Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis

International Journal for Parasitology 32 (2002) 53–63 www.parasitology-online.com

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis q R.M. Ventura a, G.F. Takeda a, R.A.M.S. Silva b, V.L.B. Nunes c, G.A Buck d, M.M.G. Teixeira a,* a

Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 1374, 05508-900, Sao Paulo, SP, Brazil b Laboratory of Animal Health, Embrapa, Centro Nacional de Pesquisa de Suı´nos e Aves, SC, Brazil c Universidade para o desenvolvimento do Estado e da Regia˜o do Pantanal, MS, Brazil d Department of Microbiology, Medical College of Virginia Commonwealth University, Richmond, VA 23298-0678, USA Received 11 July 2001; received in revised form 3 September 2001; accepted 10 September 2001

Abstract In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Trypanosoma evansi; South American trypanosomiasis; Surra disease; Randomly amplified polymorphic DNA; Polymerase chain reaction; Diagnosis

1. Introduction Trypanosoma evansi is a species of the subgenus Trypanozoon, Salivaria, with wider geographical distribution and mammalian host range than any other pathogenic species of Trypanosoma. Unlike Trypanosoma brucei, which is cyclically transmitted by tsetse flies, T. evansi is transmitted mechanically by blood-sucking insects. Trypanosoma evansi and Trypanosoma equiperdum, although non-tsetse transmitted and not restricted to Africa, are tightly clustered into the T. brucei-clade, and are considered to be recent derivatives of T. brucei which have been able to spread outside Africa. The T. brucei-clade is comprised of highly q Nucleotide sequence of the Te664 DNA fragment is available in the GenBank database under the accession number AF397194. * Corresponding author. Fax: 155-11-3818-7417. E-mail address: [email protected] (M.M.G. Teixeira).

phylogenetically related organisms well separated from other trypanosome species (Stevens et al., 1999, 2001). Although all members of this clade share morphological and genetic similarities, they are considered separate species based on the absence (T. evansi) or deletion (T. equiperdum) of the kDNA maxicircle molecules, and on differences in both their routes of transmission and pathology (Gardiner and Mahmoud, 1992). Trypanosoma evansi is widespread outside the tsetse belt in Africa, the Middle East, Asia and Latin America. In South America, T. evansi is transmitted by flies and vampire bats that bite and is found in Venezuela, Colombia, Argentina, Bolivia and Brazil. This parasite causes a wasting disease named Surra in Africa and ‘Mal de Cadeiras’ in Brazil (Hoare, 1972). Trypanosoma evansi was first reported in Brazil in 1839 in horses on the island of Marajo´, Para´ State, Northern region, and today is reported only in the

0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(01)00314-9

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Pantanal Region, a very important livestock region in the centre of Brazil (Shaw, 1977; Silva et al., 1995; Queiroz et al., 2000; Ventura et al., 2000). Many species of both domestic and wild animals are susceptible to infection with T. evansi, with symptoms ranging from acute and fatal to chronic and asymptomatic diseases. Chronic disease leading to general wasting is very common in camels, whereas horses show an acute and fatal disease and buffaloes and cattle exhibit a chronic disease without clinical signs (Gardiner and Mahmoud, 1992). American stocks of T. evansi are fatal to horses and dogs, whereas cattle and wild mammals such as coatis and capybaras rarely exhibit the characteristic clinical symptoms of the disease despite easy detectable parasitaemias (Nunes and Oshiro, 1990; Nunes et al., 1993; Franke et al., 1994a,b; Silva et al., 1995). Thus, wild animals and cattle may serve as reservoirs of T. evansi, maintaining an enzootic stability of infection in Pantanal, Brazil. In South America, Trypanosoma vivax is the most important pathogen of cattle. In contrast to the limited diversity existent among trypanosome species infecting horses and cattle, Trypanosoma cruzi and several species of the Megatrypanum and Herpetosoma subgenera can also be harboured by the wild hosts of T. evansi, commonly as mixed infections. The most reliable method for the diagnosis of T. evansi is still based on parasite identification in blood smears or by microhaematocrit and buffy-coat methods. However, besides the low sensitivity of these methods, their specificity depends on accurate morphological identification of T. evansi (Gardiner and Mahmoud, 1992). Diagnosis of T. evansi based on antibody detection is hampered by the inability to discriminate between prior and current infection, by antigenic variation and by cross-reactivity with other Trypanosoma spp. To avoid these problems, sensitive methods based on antigen detection have been used (OlahoMukani et al., 1993; Monzon et al., 1995), although these can still produce false positive results (Kashiwasaki et al., 1998). Hybridisation with probes based on repetitive DNA sequences of T. evansi (Viseshakul and Panyim, 1990) requires a large number of parasites. Most polymerase chain reaction (PCR) methods proposed for detection of T. evansi could not distinguish this species from T. brucei (Wuyts et al., 1995; Ijaz et al., 1998). Kinetoplast DNA minicircle based PCR can distinguish these trypanosomes (Masiga and Gibson, 1990), but is unsuitable for detection of akinetoplastic stocks. Moreover, none of these methods have been evaluated regarding cross-reactivity with Stercorarian species. Therefore, we still lack a specific, sensitive and simple diagnostic method, suitable for epidemiological purposes for all T. evansi stocks. Such a test is particularly important in Latin America, where mixed infections with Salivarian and Stercorarian species are common in both domestic and wild hosts. Molecular characterisation of T. evansi suggested that this species became homogeneous probably due to its inability to undergo cyclical development in the tsetse fly

where genetic exchange occurs (Gibson and Stevens, 1999). Natural populations of T. evansi infecting different hosts in distant countries did not present significant genetic variability when submitted to zymodeme analysis (Gibson et al., 1980; Boid, 1988; Godfrey et al., 1990). Isoenzyme comparison of Brazilian T. evansi stocks showed that they are very closely related to one another as well as to Old World stocks (Stevens et al., 1989; Godfrey et al., 1990; Queiroz et al., 2000). Likewise, kDNA minicircle sequences have shown only microheterogeneity among T. evansi isolates (Borst et al., 1987; Songa et al., 1990; Ou et al., 1991; Lun et al., 1992). The akinetoplasty of Brazilian stocks precludes investigation of minicircle variability (Ventura et al., 2000). The random amplification of polymorphic DNA (RAPD) is a highly sensitive method currently used to assess genetic relatedness and to discriminate very closely related species, subspecies and stocks of trypanosomes (Stevens and Tibayrenc, 1995; Tibayrenc, 1995; Kanmogne et al., 1996; Brisse et al., 2000). This method has also been helpful as source of DNA sequences useful as diagnostic and taxonomic markers for trypanosomatids (Noyes et al., 1996; Oury et al., 1997; Serrano et al., 1999). In this study we employed the RAPD method to analyse T. evansi stocks with the following aims: (a) to investigate the genetic relatedness of Brazilian T. evansi stocks by comparing them with one another and to Old World stocks of T. evansi, T. equiperdum and T. brucei spp.; (b) to assess the genetic polymorphism among Brazilian stocks from distinct domestic and wild host mammals showing marked differences in virulence for mice; (c) to compare stocks with (Old World stocks) or without (Brazilian stocks) kDNA minicircles; (d) to investigate a RAPDderived DNA sequence useful as taxonomic marker and for species-specific diagnosis of T. evansi.

2. Materials and methods 2.1. Organisms and blood samples from infected animals Trypanosoma evansi stocks and other Trypanosoma spp. used in this study are listed in Table 1. Trypanosoma evansi and T. brucei were recovered from mice and T. vivax (TviMi) was expanded in experimentally infected sheep as described (Ventura et al., 2000, 2001). Other trypanosomes were cultured at 288C in liver infusion tryptose (LIT) medium with 10% foetal calf serum. Blood samples from mice infected with T. evansi and T. brucei and containing a known number of parasites were collected as blood smears, dropped on filter paper and ,1.0 ml was also collected in microfuge tubes without anticoagulant. Blood samples from the same field-recovered animals were collected on both blood smears and filter paper. Blood smears were Giemsa-stained and the presence of trypanosomes was verified by microscopic investigation.

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Table 1 Randomly amplified polymorphic DNA and PCR amplification assays developed for the detection of Trypanosoma evansi using as template DNA either purified DNA or crude preparation of blood samples Trypanosoma

RAPD-Te615 template DNA

PCR-Te315 template DNA

pDNA a

Filter paper

Blood smears

pDNA

Filter paper

Blood smears

T. evansi stocks from blood of laboratory-infected mice Brazil Ted1, Ted2, Ted3, Ted4, Ted5 b Dog Tec1, Tec2 b Capybara Brazil Ter1 b Wild rat Brazil Horse Brazil Teh1, Teh2, Teh3 b Tect1, Tect2, Tect3 b Coati Brazil TeSH c Bovine China TeMA c Horse Philippines Camel Ethiopia TeET c TeC13 d Camel Kenya

1 1 1 1 1 1 1 1 ND

1 1 1 1 1 ND ND ND ND

1 1 1 1 1 ND ND ND 1

1 1 1 1 1 1 1 1 ND

1 1 1 1 1 ND ND ND ND

1 1 1 1 1 ND ND ND 1

Trypanosoma spp. T. equiperdum BoTat 1.1 e T. b. brucei 8195 b T. b. brucei 427 f T. b. brucei KP2 f T. b. brucei B8/18 f T. b. brucei LM184 f T. b. brucei AnTat 1.1 f T. b. gambiense (Types 1, 2) c T. b. gambiense TB26 f T. b. rhodesiense AnTat 1.12 c T. vivax Y486 c T. vivax TviMi g T. cruzi G b T. cruzi M226 b T. rangeli (San Augustin) b T. theileri (Th1) b T. theileri (Th2) b T. conorhini b

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

ND 2 ND ND ND ND ND ND ND ND ND 2 ND ND ND ND ND ND

ND 2 ND ND ND ND ND ND ND ND ND 2 ND ND ND 2 2 ND

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

ND 2 ND ND ND ND ND ND ND ND ND 2 ND ND ND ND ND ND

ND 2 ND ND ND ND ND ND ND ND ND 2 ND ND ND 2 2 ND

a b c d e f g

Host origin

Horse – Sheep Fly Pig Pig Antelope Man Pig Man Bovine Bovine Opossum Rodent Man Bovine Buffalo Rat

Geographic origin

– – Uganda Ivory Coast Nigeria Ivory Coast Uganda – Congo – Nigeria Brazil Brazil Brazil Colombia Brazil Brazil –

pDNA, purified DNA obtained by phenol–chloroform extraction. Cultures cryopreserved in the Trypanosomatidae Culture Collection (TCC) of the Department of Parasitology, USP, Brazil. pDNA donated by The´ o Baltz, University of Bordeaux, France. Infected mice blood smears on glass slides, donated by Wendy Gibson, Bristol University, UK. Sample of DNA from Institute Pasteur, Paris, France. pDNA donated by Wendy Gibson, Bristol University, UK. Isolated in Pantanal of Miranda, Brazil, cryopreserved in the TCC.

2.2. DNA preparation, RAPD analysis and T. evansispecific PCR-Te315 Genomic DNA from cultured parasites, from blood forms of T. evansi, T. brucei and T. vivax, and from blood of uninfected mouse, horse and cattle was prepared by phenol–chloroform extraction. DNA templates from blood smears on glass slides or from filter paper (Table 2) were prepared as described (Serrano et al., 1999; Ventura et al., 2001). Clotted blood samples were processed for DNA template preparation as described (Junqueira et al., 1996). For RAPD analysis of all T. evansi stocks, we selected eight primers which yielded the most discriminating patterns in the initial screening using 20 primers to amplify DNA from five T. evansi isolates. The selected primers generated well defined and reproducible DNA bands in three different reactions: 601 (5 0 CCG CCC ACT G 3 0 ),

606 (5 0 CGG TCG GCC A 3 0 ), 615 (5 0 CGT CGA GCG G 3 0 ), 625 (5 0 CCG CTG GAG C 3 0 ), 649 (5 0 AAT GCT GGA C 3 0 ), 657 (5 0 GTC CTT TAG C 3 0 ), 664 (5 0 GCC TGA AAA C 3 0 ) and ILO525 (5 0 CGG ACG TCG C 3 0 ). Amplifications were performed in reaction mixtures of 50 ml, using 50 ng of DNA, 2.5 units of Taq DNA polymerase (GIBCO/BRL), 0.2 mM each dATP, dCTP, dGTP, dTTP, PCR buffer containing 1.5 mM MgCl2 and 200 pM of primer. Reactions were cycled 29 times as follows: 1 min at 958C, 2 min at 378C and 2 min at 728C. In the first cycle, the time of denaturation at 958C was 3 min, and in the last cycle the time of extension at 728C was 10 min. Amplified products were separated on 2.0% agarose gel and stained with ethidium bromide (EtBr). A total of 180 reproducible DNA bands generated by all selected primers were used to produce a pairwise distance matrix using the Jaccard similarity index, which was employed to construct dendrograms

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by Neighbour-Joining (PHYLIP 3.5 package). Data were also analysed using the mix program of PHYLIP, based on the Wagner parsimony method. The SeQBoot program (PHYLIP 3.5 package) was used to generate 100 bootstrap replicates for assessment of the significance of the branching pattern in the Wagner network. The oligonucleotides Te664a (5 0 AAA CCC GTC CTC TTG GAG G 3 0 ) and Te664b (5 0 ATC CAT CTA AGA GTT GT 3 0 ) were designed for PCR amplification of a 315 bp DNA fragment (PCR-Te315) internal to the RAPD-Te664 sequence. PCR reactions were cycled 25 times and performed as above for RAPD, using 568C as primer annealing temperature and 50 ng of purified DNA or 5–20 ml of template recovered from glass slides or filter paper. 2.3. Sequencing and data analysis of RAPD-Te664 sequence The RAPD-Te664 amplified fragment of T. evansi Ted1 was cloned (pMOS Blue blunt ended cloning kit – Amersham Pharmacia) and three clones were sequenced in both directions by automated sequencing. Homology searches of the RAPD-Te664 sequence were performed using the BLAST program of NCBI. 2.4. Hybridisation analysis on Southern and slot-blots Slot-blots of total genomic DNA (3.0 mg) were prepared

on Nylon membranes (Hybond-N, Amersham) as described (Teixeira et al., 1996). The RAPD-Te664 amplified fragment of T. evansi Ted1 was labelled by ‘random primed synthesis’ with [a- 32P] dCTP (Du Pont 3000 Ci/mmol) using the ‘Ready to Go’ kit (Pharmacia) and used as probe. The oligonucleotide Te664a was 5 0 end-labelled with [g- 32P] dATP (3000 Ci/mmol; Amersham) using the 5 0 DNA terminus labelling system (GIBCO-BRL). Southern and slot-blots were prehybridised at 378C for 1 h in 2 £ standard saline citrate (SSC), 1% sodium dodecyl sulfate (SDS), 2.0 mM sodium pyrophosphate, 50% formamide and 40 mg/ml salmon sperm DNA and hybridised for 16– 18 h with Te664 probe at 428C in the same solution. Blots were also hybridised with Te664a probe at 408C in 3 £ SSC, 0.1% SDS, 4.0 mM sodium pyrophosphate and 40 mg/ml salmon sperm DNA. Blots of genomic DNA were washed once for 15 min at room temperature and twice for 15 min at 508C in 2 £ SSC, 0.1% SDS and 2.0 mM sodium pyrophosphate, or at 658C in 0.1 £ SSC containing 1% SDS. Southern and slot-blots containing RAPD and PCR products were washed three times at 658C with 0.1 £ SSC containing 1% SDS. 2.5. Analysis by pulsed field gel electrophoresis (PFGE) Chromosome blocks were prepared by embedding 10 7

Table 2 Diagnosis of Trypanosoma evansi using blood samples recovered directly from domestic and wild animals collected on filter paper a Blood sample

MI bGiemsa-stained blood smears

Amplification assays RAPD-Te615

Domestic animals Horses 1, 18 4–6, 12, 33 20, 36, 52, 78 75 2, 3, 7–10, 13–17, 19, 21–24, 27, 30, 41, 54, 60, 71, 72, 82–87 Dogs 1–4 Pig 1 Bovine 1, 2 Buffalo 1, 2 Wild mammals Capybaras 1–4 Coatis 1, 2 a b c d

PCR-Te315

EtBr c

Te664 probe EtBr

Te664a probe

1 2 2 2 2

1 1 2 2 2

1 1 1 1 2

1 1 1 2 2

1 1 1 1 2

1

1

1

1

1

1

1

1

1

1

2d

2

2

2

2

2d

2

2

2

2

1

1

1

1

1

1

1

1

1

1

All blood samples were collected from animals of Brazilian Pantanal. Microscopic examination. Ethidium bromide. Trypanosoma theileri (on blood smears of animals from which was isolated T. theileri).

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parasites in 1.2% low melting agarose. After incubation in 0.5 M ethylene diamine tetraacetic acid (EDTA) pH 8, 1% sarcosyl, and 2 mg/ml proteinase K at 508C for 48 h, blocks were stored in 0.5 M EDTA pH 8, 1 M Tris–HCl pH 7.5 at 48C. PFGE was performed in a contour clamped homogeneous electric field apparatus (CHEF Mapper, BioRad), using agarose gel (1.0 %) prepared in 0.045 M Tris-borate pH 8, 1 mM EDTA (0.5 £ Tris-borate/EDTA electrophoresis buffer (TBE)) under the following conditions: 68–150 s linear ramped pulse times, 21 h, 6 V/cm 3, at 148C. Chromosome bands were visualised by EtBr-staining and the gel was blotted (chromoblot) onto Hybond N 1 Nylon membranes (Amersham), prehybridised at 378C for 4 h in 5 £ SSC, 7% SDS, 20 mM sodium pyrophosphate, 10 £ Denhardt’s solution, 10% dextran sulfate and 40 mg/ml salmon sperm DNA, and hybridised for 16–18 h with Te664 probe at 428C in the same solution. Chromoblots

57

were washed once for 15 min at room temperature and twice for 15 min at 478C in 2 £ SSC, 0.1% SDS and 2.0 mM sodium pyrophosphate.

3. Results 3.1. Analysis of T. evansi stocks by RAPD Eight primers were selected to compare 14 Brazilian T. evansi stocks with T. evansi from China, the Philippines and Ethiopia, as well as with other Trypanosoma species. RAPD patterns disclosed high polymorphism among most species of Trypanosoma spp. (Fig. 1A). However, low genetic variability was observed within the subgenus Trypanozoon, for which most primers generated similar patterns. Despite this general homogeneity, different profiles were observed

Fig. 1. Randomly amplified polymorphic DNA patterns of Trypanosoma evansi isolates and other Trypanosoma species obtained with primers 606 (A) and 649 (B). Agarose gel (2.0%) stained with ethidium bromide. Te, T. evansi; Tbb, T. brucei brucei; Teq, T. equiperdum; Tbg, T. brucei gambiense; Tbr, T. brucei rhodesiense; Tvi, T. vivax; Tcr, T. cruzi; Tra, T. rangeli. DNA of BALB/c mice leukocytes (LBALB/c) was used as negative control. f, control without DNA.

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Fig. 2. Dendrogram based on randomly amplified polymorphic DNA profiles to illustrate the genetic relatedness of Brazilian Trypanosoma evansi stocks by comparing them to one another, to Old World stocks of Trypanosoma evansi and to other Salivarian species. The numbers refer to the bootstrap values of the clusters in 100 replicates.

among T. brucei spp. (Fig. 1A). Only microheterogeneities were observed among Brazilian T. evansi stocks (Fig. 1B). The dendrogram constructed based on these RAPD patterns illustrates the genetic relationship of all T. evansi isolates (Fig. 2). All stocks clustered together in 100% of the bootstrap replicates and the Brazilian stocks showed a closer relation with stocks from China and Ethiopia than with those from the Philippines (isolate MA), which were more distant from all other stocks. Thus, data from this study confirmed the close relationships of T. evansi from different host species and continents. 3.2. Evaluation of a T. evansi synapomorphic fragment as a tool for species-specific diagnosis Although RAPD patterns generated with most primers revealed low genetic polymorphism between T. evansi and T. brucei, it was possible to identify a T. evansi-specific profile generated by primer 664 which consisted of a single DNA fragment of 615 bp. This DNA fragment (RAPD-

Te664) proved to be monomorphic for all 14 Brazilian stocks of T. evansi from domestic and wild hosts and was also amplified from DNA from African (Ethiopia) and Asian (China and the Philippines) T. evansi (Fig. 3). The RAPD-Te664 fragment was synapomorphic for the T. evansi-clade since it was not detected in any other species (Fig. 3). Complex patterns of faint bands were observed for all other species of trypanosomes tested using primer 664 at a low annealing temperature (378C) (data not shown). We standardised a T. evansi-specific reaction by gradually ramping the primer annealing temperature up to 448C (Fig. 3). Thus, the specificity of the RAPD-Te664 fragment was confirmed using DNA from several other trypanosome species, including: (a) the very closely related members of subgenus Trypanozoon, T. equiperdum and T. brucei spp., (b) T. vivax and (c) the Stercorarian species Trypanosoma theileri, T. cruzi, Trypanosoma conorhini and Trypanosoma rangeli (Table 1). The sensitivity of RAPD-Te664 in detecting T. evansi was 50 pg of purified DNA or ,50 cells in crude DNA preparations by EtBr-staining or ,1.0 pg of

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conserved blocks with T. brucei sequences, as previously suggested by our hybridisation analysis, whereas other blocks are divergent from all known T. brucei sequences.

3.4. Evaluation of the species-specificity and sensitivity of Te664 probe

Fig. 3. Specificity of RAPD-Te664 and PCR-Te315 assays. Agarose gel (2.0%) stained with ethidium bromide of amplified DNA fragments. Te, T. evansi; Teq, T. equiperdum; Tbb, T. brucei brucei; Tbg, T. brucei gambiense; Tbr, T. brucei rhodesiense; Tvi, T. vivax. DNA of BALB/c mice leukocytes (LBALB/c) was used as negative control. f, control without DNA.

purified DNA, which corresponds to ,10 parasites, after hybridisation of the amplified products with the radiolabelled Te664 probe (data not shown). 3.3. Characterisation of the RAPD-Te664 synapomorphic DNA fragment Since the size identity of PCR fragments does not prove that the sequences are identical, the homology among the DNA fragments from different stocks was assessed by crosshybridising a Southern blot containing amplified products of all stocks with the fragments taken from the Ted1 and Tec1 stocks (data not shown). Similar hybridisation signals were observed in all slots under highly stringent hybridisation conditions. The similarity of Te664 bands was also demonstrated by the same restriction patterns observed in the PCRrestriction fragment length polymorphism (RFLP) analysis of the amplified fragments using 10 enzymes (data not shown). Since all the results suggested that sequences of amplified fragments from different stocks were very similar, we cloned and sequenced the Te664 fragment from Ted1 (GenBank accession number AF397194). Hybridisation of chromosomal-sized DNA molecules from bloodstream T. evansi (Ted1 and Tec1), separated on PFGE and blotted onto a Nylon membrane, using the Te664 probe showed strong signals in the region containing intermediate and minichromosome bands, indicating that the Te664 sequence is a repetitive sequence dispersed in several chromosomes (Fig. 4). The Blast search for homology revealed that the Te664 sequence showed strong nucleotide similarity (85–100%) to several short (20–150 bp) sequences in T. brucei (ACO84397) and 65–70% of amino acid similarity (block 92–212 bp) with T. b. brucei ESAG-2 (AAA63784) and ESAG-3 (B31847). Thus, the Te664 sequence shares

To investigate whether the RAPD-Te664 fragment generated from T. evansi consists of a species-specific DNA sequence, the fragment taken from one T. evansi isolate (Ted1) was used as probe for slot-blot hybridisation of genomic DNA from 17 T. evansi stocks and nine other trypanosome species belonging to various subgenera. The results revealed the absence of cross-hybridisation with species other than those of the subgenus Trypanozoon. However, whereas the signal intensity was strong and quite similar among T. evansi stocks, faint signals were detected for T. b. brucei and T. equiperdum using stringent hybridisation conditions (data not shown). On the other hand, hybridisation using the Te664a probe, a 19 nucleotide primer complementary to the internal sequence of the Te664 fragment, proved to be T. evansi-specific (Fig. 5). Although slot-blot hybridisation of a serial dilution of T. evansi DNA revealed that neither probe is very sensitive, detecting ,100 pg of DNA or ,1000 parasites (data not shown), this level of

Fig. 4. Pulsed-field gel electrophoresis analysis of chromosomal DNA of Trypanosoma evansi isolates Ted1 and Tec1. Agarose gel (1%) stained with ethidium bromide (EtBr) and chromoblot of the same gel hybridised with 32 P-labelled Te664 probe. Saccharomyces cerevisiae chromosomes (New England Biolabs) were used as chromosome band size markers.

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Fig. 5. (A) Specificity of Te664a probe on Trypanosoma evansi detection by slot-blot hybridisation of DNA (3.0 mg/slot) of Trypanosoma spp.: 1a, Ted1; 1b, Tec2; 1c, Tect2; 1d, Tbg 1; 1e, Tra; 2a, Ted2; 2b, Ter1; 2c, Tect3; 2d, Tbg 2; 2e, Tth 1; 3a, Ted3; 3b, Teh1; 3c, TeET; 3d, Tbr; 3e, Tco; 4a, Ted4; 4b, Teh2; 4c, Teq; 4d, TviY486; 4e, negative control with DNA of bovine leukocyte; 5a, Ted5; 5b, Teh3; 5c, Tbb 8195; 5d, TcrG; 5e, negative control with DNA of horse leukocyte; 6a, Tec1; 6b, Tect1; 6c, Tbb 427; 6d, TcrM; 6e, negative control with DNA of BALB/c mice leukocyte. (B) The same slot-blot membrane hybridised with 32P-labelled TeSSU probe. Te, T. evansi; Teq, T. equiperdum; Tbb, T. brucei brucei; Tbg, T. brucei gambiense; Tbr, T. brucei rhodesiense Tvi, T. vivax; Tcr, T. cruzi; Tth1, T. theileri; Tco, T. conorhini; Tra, T. rangeli.

detection is not very limiting because the typical T. evansi infection shows high parasitaemia. 3.5. Specificity and sensitivity of the T. evansi-specific PCRTe315 assay Unlike conventional PCR, RAPD has been used only with purified parasites free from mixed samples with more than one species/isolate or from contaminating host DNA. Moreover, RAPD is highly sensitive to minor variations in the amplification reactions, a fact limiting its general application with crude materials as the source of DNA templates. To avoid these shortcomings and improve the detection method, we developed a conventional PCR assay based on the Te664 sequence aligned with T. brucei sequences showing significant nucleotide similarity on the Blast search for homology. Oligonucleotides Te664a and Te664b were designed to amplify a T. evansi-specific 315 bp sequence (PCR-Te315) internal to Te664 DNA fragment. PCRTe315 was amplified from all T. evansi stocks but not from any other trypanosome species tested (Fig. 3; Table 1). The sensitivity of PCR-Te315 was evaluated using purified or crude DNA templates. Both assays proved to be very sensitive detecting ,10 pg or ,25 cells by EtBr-staining and ,1.0 pg or 10 cells after hybridisation with Te664a probe (Fig. 6).

filter paper, or clotted samples. To assess the specificity of these methods, we first ascertained the lack of amplification of DNA from leukocytes of uninfected mice, horses and cattle. Results with laboratory samples revealed that both amplification methods and blood collection protocols are suitable substitutes to purification of DNA from preserved blood. Thus, these methods are very convenient for field studies. After standardisation of both RAPD-Te664 and PCRTe315 using blood of laboratory-infected mice, we evaluated their potential for detection of T. evansi in fieldcollected blood samples from domestic and wild animals. The results proved the suitability of these methods using crude template DNA from blood samples of field-captured animals, collected on filter paper (horses) or blood smears (horses, dogs, a pig and wild mammals) (Fig. 7; Table 2). Tests on 41 samples from horses detected T. evansi in 12 animals (Table 2). Microscopic investigation of trypano-

3.6. Evaluation of RAPD-Te664 and PCR-Te315 for T. evansi detection under field conditions To evaluate the usefulness of RAPD-Te664 and PCRTe315 amplification for T. evansi detection directly in their natural hosts, we standardised these methods using crude DNA templates. We initially evaluated simplified methods for preparation of template DNA directly from the blood of laboratory-infected mice from glass slides,

Fig. 6. Sensitivity of the PCR-Te315 assay using (A) purified DNA and (B) whole cells of T. evansi Ted1 treated with proteinase K. Agarose gels (2.0%) stained with ethidium bromide (EtBr) of amplified DNA fragments and Southern blots of the same gels hybridised with 32P-labelled Te664a probe. f, control without DNA.

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Fig. 7. Trypanosoma evansi detection on field-collected blood from animals through (A) randomly amplified polymorphic DNA-Te664 assay and (B) PCRTe315 assay using DNA templates from filter paper and from blood smears. Ethidium bromide-stained 2.0% agarose gel (EtBr) and Southern blots of the gels hybridised with 32P-labelled Te664 probe in (A) and with Te664a probe in (B). Blood samples selected to illustrate the results are named or numbered according to corresponding animals listed in Table 2. f, control without DNA.

somes in blood smears prepared using the same blood samples revealed trypanosomes in only two samples (Table 2).

4. Discussion We had previously classified Brazilian T. evansi stocks from domestic and wild hosts from Pantanal by morphological, biological and molecular methods. Mouse inoculation and morphology depicting akinetoplasty were used for specific diagnosis of these stocks (Ventura et al., 1997, 2000). Aiming at molecular identification of Brazilian T. evansi stocks, we previously evaluated a diagnostic method based on repetitive DNA sequence (Wuyts et al., 1994). However, our results demonstrated that this method did not distinguish T. evansi from T. brucei and T. equiperdum (data not shown). Methods based on kDNA are not useful for Brazilian stocks since all stocks analysed are akinetoplastic (Ventura et al., 2000). In this study we employed the RAPD technique to assess the genetic variability among these stocks and to compare Brazilian with the Old World stocks. This method has been used for fine level detection of polymorphism among a small number of T. evansi stocks. Although previous attempts using the RAPD method could not differentiate African stocks of T. evansi (Waitumbi and Murphy, 1993; Waitumbi et al., 1994), this method demonstrated intraspecific differences in T. evansi stocks from Thailand (Watanapokasin et al., 1998) and from India (Basagoudanavar et al., 1999). In the present study, RAPD analysis did not reveal significant heterogeneity among T. evansi stocks. The microheterogeneity detected among T. evansi stocks could not be associated with any features such as host-species, geographic origin, kDNA status or virulence for mice. Brazilian T. evansi stocks behave differently in mice, ranging from highly virulent stocks producing high parasitaemias and killing 100% of mice in ,7–10 days (Ted1, Ted2) to stocks that gave low parasitaemia and were not

lethal (Tec1, Tec2) (Ventura et al., 1997). Analysis of both Brazilian akinetoplastic stocks and dyskinetoplastic stocks from Old World showed that the kDNA status could not be correlated with polymorphic patterns generated by RAPD. According to our data, most RAPD patterns are shared by all species of the subgenus Trypanozoon, but showed marked differences in trypanosome species of other subgenera. We constructed a dendrogram to assess the degree of relationship among stocks of T. evansi from New and Old World. As expected, all stocks clustered tightly, although the isolate MA from the Philippines was found to be the most distant from all other stocks. Previous analysis based on restriction patterns of repetitive DNA sequences suggested that three stocks from Brazil are closely similar to Chinese and Ethiopian stocks, but differ from stocks from different countries in Africa and the Philippines (Zhang and Baltz, 1994). Recently, analysis of microsatellite loci also revealed that South American stocks are more closely related to stocks from China and Ethiopia than to those from other African regions (Biteau et al., 2000). Aside from Brazilian isolates, only Chinese stocks of T. evansi have been described as being totally akinetoplastic as natural isolates (Lun et al., 1992; Ventura et al., 2000). The lack of kDNA minicircles in Brazilian isolates contrasts with the previous report of kDNA minicircles-bearing Colombian stocks (Songa et al., 1990) and support the idea of independent introduction of T. evansi in South America (Shaw, 1977). These data provide insight into the lineage origins of T. evansi stocks and dynamics of the parasitic populations dispersed around the world. Our RAPD-derived dendrogram is compatible with those based on isoenzymes (Stevens et al., 1992; Queiroz et al., 2000) and ribosomal sequences (Stevens et al., 1999, 2001). All molecular markers so far investigated demonstrate high genetic homogeneity among all species of the subgenus Trypanozoon, suggesting that it will be difficult to identify sequences of use as species-specific markers. Despite the limited variability, RAPD patterns permitted us to identify a T. evansi-specific DNA band (Te664) that seems mono-

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morphic for all T. evansi stocks. This fragment was found to be synapomorphic for the T. evansi cluster, since it was generated for all T. evansi stocks, and differed from patterns obtained for all other trypanosome species. Identification of DNA sequences from a specific organism to distinguish it from closely related organisms is labourious and time consuming. The RAPD method permits quick identification of short DNA sequences unique for a single species. Thus, we evaluated the usefulness of the RAPD-Te664 amplified DNA fragment for T. evansi detection directly in their natural hosts. Our results suggest that in addition to the length of the RAPD-Te664 fragment, its sequence is species-specific and useful to detect T. evansi by DNA probing or by PCR. Characterisation of the Te664 sequence by PFGE indicated that this sequence is present in several copies in the genome of T. evansi, dispersed through several intermediate and minichromosomes. RAPD is sensitive to minor variations in reaction parameters, some of which are difficult to control in rudimentary conditions using crude materials as a source of DNA. To improve the sensitivity and to assure the specificity of T. evansi detection, the sequence of the Te664 RAPD-derived fragment was used to design primers for use in conventional PCR reaction that will amplify a T. evansi-specific internal fragment. For diagnosis of T. evansi directly in its natural hosts, we also evaluated simplified methods for preparation of template DNA directly from blood of infected animals. The methods developed do not require well-preserved parasites or purified DNA samples, which are essential requirements for epidemiological surveys. Overall, our results suggest that both RAPD-Te664 and PCR-Te315 may be suitable as sensitive diagnostic tools for T. evansi, generating species-specific fragments of identical size and high homology for all T. evansi stocks from Brazil or elsewhere. Outbreaks of T. evansi have been reported frequently, but existing diagnostic approaches have failed to determine the sources of infection, i.e. asymptomatic animals and wild reservoirs associated with such outbreaks. Given the low specificity of current diagnostic methods, a rapid and sensitive assay as described here would greatly favour the early detection of asymptomatic or mild T. evansi infection. This would improve clinical management of the disease with the option of initiating chemotherapy before the onset of symptoms and may result in fewer cases progressing to severe and often chronic disease. Moreover, this method can be useful to assess the efficacy of drug treatment. The method developed here will permit more accurate molecular-epidemiological studies conducted to determine not only the true prevalence but also the risk factor associated with infection.

Acknowledgements We are grateful to Theo Baltz (University of Bourdeaux, Bordeaux, France) and Wendy Gibson (University of Bristol, Bristol, UK) for supplying us with DNA of T. evansi

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