!
Acknowledgments
This PhD thesis would not have been successfully conducted without:
• My promoter, Dr Dirk Geysen (Institute of Tropical Medicine of Antwerp /Instituut voor Tropische Geneeskunde) and co-promoters Dr Phelix A.O.
Majiwa (International Livestock Research Institute - African Agricultural Technology Foundation / ILRI – AATF) and Dr Luc Vanhamme (Université Libre de Bruxelles).
• The Institute of Tropical Medicine of Antwerp / Instituut voor Tropische Geneeskunde Antwerpen (ITM/ITG) with a special thought to Pr. Stanny Geerts for initiating this work and allowing my collaboration, to Dr Dirk Geysen for his advise and continuous support, to Dr Jef Brandt for his support especially during the operations in Zambia, to Jacobus De Witte, Marc
Jochems, Frank Ceulemans, Bjorn Victor, Louis Van Tiggel for their kind technical assistance.
• The Université Libre de Bruxelles with a grateful thought to Dr Luc Vanhamme who accepted the supervision of this thesis.
• The International Livestock Research Institute for providing the isogenic clones of T. congolense and particularly Dr Phelix A.O. Majiwa for helpful advice, Mrs Mary Maina for technical assistance and Paul Spooner for logistical assistance.
• The Europeean Union and INCO-DC (International Cooperation with Developing countries, Contract Number ERBIC18CT95-006) and the
University of Glasgow (Department of Veterinary Physiology) with particular acknowledgment to Dr Marc Charles Eisler.
• The Frei Universität Berlin, Dr. Peter-Henning Clausen and Dr. Yohannes Afewerk for providing T.congolense strains characterized for isometamidium resistance.
• The Government of the Republic of Zambia for its long term commitment in Animal Health and Production and Agricultural enhancement.
• The Belgian Cooperation, the Assistance to the Veterinary Services of Zambia, the Veterinary Services of Zambia and all the staff with a grateful thought to Kalinga Chilongo, Anton Chupa, Romanos Besa, George Chaka, L.
Mataa, Koen Geeraerts, John Kwenda may he rest in peace, Gabriel Mitti, Reuben Zulu, Michel Billiouw, Rik Elyn, Fridah Mukuka, Casiano Hapoma, Prosper Van Kerkhoven, Dryson Daka, Jennifer Phiri, Nelson Banda and E.
Chanda.
• Het Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (FWO) for financing the work performed in molecular biology.
• I have been honoured to gain the collaboration of Senior Chief Kalindawalo, Chief Kawasa, Chieftainess Nyanje, Chief Sandwe Chief Mba’Gombe and their people.
It is impossible to mention individually all the persons who helped in some way but I would like to express my gratitude for all the assistance, both professional and personal, I received from the various people I forgot to mention.
Table of contents
Acknowledgments...3
Table of contents...5
Published data ...10
Summary ...11
List of abbreviations ...12
1. Introduction...13
1.1. Animal trypanosomosis ...13
1.1.1. Life cycle ...13
1.1.2. Phylogenetic tree ...15
1.2. Trypanosomosis diagnosis...17
1.2.1. A gold standard?...17
1.2.2. Clinical diagnosis ...17
1.2.3. Parasitological diagnosis by direct examination ...18
1.2.3.1. Wet blood film ...18
1.2.3.2. Thick blood film ...19
1.2.3.3. Thin blood smear technique...19
1.2.3.4. Parasite concentration techniques ...20
1.2.4. Parasitological diagnosis by indirect diagnosis...21
1.2.4.1. The indirect fluorescent antibody test (IFAT) ...21
1.2.4.2. The indirect enzyme-linked immunosorbent assay...22
1.2.4.3. Card agglutination test ...23
1.2.4.4. Antigen-detecting tests...23
1.2.4.5. Polymerase Chain Reaction and Restriction Fragment Length Polymorphism...24
1.2.4.6. Proteomic signature analysis...24
1.3.4. Monitoring and treatment of individual infected animals...27
1.3.5. Monitoring and treatment of clinical cases ...27
1.4. Resistance against trypanocidal drugs...28
1.4.1. Possible strategies for drug resistance...28
1.4.2. Chemotherapy of animal trypanosomosis ...29
1.4.3. Isometamidium resistance ...31
1.4.4. Diminazene resistance...35
1.5. Diagnosis of trypanocidal resistance ...38
1.5.1. Tests in ruminants ...38
1.5.2. Tests in mice...39
1.5.3. In vitro assays...40
1.5.4. Trypanocidal drug ELISAs ...40
1.5.5. Longitudinal parasitological data ...42
1.5.6. Early diagnosis of treatment failure ...42
1.5.7. New tests for detection of resistance to isometamidium...42
2. Results...46
2.1. Disease prevalence and drug resistance...47
2.1.1. Specific objective of this chapter ...47
2.1.2. Introduction ...47
2.1.3. Prevalence of bovine trypanosomosis ...48
2.1.4. Investigation of trypanocidal drug resistance in mice...49
2.1.5. Investigation of trypanocidal drug resistance in calves ...50
2.1.6. Investigations using isometamidium-ELISA ...51
2.1.7. Summarized data per district...52
2.2. Drug use...54
2.2.1. Specific objective of this chapter ...54
2.2.2. Introduction ...54
2.2.3. Questionnaire ...54
2.2.4. Cattle bodyweight estimation...57
2.2.5. Serum isometamidium concentration...58
2.3. Diagnosis of trypanosome infections in cattle by PCR-RFLP ...60
2.3.1. Specific objective of this chapter ...60
2.3.3. Results ...61
2.4. Use of PCR-RFLP for the diagnosis of mixed trypanosome infections in cattle ...68
2.4.1. Specific objective of this chapter ...68
2.4.2. Introduction ...68
2.4.3. Results ...68
2.5. AFLP analysis of sensitive and resistant T.congolense clones...75
2.5.1. Specific objective of this chapter ...75
2.5.2. Introduction ...75
2.5.1. Similarity of AFLP profiles of the isogenic clones...75
2.5.2. Sequencing and analysis of the fragments ...78
2.5.3. Resistant phenotype and RFLP results...82
3. Discussion...84
3.1. Epidemiological background and drug use ...85
3.2. Improvement of the diagnosis of trypanosome infections...90
3.3. AFLP analysis of sensitive and resistant T.congolense clones...94
3.4. Final recommendations...96
3.4.1. Single Drug Resistance ...96
3.4.2. Multiple drug resistance Resistance to both isometamidium and diminazene is present at the level of individual trypanosomes may be demonstrated by testing cloned populations in mice or in ruminants (1). If resistance to both isometamidium and diminazene is present at the level of individual trypanosomes, the following guidelines should be followed: ...97
3.4.3. Recommendations on the use of isometamidium prophylaxis:...97
4. Materials and methods ...98
4.1. Epidemiological survey ...98
4.1.1. Cross-sectional study...98
4.1.2. Longitudinal study...99
4.1.3. Staining and examinations of blood smears...99
4.1.7. Isometamidium-ELISA ...101
4.2. Drug use survey...102
4.2.1. Analysis of the use of trypanocides by farmers in Eastern Province ...102
4.2.2. Information on drug use ...103
4.2.3. Monitoring of cattle bodyweight...103
4.2.4. Isometamidium administration...103
4.2.5. Isometamidium-ELISA ...103
4.3. Diagnosis of trypanosome infections in cattle by PCR-RFLP ...104
4.3.1. DNA reference samples ...104
4.3.2. Filter paper samples ...105
4.3.3. DNA amplification...106
4.3.4. Primers used ...106
4.3.5. Restriction Fragment Length Polymorphism ...107
4.4. Use of PCR-RFLP for the diagnosis of mixed trypanosome infections in cattle ...107
4.4.1. DNA reference samples ...107
4.4.2. Field samples...110
4.4.3. DNA amplification...110
4.4.4. Primers used ...110
4.4.5. Restriction Fragment Length Polymorphism ...111
4.5. AFLP analysis of sensitive and resistant T.congolense clones...111
4.5.1. Trypanosomes and DNA samples ...111
4.5.2. AFLP ...113
4.5.3. Polyacrylamide Gel Electrophoresis (PAGE) ...113
4.5.4. DNA extraction from the polyacrylamide gel...114
4.5.5. PCR amplification of the purified fragments of DNA ...114
4.5.6. Cloning and sequencing ...114
4.5.7. DNA amplification of field strains...114
4.5.8. Primers ...115
4.5.9. Restriction Fragment Length Polymorphism ...115
4.5.10. Single dose mouse test ...115
5. References...116
7. Tables...139 8. Index ...140
Published data
Delespaux V, Geerts S, Brandt J, Elyn R and Eisler MC. Monitoring the correct use of isometamidium by farmers and veterinary assistants in Eastern Province of Zambia using the isometamidium-ELISA. Veterinary Parasitology 110:
117-122, 2002.
Delespaux V, Ayral F, Geysen D and Geerts S. PCR-RFLP using Ssu-rDNA
amplification: applicability for the diagnosis of mixed infections with different trypanosome species in cattle. Veterinary Parasitology 117: 185-193, 2003.
Geysen D, Delespaux V and Geerts S. PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle. Veterinary Parasitology 110: 171-180, 2003.
Sinyangwe L, Delespaux V, Brandt J, Geerts S, Mubanga J, Machila N, Holmes PH and Eisler MC. Trypanocidal drug resistance in eastern province of Zambia. Veterinary Parasitology 119: 125-135, 2004.
Delespaux V, Geysen D, Majiwa PAO and Geerts S. (2004). Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense. International Journal for Parasitology (in press).
Summary
The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of
trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense.
After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented.
The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.
List of abbreviations
ABC ATP binding cassette
AFLP Amplified Fragment Length Polymorphism
Ag Antigen
ATP Adenosine triphosphate
CATT Card Agglutination Test for Trypanosomiasis CD Curative dose
DIM Diminazene aceturate ED Effective dose
ELISA Enzyme Linked Immunosorbent Assay IFAT Indirect Fluorescent Antibody Test ISMM Isometamidium chloride
KIVI Kit for In Vitro Isolation PCR Polymerase Chain Reaction PCV Packed Cell Volume
RBC Red Blood Cell
rDNA ribosomal Deoxyribo Nucleic Acid
1. Introduction
1.1. Animal trypanosomosis
In Africa, Trypanosoma brucei, T. vivax and T. congolense occur wherever the tsetse fly vector is found. The clinical signs of the disease caused by these organisms vary according to the trypanosome species, the virulence of the particular isolate and the species of host infected. Acute disease is characterized by anaemia weight loss, abortion and, if not treated, possibly death. Animals that survive are often infertile (Al Qarawi et al., 2004; Sekoni et al., 2004) and of low productivity. In some instances, infected animals show no evident signs of disease but can perish if stressed, for example, by work, pregnancy, milking or adverse environmental conditions (Luckins, 1988). In southern Africa the disease is widely known as Nagana, which is derived from a Zulu term meaning 'to be in low or depressed spirits which is a very pertinent description of the disease. The impact of the tsetse-associated disease extends in sub- Saharan Africa over some 10 million km2 (a third of the continent). Of these 10 million km2 some 3 million are covered by equatorial rain forest; the remaining area contains some very good grazing areas, which perhaps fortunately have been
protected so far by the tsetse fly against overgrazing (Uilenberg, 1998).
1.1.1. Life cycle
Transmission and Epidemiology:
Most tsetse-transmission is cyclical, and begins when blood from a trypanosome- infected animal is ingested by the tsetse fly (Figure 1). The trypanosome loses its surface coat, multiplies in the fly, then reacquires a surface coat and becomes infective. Trypanosoma brucei species 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. The life cycle in the tsetse may be as short as 1 week with T. vivax or extend to a few weeks for T. brucei species.
environments; G. morsitans usually is found in savanna country, G. palpalis prefers areas around rivers and lakes, and G. fusca lives in high forest areas. All three species transmit trypanosomes, and all feed on various mammals.
Figure 1 Life cycle of Trypanosoma brucei
Mechanical transmission can occur through tsetses or other biting flies. In the case of T. vivax , Tabanus spp. and other biting flies seem to be the primary mechanical vectors outside the tsetse areas, as in Central and South America. Mechanical transmission requires only that blood containing infectious trypanosomes be transferred from one animal to another.
Cattle, sheep, and goats are infected, in order of importance, by T.congolense, T.vivax and T. brucei brucei . In pigs, T simiae is the most important. In dogs and cats, T.
brucei is probably the most important. It is difficult to assign an order of importance for horses and camels (see Table 1).
The trypanosomes that cause tsetse-transmitted trypanosomiasis (sleeping sickness) in man, T. brucei rhodesiense and T. brucei gambiense , closely resemble T.brucei brucei from animals; the animal isolates of T brucei are lysed by human serum. There are indications that changes in resistance to human serum occur in some isolates of T.
brucei species; therefore, reasonable precautions should be taken when working with
Domestic hosts Reservoir Laboratory animals T.vivax Cattle, buffaloes,
small ruminants, horses, camels.
Refractory: pig, dog, cat
Wildlife, antelopes, girafs
none
T.congolense Cattle, small ruminants, horses, camels, pigs, dogs
Wildlife (wide range)
Rodents(*), rabbits
T. brucei brucei Horses, camels, dogs: very sensitive Cattle and small ruminants
Wildlife (wide range
Rodents, rabbits
(*) Some strains do not grow in rodents
Table 1 Host range of the principal animal trypanosomes 1.1.2. Phylogenetic tree
Species of trypanosome infecting mammals fall into two distinct groups and, accordingly, have been divided in two sections (Hoare, 1972): (A) the Stercoraria (subgenera Schizotrypanum, Megatrypanum and Herpetosoma) in which
trypanosomes are typically produced in the hindgut and are then passed on by contaminative transmission from the posterior; and (B) the Salivaria (subgenera Duttonella, Nannomonas and Trypanozoon), in which transmission occurs by the anterior station and is inoculative.
A detailed phylogenetic tree based on the analysis of the 18S SSU ribosomal rna gene sequences is presented in Figure 2.
Figure 2 Phylogenetic tree based on bootstrapped maximum parsimony analysis of 18S SSU ribosomal RNA gene sequences.
The tree represents an extended analysis of Stevens et al. (1999, 2001) and is based on an alignment of 1809 nucleotide positions, being one of three alignments tried (Morrison and Ellis, 1997; Stevens et al., 1999). It contains 61 Trypanosoma taxa and shows the genus to be monophyletic. Nannomonas suggroups are denotated by (s) = savannah, (r-f) = riverine-forest, (Kc) = Kenya coast and (T) = Tsavo;
sequence accession numbers are given in Haag et al., 1998 and Stevens et al., (1999, 2001).
1.2. Trypanosomosis diagnosis
A disease may be diagnosed on the basis of the clinical signs and symptoms, by demonstration of the causative organism or by reactions to diagnostic tests. In some situations, the clinical manifestations of trypanosomosis, particularly anaemia might provide sufficient grounds for a putative diagnosis. Diagnosis refers to methods for detecting infection, either by identifying the parasites themselves or by interpretation based on the results of other tests (Luckins, 1992).
1.2.1. A gold standard?
The type of diagnostic test used will vary according to the epidemiological
characteristics of the disease and the strategy for control. Where disease prevalence is high, even tests of low sensitivity will suffice if chemotherapy or chemoprophylaxis is administered on a herd basis. However, in many situations drugs are often
administered therapeutically to individual infected animals and it is essential that more sensitive diagnostic tests are used in order to detect active infections. Similar considerations also apply after control campaigns. As the disease prevalence declines, the need for individual treatment as opposed to block treatment becomes an important issue. When chemotherapy has been applied in areas where drug resistance is known to exist, it is also necessary to detect rapidly any failure in treatment. Whatever the requirements of the particular situation, the tests themselves need to fulfill a number of criteria to be of practical use. Such criteria include high diagnostic specificity and sensitivity, easy reproducibility, simplicity, economy and ease of interpretation.
Ideally, the tests should be able to be used in the field. To improve diagnostic efficiency, many parasitological and serological techniques have been developed (Luckins, 1992). Diagnostic tests must be adapted to each particular situation. Tools for an epidemiological survey will be different from the individual diagnosis of a single cow owned by a small scale farmer in a zero-input system. The panel of methods is wide and the choice must be adapted depending on the aim and resources (financial and technical) available.
The incubation period is usually 1-4 wk. The primary clinical signs are intermittent fever, anaemia, and weight loss. Cattle usually have a chronic course with high mortality, especially if there is poor nutrition or other stress factors. Ruminants may gradually recover if the number of infected tsetse flies is low; however, stress results in relapse. Cases progressing to a more steady chronic state may be characterized by anaemia, cachexia, poor productivity and infertility. Necropsy findings vary and are not pathognomonic. In acute, fatal cases, extensive petechiation of the serosal
membranes, especially in the peritoneal cavity, may occur. Also, the lymph nodes and spleen are usually swollen. In chronic cases, swollen lymph nodes, serous atrophy of fat, and anaemia are seen.
Recently a comparative study on the clinical, parasitological and molecular diagnosis of bovine trypanosomosis was carried out by Magona et al. (2003). Clinical diagnosis was found to have a good sensitivity (78%) but a low specificity (27%) when
compared to parasitological tests. From this study, it appears that treatment of cattle based on clinical examination may clear up to 87, 5% or 78% of the cases that would be positive by either molecular or parasitological diagnosis, respectively. Under field conditions, in the absence of simple and portable diagnostic tools or access to
laboratory facilities, veterinarians could rely on clinical diagnosis to screen and treat cases of bovine trypanosomosis presented by farmers.
1.2.3. Parasitological diagnosis by direct examination 1.2.3.1. Wet blood film
These are made by placing a drop of blood on a microscope slide and covering with a cover-slip. The blood is examined microscopically using an x40 objective lens.
Approximately 50-100 fields are examined. Trypanosomes can be recognized by their movement among the red blood cells. The method is simple, inexpensive and gives immediate results. Depending on the trypanosome size and movements a presumptive diagnosis can be made of the trypanosome species. Final confirmation of the species is made by the examination of the stained preparation. The diagnostic sensitivity of the method is generally low but depends on the examiner’s experience and the level of parasitaemia. Sensitivity can be improved significantly by lysing the RBCs before examination using a haemolytic agent such as sodium dodecyl sulfate (Ndao et al.,
1.2.3.2. Thick blood film
The method is simple and relatively inexpensive, but results are delayed because of the staining process. Trypanosomes are easily recognized by their general
morphology, but may be damaged during the staining process. This may make it difficult to identify the species.
1.2.3.3. Thin blood smear technique
Usually, both a thin and thick smear is made from the same sample. Thick smears contain more blood than thin smears and, hence, have a higher diagnostic sensitivity.
Thin smears on the other hand allow trypanosome species identification.
Trypanosome species can be identified by the following morphological characteristics (Criteria of the Office International des Epizooties):
-Trypanosoma vivax: 20-27 µm long, undulating membrane is not obvious, free flagellum present at the anterior end, posterior end rounded, kinetoplast large and terminal.
-Trypanosoma brucei is a polymorphic trypanosome species. Two distinctly different forms can be distinguished, i.e. a long slender form and a short stumpy form. Often, intermediate forms, possessing characteristics of both the slender and stumpy forms, are observed. The cytoplasm often contains basophilic granules in stained specimens.
-Trypanosoma brucei (long slender form): 17-30 µm long and about 2.8 µm wide, undulating membrane is conspicuous, free flagellum present at the anterior end, posterior end pointed, kinetoplast small and subterminal.
-Trypanosoma brucei (short stumpy form): 17-22 µm long and about 3.5 µm wide, undulating membrane is conspicuous, free flagellum absent, posterior end pointed, kinetoplast small and subterminal.
-Trypanosoma congolense: 8-25 µm (small species), undulating membrane not obvious, free flagellum absent, posterior end rounded, kinetoplast is medium sized and terminal, often laterally positioned. Although T. congolense is considered to be monomorphus, a degree of morphological variation is sometimes observed.
-Trypanosoma theileri 60-70 µm (large species), undulating membrane is
1.2.3.4. Parasite concentration techniques
The probability of detecting trypanosomes in a sample from an infected animal depends largely on the amount of blood examined and the level of parasitaemia. The amount of blood examined with direct examination techniques is low and parasites are often very scanty in the blood of an infected animal. Both of these factors contribute to the low sensitivity of direct examination techniques. Sensitivity can be improved by increasing the volume of blood to be examined and by concentrating the
trypanosomes.
-Microhaematocrit centrifugation technique (Woo, 1970)
The microhaematocrit centrifugation technique, or the Woo method, is widely used for the diagnosis of trypanosomosis . The sensitivity of the test for known, artificially created parasitaemia was 100% above 700 parasites/ml, about 80% between 300 and 700, 50% between 60 and 300, and was negligible below 60 parasites/ml
(Desquesnes, 1998). It is based on the separation of the different components of the blood sample depending on their specific gravity. The microhaematocrit
centrifugation technique is more sensitive than the direct examination techniques (Kratzer, 1989). Identification of trypanosome species is difficult. As the specific gravity of T. congolense is similar to that of RBCs, parasites are often found below the buffy coat in the RBC layer. To improve the separation of RBCs and parasites, and increase the sensitivity for T. congolense, the specific gravity of RBCs can be increased by the addition of glycerol (Walker et al., 1972). A modification of the Woo method is the quantitative buffy coat method (QBC) (Bailey et al., 1992). The method has been used for the diagnosis of T. b. gambiense infections. The latter method is probably too expensive for the routine large-scale use in animal trypanosomosis surveys but is more sensitive the Woo technique. Results showed that the sensitivity of the QBC test was 95% down to a concentration of 450 trypanosomes/ml. In comparison 95% sensitivity of the Woo test was observed only down to 7500 trypanosomes/ml and reading time was two fold longer (Ancelle et al., 1997).
-Dark-ground/phase-contrast buffy coat technique
The buffy coat technique or Murray method (Murray et al., 1977) represents an
collected in capillary tubes following a standardized procedure, after which the capillary tube is cut, with a diamond tipped pencil, 1 mm below the buffy coat, to include the top layer of RBCs. The buffy coat and the uppermost layer RBCs are extruded on to a clean microscope slide and covered with a cover-slip (22 x 22 mm).
Approximately 200 fields of the preparation are examined for the presence of motile trypanosomes with a dark-ground or a phase-contrast microscope with an x40 objective lens.
1.2.4. Parasitological diagnosis by indirect diagnosis
The aim of serological tests is to detect specific antibodies, developed by the host against the infection or, inversely, to demonstrate the occurrence of circulating parasitic antigens in the blood by the use of specific antibodies. The detection of antibodies indicates that there has been infection, but as antibodies persist for some time (up to 13 months, Van Den Bossche et al., 2000) after all trypanosomes have disappeared from the organism (either by drug treatment or self-cure) a positive result is no proof of active infection. In human African trypanosomosis, the measurement of immunoglobulin and trypanosome specific antibody concentrations in serum and CSF allows calculation of antibody synthesis and is a possible tool for determining the clinical stage of sleeping sickness. Such tests are not available for animal
trypanosomosis and it seems that the first IgM response is polyspecific (Taylor et al., 1998) and does not allow a precise diagnostic of the disease. Results of Buza et al., 1999, shows that the polyspecific IgM antibodies were also present in pre-infection sera, it is probable that they were natural antibodies that were not induced but only amplified by the trypanosome infection (Buza et al., 1999).
1.2.4.1. The indirect fluorescent antibody test (IFAT)
A smear of blood containing fixed trypanosomes constitutes the antigen. The cattle serum to be examined is put into contact with the smear, and immunoglobulins (antibodies) against the trypanosomal antigens attach themselves to the trypanosomes in the smear, and remain stuck to the smear even when the serum is washed off. In order to show that antibodies have reacted with the antigen, a commercial preparation
the presence of anti-trypanosome cattle immunoglobulins which have reacted with the antigen slide. In order to show the presence or absence of the rabbit immunoglobulins, and thus indirectly the presence or absence of specific antibodies against
trypanosomes in the bovine serum, the rabbit anti-bovine immunoglobulins in the commercial preparation are conjugated with a fluorescent dye, usually fluoresceine, which can be detected by looking at the smear with ultraviolet light. The combination of immunoglobulins and fluoresceine is called a conjugate, and in this case it is a rabbit anti-bovine conjugate. If the microscopic examination shows the trypanosomes in the antigen smear to be fluorescent, the test result is positive. It is possible to titrate the cattle serum by using serial dilutions of the serum and determining the end point, the highest dilution still giving a positive test result.
1.2.4.2. The indirect enzyme-linked immunosorbent assay
The principle of this test is in fact very similar to that of the IFA test. The binding of anti-trypanosomal antibodies to the antigen is shown by a conjugate of antibovine (if the test serum is bovine) immunoglobulins labelled with an enzyme, which can be visualized by adding an appropriate chromogenic substrate (i.e. the interaction between enzyme and substrate will create a colour). The use of substrate can be compared to the use of UV rays to visualize the fluorescent conjugate in the IFA test.
Usually solubilized antigens obtained from disrupted trypanosomes (successive freezing and thawing cycles or ultrasound) are used and the soluble antigens are coated in the wells of microtrays. Each microtray contains usually 96 wells. This makes it possible to process many sera at the same time, using multichannel pipettes.
Only small quantities of sera and conjugate are used. An ELISA reading instrument will quickly give the optical density (OD) of each well (showing quantitatively the intensity of the interaction between the enzyme and the substrate), thus helping to speed up the processing of large numbers of sera.
Nevertheless, the use of antigens derived from whole trypanosomes means that the test is not necessarily more species-specific than the IFAT.
This technique used for the detection of anti-trypanosome antibodies in bovine serum was recently adapted and evaluated for serodiagnosis in goats (Lejon et al., 2003).
Various ELISA systems have been constructed exploiting different reagents for detection of antibodies but still require laboratory and field validation studies to be
further assessed for their capacity to improve diagnosis of African trypanosomosis (Rebeski et al., 1999).
Recently, the ability to use mitochondrial heat shock protein 70 (MTP) of
T.congolense as a diagnostic antigen was examined by Boulange et al., 2002 and Bannai et al. (2003) with encouraging results but the technique still needs to be further validated and evaluated for natural infections in cattle.
1.2.4.3. Card agglutination test
This has been developed from a test for the diagnosis of human sleeping sickness (Magnus et al., 1979), into a commercial kit for T. evansi, CATT test T. evansi®
(Diall et al., 1994; Pathak et al., 1997). For the detection of antibodies to surra (T.
evansi infection) serum samples are mixed on a plastic card with fixed and stained trypanosomes as antigen and the test is positive when the antigen agglutinates. A titre can be determined by serial dilutions of the serum. The great advantage of this test is that in principle it is easy to carry out even in the field. Its specificity and sensitivity appear to need further evaluation, and in the experience of the author, reading the test results is not always easy. The CATT/T. evansi was recently evaluated for the
detection of Trypanosoma evansi infection in water buffaloes (Bubalus bubalis) in Egypt and showed positive results as early as eight days post infection (Hilali et al., 2004). False negative results (parasitologically positive camels negative by the antibody detection test) were reported by Ngaira et al., 2004 in Kenya. Those were correlated to the absence of the RoTat 1.2 VSG gene from some T. evansi
trypanosomes of the area.
1.2.4.4. Antigen-detecting tests
These tests have been developed for the detection of circulating trypanosomal antigens but are not reliable (Eisler et al., 1998; Davison et al., 2000). Consideration of why the Ag-ELISA fails to detect trypanosomal antigen(s) in serum samples is worthwhile and must take into account the following five factors: (1) the reactivity of the reagents (number of available epitopes of the antigenic target); (2) the specificity
an understanding of the fluctuations in trypanosomes in the systemic circulation (Rebeski et al., 1999).
1.2.4.5. Polymerase Chain Reaction and Restriction Fragment Length Polymorphism
Molecular biology provides tools for sensitive and specific diagnosis based on DNA sequence recognition and amplification. The polymerase chain reaction (PCR) permits identification of parasites at levels far below the detection limit of the commonly used parasitological techniques. PCR assays for trypanosome detection have been
developed (Kukla et al., 1987; Moser et al., 1989; Desquesnes and Tresse, 1996; de Almeida et al. 1997) using species specific DNA hybridisation probes (Majiwa et al., 1985; Majiwa and Webster, 1987). This method requires either prior knowledge of the species to be found or the use of several probes for each sample to be tested. A 'pan- Trypanosoma' test based on the ITS 1 region of the ribosomal genes has been recently described by Desquesnes et al. (2001) replacing the various PCR with a single assay.
The authors concluded in their paper that the test is not applicable to the field situation as it lacks sensitivity especially for T. vivax.
Hence the need for an easy universal and sensitive test to identify trypanosome infection in field samples coupled with species differentiation is still highly desirable.
The association of PCR with Restriction Fragment Length Polymorphism (RFLP) provides an elegant, sensitive and specific tool to meet this demand. The 18S
ribosomal subunit is an ideal target sequence as it is a mosaic of highly conserved and species specific sequences, present as a multi-copy locus. Albeit PCR and RFLP need a well equipped laboratory, the collection of field samples on filter paper is very simple and the sample transportation and conservation easy. This in association with the ease of the technique makes PCR-RFLP an important diagnostic tool well suited for large scale surveys.
1.2.4.6. Proteomic signature analysis
A novel test that can diagnose human African trypanosomosis with high sensitivity and specificity is reported by Papadopoulos et al. (2004) and seems very promising for humans but is too sophisticated and expensive to be used at present in veterinary medicine. The technique reveals the presence of a characteristic protein marker for individuals known to have the disease even before high levels of the pathogen are
individuals and compared them with blood samples from 146 patients who had other parasitic or non-parasitic infections. Roughly half the samples were used to calibrate the diagnostic test; the other half served as the main research sample. Mass
spectrometry (analysis at 2-100kDa) produced distinct proteomic signatures that were clearly characteristic of human African trypanosomosis and as such the authors claim 100% sensitivity and a specificity of 98.6%.
Resistance against trypanocidal drugs
1.3. Strategies for trypanocidal drug usage
1.3.1. Routine block treatments
These are generally carried out using prophylactic drugs, notably isometamidium chloride, at predetermined intervals based on the perceived duration of prophylaxis (0,5 to 1 mg/kg BW, intramuscularly). All animals in a herd may be treated or treatment may be targeted at a particular group of valuable or at risk animals. When routine block treatment with isometamidium is practised, it is recommended that once a year, the animals are separately treated with diminazene in order to delay the
development of drug resistance following the concept of sanative pair (Whiteside, 1962).
1.3.2. Strategic block treatments
These are generally carried out using prophylactic drugs, though curative drugs may also be used. All animals in a herd, or particularly valuable or at risk stock, are treated when challenge (as measured by the number of animals succumbing to infection) reaches a predetermined threshold.
1.3.4. Monitoring and treatment of individual infected animals
Cattle are monitored using standard parasitological methods. Treatment of infected animals is generally conducted using a therapeutic drug, usually diminazene aceturate (3,5 to 7 mg/kg BW, intramuscularly) .
1.3.5. Monitoring and treatment of clinical cases
This is similar to monitoring and treatment of individual infected animals but not all infected animals are treated. Cattle are treated usually with a curative trypanocide, only if the PCV falls below a predetermined threshold or if clinical signs of
trypanosomose are detected. The rationale for this strategy is the belief that cattle may acquire resistance to locally circulating strains of trypanosomes, and that treatment of animals with suclinical infections is unnecessary and may interfere with the process.
1.4. Resistance against trypanocidal drugs
1.4.1. Possible strategies for drug resistance
Cells under drug pressure tend to develop different strategies with a single and ultimate goal: survive (Figure 3). Some of these mechanisms, such as loss of a cell surface receptor or transporter for a drug, specific metabolism of a drug, or alteration by mutation of the specific target of a drug, result in resistance to only a small number of related drugs. In such cases, use of multiple drugs with different mechanisms of entry into cells and different cellular targets allows for effective chemotherapy and high cure rates. All too often, however, cells express mechanisms of resistance that confer simultaneous resistance to many different structurally and functionally unrelated drugs. This phenomenon, known as multidrug resistance, can result from changes that limit accumulation of drugs within cells by limiting uptake or enhancing efflux, activate a general response mechanism that detoxify drugs and/or repair damage caused by certain drugs to DNA, all those mechanisms rendering cells relatively resistant to the cytotoxic effects (Gottesman, 2002).
Figure 3 Summary of possible strategies of cells to develop resistance to drugs. (Gottesman, 2002)
1.4.2. Chemotherapy of animal trypanosomosis
Drugs currently recommended for chemotherapy of animal trypanosomosis belong to only three closely related groups. These are the two phenanthridines isometamidium (Figure 4) and homidium (Figure 6) and the aromatic diamidine, diminazene (Figure 7). Induction of cross resistance is possible between the two phenanthridines but not between phenanthridines and diamidines. (Whiteside, 1960).
Figure 4 Molecular structure of isometamidium
Quinapyramine (Figure 5), a trypanocide widely used in cattle in Africa during the period 1950-1970, was withdrawn from sale in 1976 because of problems with toxicity and resistance development. However, it is still available for use in camels and it is likely that it is still used in cattle in some situations in Africa where both species exist in the proximity to the margins of the tsetse belts. Its use in cattle should absolutely be banned because quinapyramine confers quickly resistance to both diamidines and phenanthridines.
Figure 5 Molecular structure of quinapyramine
Figure 6 Molecular structure of homidium
Figure 7 Molecular structure of diminazene aceturate
Diminazene, isometamidium and the homidium salts have been in use for more than 40 years and it is estimated that about 35 million doses per year are currently used in Africa. These drugs remain popular with livestock owners and veterinarians because they are generally affordable and effective. There is no indication that new products will become available in the near future even if good candidates have been identified (Hoet et al., 2004; Olbrich et al., 2004; Sturk et al., 2004; Lanteri et al., 2004) with, among others, five new diamidines exhibiting excellent in vivo activity in the trypanosomal STIB900 mouse model (Ismail et al., 2004). Altough there is a
consistent demand for trypanocides by African farmers, the total value of the market (about 24 million €) is not considered sufficient to justify investment by large pharmaceutical companies in the development and licensing of new animal
trypanocides, the costs of which may exceed 200 million € for a single compound. It is therefore of utmost importance that measures are taken to avoid or delay the
development of resistance and to maintain the efficacy of the currently available drugs (Holmes et al., 2004).
1.4.3. Isometamidium resistance
The amphiphilic cationic phenanthridine, isometamidium chloride, known as 8- [ (m- amidinophenyl-azo) amino]-3-amino-5-ethyl-6-phenylphenanthridinium chloride hydrochloride (C28H25ClN7HCl; MW: 531.5), has been used in the field for several decades in the treatment of livestock suffering from trypanosomosis due to infection with Trypanosoma congolense (Leach et al., 1981). Isometamidium chloride is widely available to livestock keepers through different marketing networks, since the
liberalisation of veterinary services in a number of African countries (Delespaux,
2004). The authenticity of the resistance phenotype in some of these field isolates of trypanosomes has been confirmed by in vivo testing of individual clones derived from the isolates (Codija et al., 1993; Zhang et al., 1993; Sinyangwe et al., 2004). The therapeutic failure is thought to arise inter alia as a consequence of underdosage of the infected animals with the trypanocides and could induce acquired resistance
(Delespaux et al., 2002). In order to avoid the spread of this phenomenon, which is an additional threat to the already poor livestock industry in Africa, an effective
management of trypanocidal drugs is essential (Geerts and Holmes, 1998; Holmes et al., 2004).
Biological effects of isometamidium were firstly described by a team of researchers in cancerology, Henderson et al., 1977 who observed that isometamidium induced the breakdown of intracellular adenosine triphosphate in Ehrlich ascites tumor cells incubated in vitro. In cells treated with isometamidium, purine nucleoside monophosphates accumulate.
In 1990, Shapiro and Englund suggested that the main mode of action of
isometamidium chloride was the cleavage of kDNA-topoisomerase complexes. The same progressive network shrinking effect was observed when silencing the
mitochondrial topoisomerase II by RNA interference by Wang et al. (2000) and by Wang and Englund (2001) or by the use of specific topoisomerase II inhibitors (Cavalcanti et al., 2003; Meresse et al., 2004). Topoisomerase II-targeting drugs are very effective to kill cells with a high rate of division as it is the case for tumoral cells (Lin et al., 2001) as well as for trypanosomes or Leishmania (De Sousa et al., 2003;
Hanke et al., 2003). Parasite mitochondria are promising targets for chemotherapy of multiple parasites as the mitochondrion goes through deep modifications in
morphology and components throughout the life cycle (Kita et al., 2001; Das et al., 2004). The toxic effect of isometamidium chloride within the kinetoplast was supported by Wilkes and Peregrine (1995) who showed that the trypanosome kinetoplast is the primary site of isometamidium chloride accumulation. This was later confirmed for the hemoflagellate Cryptobia salmositica (Kinetoplastida, bodonina) by Ardelli (2001) and by Woo (2003) and for T. brucei by Boibessot (2002) using more sophisticated chromatographical and microscopical techniques.
kinetoplast than ethidium bromide which was more diffuse within the trypanosome.
The mechanism of resistance to isometamidium chloride, however, is less clear.
Decreased levels of drug accumulation have been observed in drug-resistant
populations of T. congolense (Sutherland et al., 1991) and later work found indirect evidence of an increased efflux of drug from resistant trypanosomes (Sutherland and Holmes, 1993). More recently, Mulugeta et al. (1997) showed that the maximal uptake rates (Vmax) of isometamidium chloride in resistant T. congolense was significantly lower than in sensitive populations. It appears that isometamidium chloride freely crosses the plasma membrane, probably by facilitated diffusion and is then actively accumulated within the kinetoplast using the mitochondrial potential as a driving force. When resistant T.congolense are placed in an isometamidium chloride free environment the isometamidium chloride rapidly diffuses from the trypanosome in the isometamidium chloride free medium. The same phenomenon occurs in sensitive T.congolense where free isometamidium chloride rapidly diffuses in the isometamidium chloride free medium but a large proportion of the drug remains concentrated, sequestered in the kinetoplast (Wilkes et al., 1997). The same
phenomenon was observed with pentamidine in Leishmania Mexicana by Basselin et al., (2002). Changes in mitochondrial electrical potential have been demonstrated in isometamidium chloride resistant T. congolense by Wilkes et al. (1997) who showed a decreased mitochondrial electrical potential in resistant T.congolense.
Microscopically, the effect of isometamidium chloride on the kinetoplast is obviously different when considering a sensitive or a resistant T.congolense strain. Figure 8 shows a Giemsa stained smear of T.congolense in absence of isometamidium. The unique kinetoplast is well visible at the opposite side of the flagellum of the trypanosome.
Figure 8 Giemsa stained T.congolense sampled from a mouse before treatment with isometamidium chloride.
Figure 9 shows the effect of 10mg/kg of isometamidium chloride on a sensitive trypanosome (IL1180) 24 hours after the treatment. The kinetoplast has completely disappeared and cannot be distinguished in the cytoplasm anymore.
Figure 9 Giemsa stained sensitive T.congolense IL1180 sampled from a mouse 24 hours after treatment with 10mg/kgisometamidium chloride.
Figure 10 shows the effect of 10mg/kg of isometamidium chloride on a resistant trypanosome (TRT57c1) 24 hours after the treatment: several kinetoplast-like structure are well visible. Hammarton et al. (2003) describe the same kind of
phenomenon in T. brucei with one nucleus and multiple kinetoplasts when a mitotic block is induced by RNA interference which inhibits the mRNAs of the CYC6 gene (mitotic cyclin gene). A similar action could be induced in the resistant T.congolense but was never reported in the literature.
Figure 10 Giemsa stained resistant T.congolense TRT57c1 sampled from a mouse 24 hours after treatment with 10mg/kg isometamidium chloride.
It should also be noticed here that dyskinetoplastic T.evansi and T.equiperdum (cultured material) have been reported susceptible to isometamidium chloride
indicating that in those trypanosome species, isometamidium chloride seems to exert its trypanocidal activity by interfering with other essential target(s) that remain to be elucidated (Kaminsky et al., 1997, Schnaufer et al., 2002).
Although contradictory observations have been reported on the genetic stability of isometamidium chloride resistance, field observations in Ethiopia, based on cloned populations, showed that the drug-resistant phenotype of T. congolense had not altered over a period of four years (Mulugeta et al., 1997).
1.4.4. Diminazene resistance
Although diminazene probably exerts its action at the level of the kinetoplast DNA, this has not been proven in vivo, and other mechanisms of action cannot be excluded (Peregrine and Mamman, 1993). Berger et al. (1995) showed that the accumulation of diminazene was markedly reduced in arsenical-resistant T. brucei owing to alterations in the nucleoside transporter system (P2). Increased resistance to diminazene was also observed in P2 deficient mutant of T. brucei (Matovu, 2003) and recently, RNA- interference silencing the adenosine transporter-1 gene in Trypanosoma evansi confered resistance to diminazene aceturate (Witola et al., 2004). Those results are
Similarly to isometamidium chloride, contradictory reports have also been published on the stability of resistance to diminazene. Mulugeta et al. (1997), however, showed that the phenotype of multiple drug-resistant (including diminazene) T. congolense remained stable over a period of four years.
In conclusion, it is clear that much more work is required in order to elucidate the mechanism of resistance to the three currently available trypanocidal drugs. Such studies, as well as being of great value in their own right, may also provide novel methods for the detection of drug-resistant trypanosomes in the future. The same is true for the genetics of drug resistance in trypanosomes. Hayes and Wolf (1990) distinguish three major types of genetic change that are responsible for acquired drug resistance: mutations or amplifications of specific genes directly involved in a
protective pathway; mutations in genes that regulate stress-response processes and lead to altered expression of large numbers of proteins; and gene transfer. Gene amplification under conditions of drug pressure is well known in Leishmania spp. and has also been demonstrated in trypanosomes, but until now there is no evidence that this occurs in the latter parasites as a mechanism of drug resistance under field conditions.
Diagnosis of trypanocidal resistance
1.5. Diagnosis of trypanocidal resistance
1.5.1. Tests in ruminants
The tests consist of infecting a group of cattle or small ruminants with the isolate under investigation and later, when the animals are parasitaemic, treating them with various dosages of trypanocide or following a simplified procedure with a single discriminatory dose as described by Eisler et al., 2001. The animals are then regularly monitored over a prolonged period (up to 100 days) to determine the effective dose (ED), i.e. the dose that clears the parasites from the circulation, and the curative dose (CD), i.e. the dose that provides a permanent cure (Sones et al., 1988). For these studies, the cattle or small ruminants must be kept in fly-proof accommodation or in a non-tsetse area in order to eliminate the risk of reinfection during the study. A
variation of this technique was used by Ainanshe et al.,(1992) in Somalia to examine a group of isolates from a district. Blood from a group of infected cattle was
inoculated into a single recipient calf, which was monitored, and later, when it was parasitaemic, treated with trypanocide at the recommended dose. A breakthrough infection, indicative that one of the inoculated trypanosome populations was drug- resistant, was inoculated into groups of calves and mice to determine the level of drug resistance. This technique is useful in situations where laboratory facilities are very limited but it only allows a qualitative assessment and does not indicate how many of the isolates inoculated into a single calf were resistant. Further constraints to this technique are that not all populations might grow equally well and that sensitive isolates might overgrow resistant ones when inoculated together (Sones et al., 1989).
However this is not a consistent observation (Burri et al., 1994). A useful indication of the level of resistance can be obtained from studies in ruminants (and mice) by recording the length of time between treatment and the detection of breakthrough populations of trypanosomes: the shorter the period, the greater the level of resistance (Ainanshe et al., 1992). The advantages of studies in ruminants are that most
trypanosome isolates of cattle are able to grow in these hosts and that the data obtained are directly applicable to the field. The disadvantages are the long duration (a follow-up of 100 days is necessary to allow the detection of relapses) and the cost (purchase and maintenance of the animals are expensive). Furthermore, if only one
isolate per animal is tested, it is usually impractical and too expensive to examine a large number of isolates.
1.5.2. Tests in mice
After expansion of an isolate in a donor mouse, groups of five or six mice are inoculated with trypanosomes. Twenty-four hours later, or at the first peak of parasitaemia, each group except the control group is treated with a range of drug doses. Thereafter, the mice should be monitored three times a week for 60 days. The ED50 or ED95 (the effective dose that gives temporary clearance of the parasites in 50 or 95 percent of the animals, respectively) can be calculated, as can the CD50 or CD95 (the curative dose that gives complete cure in 50 or 95 percent of the animals, respectively). Sones, Njogu and Holmes (1988) used groups of five mice, which allowed an easy calculation of ED80 and CD80 values (one out of five mice was not cleared or cured). These figures should be compared with those obtained using
reference-sensitive trypanosome strains. The advantage of the mouse assay is that it is cheaper than the test in cattle. There are several disadvantages, however:
1) Most T. vivax isolates, and also some T. congolense isolates, do not grow in mice.
2) Although there is reasonable correlation between drug sensitivity data in mice and in cattle, higher doses of drug must be used in mice (normally ten times higher) in order to obtain comparable results to those obtained in cattle because of the vast difference in metabolic size (Sones, Njogu and Holmes, 1988). However the extrapolation from mice to cattle is not always evident (Kone, 1999).
3) Precise assessment of the degree of resistance needs a large number of mice per isolate, which makes it a labour-intensive test. A discriminatory dose, above which an isolate should be considered as resistant of 1mg/kg for isometamidium and 20 mg/kg for diminazene was determined by Eisler et al. in 2001.
4) It takes as long as 60 days to evaluate the drug sensitivity of an isolate.
1.5.3. In vitro assays
Since the review of Kaminsky and Brun (1993) further progress has been made in the field of in vitro assays to determine the drug sensitivity of trypanosomes. These authors advised the use of metacyclic or bloodstream forms instead of procyclic forms in such assays. Procyclic forms are easier to grow in vitro but the comparison of drug sensitivity tests performed with procyclic forms and with metacyclic or bloodstream forms is not obvious (Elrayah et al.,1991). However, it takes up to 40 to 50 days of in vitro incubation to generate metacyclic trypanosomes (Gray et al., 1993). The
advantage of this technique is that large numbers of isolates can be examined; tests with metacyclic trypanosomes correlate well with field observations. However there are several disadvantages. In vitro cultivation of bloodstream forms is only possible using preadapted lines and not using 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 of adaptation is the possible selection against trypanosomes that have the phenotype of the original population. In vitro assays are expensive to perform and require good laboratory facilities and well- trained staff. If better techniques can be developed in order to adapt isolates more rapidly to grow in vitro, these assays may become more popular, especially in those laboratories where culture facilities are already established.
1.5.4. Trypanocidal drug ELISAs
As an alternative to the tests mentioned above, the use of trypanocidal drug enzyme- linked immunosorbent assays (ELISAs) in combination with parasite detection tests has given promising results for the detection of resistant trypanosomes. A competitive ELISA which allowed the detection of small amounts of isometamidium in serum of cattle was first described by Whitelaw et al. (1991). This technique was further improved by Eisler et al. (1993) and Eisler, Elliott and Holmes (1996) and has been validated in cattle under experimental and field conditions (Eisler, 1996; Eisler et al., 1994; 1996; 1997a). The test is both sensitive, detecting subnanogramme
concentrations, and specific. It allows the monitoring of drug concentrations in the
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