ANIMAL TRYPANOSOMOSIS: DIAGNOSIS AND GENETICS

TRYPANOSOMOSE ANIMALE: DIAGNOSTIC ET GENETIQUE

COMPARATIVE ANALYSIS OF TWO ISOGENIC CLONES OF TRYPANOSOMA CONGOLENSE THROUGH AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) AS A PUTATIVE GENETIC MARKER FOR ISOMETAMEDIUM RESISTANCE

MISE EN EVIDENCE D'UN MARQUEUR GENETIQUE DE RESISTANCE A L'ISOMETAMIDIUM PAR L'ANALYSE COMPARATIVE DE DEUX CLONES ISOGENIQUES DE TRYPANOSOMA CONGOLENSE PAR AFLP (AMPLIFIED FRAGMENT LENGTH POLYMORPHISM)

V. Delespaux*^,1, D. Geysen¹, Phelix A.O. Majiwa² and S. Geerts¹.

¹Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.

²International Livestock Research Institute, (ILRI) P. O. Box 30709, Old Naivasha Road, Nairobi 00100, Kenya.

Corresponding author: Tel.: +32-3-247 62 62; fax: +32-3-2476268.

E-mail address: vdelespaux@itg.be (Vincent Delespaux).

Résumé

La technique de l'AFLP a été utilisée pour comparer deux clones isogéniques de Trypanosoma congolense. La souche parentale, sensible à l'isométamidium, présente une CD50 de 0,018 mg/kg lors de tests sur souris, et le clone dérivé qui a été soumis à des doses croissantes d'isométamidium présente une CD50 94 fois supérieure. Pour l'analyse AFLP, 64 combinaisons de huit amorces EcoR I et de huit amorces Mse I ont été utilisées pour la détection des différences génomiques entre les deux clones. Cinquante-huit fragments polymorphiques d'ADN présents dans le clone résistant ont été isolés, purifiés et séquencés. Un de ces fragments présente la signature d'une ferrédoxine 4Fe-4S ( [C]-x-x-[C]-x-x-[C]-x-x-x-[C] ). Les ferrédoxines pourraient donc être impliquées dans la résistance de Trypanosoma congolense à l'isométamidium. A notre connaissance, il s’agit du premier rapport de l'utilisation de l'AFLP pour la comparaison de deux clones isogéniques de trypanosomes africains pathogènes pour le bétail.

Most-clés: Trypanosoma congolense, AFLP, chimiorésistance, isométamidium, ferrédoxine,

déhydrase NADH

Summary

Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD₅₀ in mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD ₅₀ that is 94-fold higher. Sixty-four combinations of eight EcoR I and eight Mse I primers were used in AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were present only in the resistant clone were purified and then sequenced. One of the fragments presents a 4Fe-4S-ferredoxin consensus pattern ([C]-x-x-[C]-x-x-[C]-x-x-x-[C]) and appears to be very similar to the subunit 8 of the NADH dehydrogenase of the Euglenozoa class. Ferredoxins might thus play a role in the resistance of Trypanosoma congolense to isometamidium chloride. To the best of our knowledge, this is the first report of AFLP fingerprinting of isogenic clones of African trypanosomes pathogenic to livestock

Keywords: Trypanosoma congolense, AFLP, drug resistance, isometamidium, ferredoxin, NADH dehydrogenase.

Introduction

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 [1]. Isometamidium chloride is widely available to livestock keepers through different marketing networks, since the liberalisation of veterinary services in a number of African countries [2], [3]. Trypanosome strains showing evidence of natural resistance to this drug have been found in many different countries [4], [5], [6], [7]. The authenticity of the resistance phenotype in these field isolates of trypanosomes has been confirmed by in vivo testing of individual clones derived from the isolates [8], [9]. The resistance is thought to arise inter alia as a consequence of underdosage of the infected animals with the trypanocides [10]. 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 [6].

The diagnosis of trypanocidal resistance has been simplified and standardised [11] but the test must still be performed either in mice or in cattle. The tests can be conducted also after growing the trypanosomes in vitro. Since both in vivo and in vitro tests for the detection of trypanocidal drug resistance are laborious and time consuming, new diagnostic methods for the detection of drug resistance are urgently needed. A polymerase chain reaction (PCR)-based test could provide a rapid and convenient tool, suitable for large-scale herds’ surveys of livestock. The development of such a test requires the identification of genetic mutations associated with isometamidium resistance in livestock-infective trypanosomes.

The trypanosomes infective only to livestock in Africa have not been as well studied at the molecular level as those that are infective to humans. Consequently, little is known about the genome of T.congolense, the trypanosome in which trypanocide resistance has been well documented. To detect genetic alterations that may be associated with resistance to isometamidium chloride, the method to be used should require no prior knowledge of the sequences of any specific genes that may be involved.

Amplified fragment-length polymorphism (AFLP) [12], [13] is a fingerprinting technology that is based on the selective amplification of a subset of genomic restriction fragments using PCR. Deoxyribonucleic acid (DNA) is first digested with restriction enzymes selected on the basis of the expected genomic complexity of the organism to be analysed. The restriction enzymes used in the analysis will influence the number of bands detectable in the fingerprint, depending on the occurrence of their respective recognition sequences. AFLP technology requires no sequence information or probe collections prior to the generation of the fingerprints. This is of particular benefit when studying organisms where little DNA sequence information is available in the existing databases as is the case for T. congolense. AFLP is widely used in plant genetics but was only recently used for genetic analysis of population relatedness [14],. [15] to study trypanosome. In this study, AFLP was used to compare the genome of two isogenic clones of T.congolense, in order to search for mutation(s) that might be responsible for resistance to isometamidium chloride.

Materials and methods

2.1. Trypanosomes and DNA samples

The trypanosomes used in the study were isogenic clones of a savannah type T. congolense derived one from the other under laboratory conditions as has been described [16]. The parent clone IL1180 has a CD₅₀ in mouse of 0.018 mg/kg [18] and the derivative isogenic clone IL3343 has a CD₅₀ of 1.7 mg/kg [16]. Thus the resistant clone IL3343 tolerates 94-fold higher levels of isometamidium chloride, compared to IL1180 (ILNat 3.1, [17]) from which it was derived. Both IL1180 and IL3343 were shown to have different kinetics of uptake of the drug [18], [19], which correlated with their drug resistance phenotype.

Trypanosomes were grown in mature rats by injecting approximately 10⁵ trypanosomes intra-peritoneally into each rat. At the first peak of parasitaemia, the rats were euthanised, and the blood collected with anticoagulant. The trypanosomes were separated from the blood elements by centrifugation in Percoll gradients [20] followed by chromatography on a column of DE-52 [21]. The purified trypanosomes were washed with 15 ml of phosphate buffered saline-glucose (PSG) pH 8.0, and then pelleted by centrifugation. The pellet was used immediately for the preparation of nucleic acids using routine procedures [22].

2.2. AFLP

AFLP was performed using the commercial kit AFLP Analysis System II ® (Gibco BRL – Life Technologies, cat no 10717-015) according to the instructions of the supplier with the standard EcoR I and Mse I restriction enzymes. The kit includes 8 EcoR I primers containing a common segment (5’GAC TGC GTA CCA ATT C) combined with 8 different selective nucleotide sequences (AA, AC, AG, AT, TA, TC, TG, TT) and 8 Mse I primers containing a common segment (5’GAT GAG TCC TGA GTA A) with 8 selective nucleotide sequences (CAA, CAC, CAG, CAT, CTA, CTC, CTG, CTT). Primers used in the selective AFLP amplification were labelled with [γ^-33P] ATP.

The sixty-four possible primer combinations were used in the selective amplification of DNA from the trypanosomes.

2.3. Polyacrylamide Gel Electrophoresis (PAGE)

After PCR amplification, an equal volume (20µl) of formamide dye (95% formamide, 10mM NaOH, 0.05% bromophenol blue, 0.05% Xylene cyanol) was added to each reaction. The samples were heated at 95°C for 5 minutes and immediately chilled on ice. They were subsequently resolved by electrophoresis under denaturing conditions in a 6% polyacrylamide gel. After electrophoresis, the gel was fixed for 5 minutes in a solution containing 80% distilled water, 10% methanol and 10% acetic acid then transferred to a filter paper and vacuum-dried for two hours. The filter paper was exposed to a x-ray film (Kodak® SB) for 72 hours at room temperature, for autoradiography.

2.4. DNA extraction from the polyacrylamide gel

A portion of filter paper containing the DNA fragment of interest was cut out with a scalpel blade and placed in an Eppendorf tube with 500µl milliQ water, then gently agitated for 30 minutes at 56°C to elute the DNA from the paper.

2.5. PCR amplification of the purified fragments of DNA

Standard PCR amplifications were conducted with 5 µl DNA eluate in 20 µl solution containing 50mM KCl, 10mM Tris-HCl (pH 8.3), 1.5mM MgCl₂, 200µM of each dNTP, 20 pmol of each primer and 0.5 U Taq polymerase (Goldstar, Eurogentec). The reaction mixture was overlaid with 50-µl fine neutral mineral oil (Sigma) and placed in a heating block of a programmable thermocycler (PTC-100 TM, M.J. Research Inc.). After a denaturation step of 4 minutes at 94°C, each of the 40 cycles consisted of sequential steps of 60sec at 94°C, 90sec at 56°C and 120sec at 72°C. A 5µl volume of each sample was electrophoresed in a 2% agarose gel for 20min and stained with ethidium bromide for 30min before photography.

2.6. Cloning and sequencing

The PCR products were cloned using Topo-cloning® kit (Invitrogen, Carlsbad CA, USA), exactly as described by the manufacturer. The recombinant plasmids containing the desired inserts were purified then completely sequenced using the Model 377-XL Sequencer (PE-Applied Biosystems, Eurogentec® Belgium).

3. Results

3.1. The isogenic clones are largely similar

The patterns of DNA fragments generated by the AFLP analysis of the isogenic clones IL1180 and IL3343 strongly indicate that they have similar fingerprints (Figure 1a). Our current interest is in those fragments appearing only in the DNA from the resistant clone. The use of the 64 possible combinations of the 8 EcoR I and the 8 Mse I primers produced at least 58 bands present only in the DNA of the resistant clone (Table 1). There were other bands than the 58 but some of these were either too faint or too close to each other, making it impractical to recover each one separately from the polyacrylamide gel.

Table.1 Number of AFLP bands observed only in the DNA of the resistant isogenic clone of T.congolense using 64 combinations of EcoR I and Mse I primers.

	M-CTG	M-CTC	M-CTA	M-CAT	M-CAG	M-CAC	M-CAA	M-CTT
E-TT	4	-	2	2	2	3	1	-
E-AA	-	1	1	1	1	1	-	3
E-AC	-	1	-	-	-	-	-	-
E-AG	-	1	2	-	2	-	2	-
E-AT	-	-	-	-	-	-	-	-
E-TA	1	-	5	-	2	2	3	1
E-TC	-	-	-	-	1	-	-	1
E-TG	4	1	1	-	1	2	2	1

With E for EcoR I primer and the two letters corresponding to the specific portion of the primer and M for Mse I primer and the three letters corresponding to the specific portion of the primer.

Figure 1a. AFLP fingerprint (detail) of T.congolense IL1180, sensitive to isometamidium chloride, in lanes labelled (S) and a derived clone IL3343, resistant to isometamidium chloride, in lanes labelled (R). C⁺ stands for DNA positive control. EcoR I-TT and Mse I-CTG primers in lane 1, EcoR I-TT and Mse I-CTC primers in lane 2, EcoR I-TT and Mse I-CTA primers in lane 3, EcoR I-TT and Mse I-CAT primers in lane 4, EcoR I-TT and Mse I-CAG primers in lane 5.

Some primer combinations did not show any difference in AFLP fingerprints of DNA from the two trypanosomes however, other primer combinations gave 1-5 well-separated fragments only in the DNA of the resistan clone (Table 1). In Figure 1b an example is shown of AFLP profiles obtained with 5 different primer combinations. A close-up of a portion of this autoradiograph is shown in Figure 1b. Plain arrows point to some of the fragments that were recovered, amplified by PCR, cloned and sequenced. The DNA was successfully extracted from the polyacrylamide gel for 35 of the 58 bands. The AFLP fragments varied in size from 28 bp to 341 bp.

Figure 1b. Close-up from Figure 1a, lane 1 with EcoR I-TT and Mse I-CTG primers. Plain arrows show bands that were extracted from the polyacrylamide gel for sequencing. Empty arrows show bands that were not further analysed