would have been better.
The moditïed study protocol is therefore the following:
- A fïrst study phase for the detection and isolation of suspected drugresistant
strains.
- A second study phase involving the laboratory characterisation of the
isolated strains, followed by field visits to treat the animais with the
identifïed proper sanative drugs and verifkation of their efkacy.
3.1. Detection and isuhztion (sec table 1)
- Visit 1: Screening of 1,000 cattle by BCT and Ag-ELISA in the villages
where suspeded strains were found in 199 l-92. The serological and BCT results Will
identifj the infected animals and the trypanosome species involved.
- Visit 2 (= Vi + 21 days):
Al1 animals found positive following Vl
(envisaged 250-300) Will be re-sampled for BCT and Ag-ELISA and treated with Samorin.
Prior to treatment however, a number of these positive animals Will be selected (on basis of
trypanosome species and animal location) and bled for subsequent subinoculation into 1 O-l 5
goats (Y. v.) or 100 mice (T.b. T.C.).
- Visit 3 (= V2 + 30 days): Sampling of a11 Vl positive animais for BCT and
Ag-ELISA to determine the effect of Samorin.
- Visit 4 (= V3 + 30 days): Sampling of a11 selected Vl positive animals for
BCT and Ag-ELISA Based on the overall (BCT and Ag-ELISA) results of V3, blood fi-om
those animals with persisting infections Will be collected for subsequent subinoculations.
- End of the fïrst part of the field work
3.2. Characterisation (sec table 2)
During the two months following V4, chemotherapeutic triais Will be carried out at
the laboratory. It is envisaged that this Will provide essential information on the nature and
extend of possible drugresistance of the isolated strains, and the identication of an effective
sanative drug. During each stage of these investigations, trypanosome stabilates Will be
stored in liquid nitrogen.
Following thc identification of ths eff4ve sanative drug(s). two field visits will
further be made; the first visit to treat the animals with that drug(s), the second visit one
month later to ver@+ the efficacy of that treatment.
3.3. Negative reference sera
In addition to the collection of sera from the tsetse-infested Sokone region, some 100
bovine sera will be collected in a tsetse and trypanosomiasis fi-ee region in northen Senegal
for subsequent screening by Ag-ELISA. The sampled animais will then be treated with
Samorin and sampled again a.fIer one month. This will provide additional information for the
verification of the specifïcity of the assay.
These above described investigations are planned for the fti year of the programme.
The results of this study Will fnst of a11 be used to formulate recommendations
for the
Government of Senegal to prohibit the use of determined non effective drug(s) in the study
area. It is envisaged however, that the findings could be used on a larger scale within the
PanaGican Rinderpest Campain (PARC) activities carried in Senegal.
Table 1. Detection and isolation of suspected drug rcsistsnt strains
Visit Duration
Action
8 days
M.SEYE, i\\. MAKE
Screening of 1000 cattle by BCT and Ag-ELISA
Subinoculations from positives of visite 1
5
M. SEYE
Samorin treatment +ve animals: expected 300 cattlr
M. SEYE
Post therapeutic verikation (BCT and Ag-ELISA)
Subinoculation from persisting cases
4
5
M. SEYE
Second post therapeutic verifkation
of Is&nion phase: sfarting of khmu’my tri& for 2 months
3rd post therapeutic verification
5
5
M. SEYE
Treatment with adopted sanative drug
6
5
M. SEYE
Verification of the V5 treatments
Table 2. Characterisation of suspected drugresistant fïeld strains
Phase i: Natare of the eventid resistmce
GROUP TREATMENT BCT ESAbiINATION
SIGNIFICATION
(Day 0)
(Day 0 + 24-48 h.)
Positive Tryps.
Resistance for 3erenil
1
BERENIL:lO.5mgkg
Negative Tryps.
Sensible for this dose
Pusdive
Resistance for Samorin
2
SAMORpN:l.Omgkg
Negative
Sensible for this dose
Posidve
Resistance for Ethidium
3
ETHlDIUM:1.5mgkg
Negative
Sensible for this dose
Phase 2: Level of sensitivi@ (cuncerns the strains with post therapeutic
negizfive resulis)
Positive
Resistance l usual dose
1
BERJWlL : 7.0 mgkg
Negative
Normal sensitivity
Pos&e
Resistauce / usual dose
2
SAMORIN: 0.5 mgkg
Negative
Normal sensitivity
Posifive
Resistauce / usual dose
3
ETHIDIUM: 1.0 mgkg
Negative
Normal sensitivity
In case of resistance to the normal dose of a drug the sanative drug for the
concerned strain will be that showing a normal efficacy.
If su& normally effective trypanocid is not found, the trials will be continued
using other compounds.
For each phase, a non- treated control group (Gr. 4) Will be constitued.
DIRECTION DES RECHERCHES SUR LES
---m-----------
PRODUCTIONS ET LA SANTE ANIMALES
MTNISTERE DE L’,L\\GRICULTURE
--_____--------
--------
LABORATOIRE NATIONAL DE L’ELEV.4GE
INSTITUT SENEGALAIS DE
ET DE RECHERCHES VETERINAIRES
RECHERCHES AGRICOLES (ISRA)
(L. N. E. R.V.) DAKAR-HANN
FIRST RESEARCH COORDINATIONMEETING
OF THE FAOLMm AIBKXV TRYPANOSOMASIS NETWORK
N’AIROBI, KENYA, 7-11 FEBRUARY 1994
(I.A.E.A./I.S.R.A RESEARCH CONTRACT No 7596iR.B)
RAPPORT N.@kPARHO. ANIM.
JANVIER 1994
*: Assistant de Recherches, Laboratoire de Parasitologie.
DETECTION AND CHARACTERISATION
OF SUSPECTED DRUG RESISTANT TRYPANOSOME
STRAINS IN SENEGAL USING B.C.T. ,4ND ,4g-ELISA
DIAGNOSTIC TECHNIQUES
BY
Mamadou SEYE
(ISRA / DRPSA / LNERV, BP 2057,Dakar, Sénégal)
SUMMARY
Animal trypanosomiasis is a major livestock disease in the southern-central region of Senegal.
The major@ of the cattle in this area are of the Diakme breed (cross Zebu-Ndama). Because of their
properties, these animals are widely used by the f%ming community for animal traction, but also for
milk and meat production. Despite their reasonable level of trypanotolerance, many of these animais
are affected by trypanosomiasis in the are& with significant tsetse challenge. For this reason,
trypanocidal drugs such as Berenil and Samorin are being used on a large scale. Their use and
particularly mis-use has lead to the occurence of drug resistant strains. In a previous study (1991-
1992) carried out in this region, results were obtained to indicating this. The proposed study to be
conducted in 1994, is to confüm these fïndings in greater details , using the BCT and Ag-ELISA, and
to identify the most appropriate trypanocidal drug(s) to be used in the Sokone region. The project
activities will involve the screening of approximately 1,000 Diakme cattle in the Sokone area,
followed by the isolation of trypanosomes from infected cattle and their subinoculation into laboratory
animals for subsequent chemotherapeutic trials with Berenil, Samorin and Ethidium. The results
obtained will be provided to the Senegalese Ministry of Agriculture
KEY WORDS
SENEGAL - BOVINE -TRYPANOSOMIASIS - DIAGNOSTIC - ELISA
TRYPANOCID - DRUG RESSISTANCE
DETECTION AND CH/l.RACTERISATION OF SUSPECTED
I>RLJG RESISTANT TRYPA!NOSOME STRAINS IN SENEGAL
LJSING B.C.T AND Ag-ELISA DIAGNOSTIC TECHNIQUES
bY
Mamadou SEYE”
1. INTRODUCTION
The efforts to produce a vaccin against trypanosomiasis bave failed so far, &spite a11
subtantial fùnding and the high level of expertise inputs allocated to this for some decades
throughout the world.
In view of this, the use of trypanocidal drugs remains the only alternative to tr-est
trypanosomiasis afI?ected animais. This bas led to a wide spread use of trypanocidal drugs in
those areas where the disease exists. However, the success of such chemotherapeutic
treatment depends on specific criteria : identification of the causitive trypanosomes, correct
deterrnination of the anima& weight, correct use of effective drug. In field practice however,
it is ofien difficult to meet those requirments. It is known that a frequently used suboptimal
dosage (under-estimated weight, expired drug,etc.) may subsequently lead to the occurence
of a resistance of the trypanosomes to the drug used. As a result, the ill-informed veterinary
technicians Will tend to increase the drug dosage with the risk of producing even more drug
resistance and eventually even reaching the toxic level of the drug. The correct approach
would be to abandon fiuther use of that particular drug and to use an other effective sanative
drug. Ifthis is not done, the occurence and subsequent distribution of such drug resistant
trypanosome strains Will eventually lead to heavy economic losses due to weight loss, loss in
overall animal production and death. The range of trypanocidal drugs available to the
veterinarian is very limited and the occurence of an eventual resistance to these few drugs
Will render a11 possible treatment in future ineffective.
In Senegal, the stock breeders in trypanosomiasis endernic areas regularly request the
local veterinarians for trypanocidal treatrnent of their animais. The conditions under which
these are oRen carried out form a permanent risk in the appearence of chemoresistant
trypanosome s-trains. The major@ of cattle in the Sokone region (southern-Central region)
belong to the Diakore breed (cross Zebu-Ndama). These animais are widely used for bath
milk and meat production and animal traction. The region is almost entirely infested with
Glassina morsitans submorsitans and G. palpalis gambiensis, vectors of Trypanosoma
bru& T. congolense and T. vivax. Since several years, the local veterinarians in the Sokone
region have reported cases of ineffective trypanocidal treatments, in particular with Berenil,
to a lesser extend with Samorin. The widespread use/mis-use of Berenil and Samorin in this
region for many years could have resulted in creating resistant trypanosome strains for one or
bath of these drugs. Results of relevant studies carried out in the region during the period -
199 1-1992 indicated the possible presence of such trypanosome strains. These results Will
bave to be confiirmed by the isolation and identification of these strains following their
subinoculation into laboratory animais and drug triais to determine the effrcacy of the
available trypanocidal drugs.
I.S.R.A/D.R.P.S.AiL.N.E.R.V.,
B.P. 2057, Dakar-Hann, Sénégal (on behalf ofDr. A DIAITE).
The proposed study Will rsquirs the use of both specific and sensitive diagnostic
techniques to enable the detection of possibly a11 infected animals. The combined use of the
BCT and the Ag-ELIS=2 proved to be salisfacto? during ths 199 l-1992 study and will
therefore be utilised again in this proposed study. In addition, suflicient funds shouid be
available to carry out the proposed field visits required to verify the chemotherapeutic trials.
Assistance Will be requested from the FAOIIAEA’s Joint Division of the IAEA to provide
these fùnds for the study.
2. STUDY MATERIALS
- Cattle :
1000 from the Sokone area, from the villages Karang and Keur
Aliou Guèye in particular, where suspected drugresistant strains
were found in 199 l-92.
-Mice:
100 for subinoculation with T. brzwi and T. congolense field strains.
- Goats:
lO- 15 for subinoculation with T. vivux fïeld strains.
- Liquid Nitrogen: 2 X 20 litres for cryopreservation of trypanosome stabilates.
- Diagnostics: FAOUEA Ag-ELISA kit and materials for the serological diagnosis
of T. brucei, T. congolense and T. vivux.
3. STUDY METHODS
The protocol used in 199 l-92 was the following:
- First field visit (= Vi): treatment with Berenil of a11 the study animais;
- Visit 2 (Vl + 15 days): subdivision of the animais into 3 groups:
Group 1: Samorin treatment
Group 2: Samorin treatment
Group 3: No more treatment following VI
- Visit 3 (V2 + 15 days):
Group 1: Ethidium treatment
Group 2: No more treatment following V2
Group 3 : No more treatment following V 1 and V2.
- FoIIow up:
Regular visits at 1 mont-h intervals for 5 months and recording of
the parasitaemic or/a.nd antigenaemic post-therapeutic persisting cases.
Tria1 to obtain natural infection of 2 sheep, but fmaIly cancelled due to
lack of fùnds.
The above described study protocol was highly interesting, however, some problems
remained to be investigated. The effrcacy of Samorin and Ethidium could not be truely
determined for the Berenil sensitive strains which were elirninated by the Berenil treatment
carried out during visit one. In addition, the use and properties of Berenil made it necessary
to plan the second visit two weeks after the first. This period is too short to distinguish
serologically between truiy persisting infections and incompletely cleared parasite
antigenaemia. It is for these reasons that the use of Samorin during the second visit, with a
prophylactic period of approximately 3 months, would have been better.
The moditïed study protocol is therefore the following:
- A fïrst study phase for the detection and isolation of suspected drugresistant
strains.
- A second study phase involving the laboratory characterisation of the
isolated strains, followed by field visits to treat the animais with the
identifïed proper sanative drugs and verifkation of their efkacy.
3.1. Detection and isuhztion (sec table 1)
- Visit 1: Screening of 1,000 cattle by BCT and Ag-ELISA in the villages
where suspeded strains were found in 199 l-92. The serological and BCT results Will
identifj the infected animals and the trypanosome species involved.
- Visit 2 (= Vi + 21 days):
Al1 animals found positive following Vl
(envisaged 250-300) Will be re-sampled for BCT and Ag-ELISA and treated with Samorin.
Prior to treatment however, a number of these positive animals Will be selected (on basis of
trypanosome species and animal location) and bled for subsequent subinoculation into 1 O-l 5
goats (Y. v.) or 100 mice (T.b. T.C.).
- Visit 3 (= V2 + 30 days): Sampling of a11 Vl positive animais for BCT and
Ag-ELISA to determine the effect of Samorin.
- Visit 4 (= V3 + 30 days): Sampling of a11 selected Vl positive animals for
BCT and Ag-ELISA Based on the overall (BCT and Ag-ELISA) results of V3, blood fi-om
those animals with persisting infections Will be collected for subsequent subinoculations.
- End of the fïrst part of the field work
3.2. Characterisation (sec table 2)
During the two months following V4, chemotherapeutic triais Will be carried out at
the laboratory. It is envisaged that this Will provide essential information on the nature and
extend of possible drugresistance of the isolated strains, and the identication of an effective
sanative drug. During each stage of these investigations, trypanosome stabilates Will be
stored in liquid nitrogen.
Following thc identification of ths eff4ve sanative drug(s). two field visits will
further be made; the first visit to treat the animals with that drug(s), the second visit one
month later to ver@+ the efficacy of that treatment.
3.3. Negative reference sera
In addition to the collection of sera from the tsetse-infested Sokone region, some 100
bovine sera will be collected in a tsetse and trypanosomiasis fi-ee region in northen Senegal
for subsequent screening by Ag-ELISA. The sampled animais will then be treated with
Samorin and sampled again a.fIer one month. This will provide additional information for the
verification of the specifïcity of the assay.
These above described investigations are planned for the fti year of the programme.
The results of this study Will fnst of a11 be used to formulate recommendations
for the
Government of Senegal to prohibit the use of determined non effective drug(s) in the study
area. It is envisaged however, that the findings could be used on a larger scale within the
PanaGican Rinderpest Campain (PARC) activities carried in Senegal.
Table 1. Detection and isolation of suspected drug rcsistsnt strains
Visit Duration
Action
8 days
M.SEYE, i\\. MAKE
Screening of 1000 cattle by BCT and Ag-ELISA
Subinoculations from positives of visite 1
5
M. SEYE
Samorin treatment +ve animals: expected 300 cattlr
M. SEYE
Post therapeutic verikation (BCT and Ag-ELISA)
Subinoculation from persisting cases
4
5
M. SEYE
Second post therapeutic verifkation
of Is&nion phase: sfarting of khmu’my tri& for 2 months
3rd post therapeutic verification
5
5
M. SEYE
Treatment with adopted sanative drug
6
5
M. SEYE
Verification of the V5 treatments
Table 2. Characterisation of suspected drugresistant fïeld strains
Phase i: Natare of the eventid resistmce
GROUP TREATMENT BCT ESAbiINATION
SIGNIFICATION
(Day 0)
(Day 0 + 24-48 h.)
Positive Tryps.
Resistance for 3erenil
1
BERENIL:lO.5mgkg
Negative Tryps.
Sensible for this dose
Pusdive
Resistance for Samorin
2
SAMORpN:l.Omgkg
Negative
Sensible for this dose
Posidve
Resistance for Ethidium
3
ETHlDIUM:1.5mgkg
Negative
Sensible for this dose
Phase 2: Level of sensitivi@ (cuncerns the strains with post therapeutic
negizfive resulis)
Positive
Resistance l usual dose
1
BERJWlL : 7.0 mgkg
Negative
Normal sensitivity
Pos&e
Resistauce / usual dose
2
SAMORIN: 0.5 mgkg
Negative
Normal sensitivity
Posifive
Resistauce / usual dose
3
ETHIDIUM: 1.0 mgkg
Negative
Normal sensitivity
In case of resistance to the normal dose of a drug the sanative drug for the
concerned strain will be that showing a normal efficacy.
If su& normally effective trypanocid is not found, the trials will be continued
using other compounds.
For each phase, a non- treated control group (Gr. 4) Will be constitued.