Experimental Parasitology 99, 181–189 (2001)

doi:10.1006/expr.2001.4670, available online at http://www.idealibrary.com on

Trypanosoma evansi: Cloning and Expression in Spodoptera fugiperda InsectCells of the Diagnostic Antigen RoTat1.2

Toyohiko Urakawa,*,1 Didier Verloo,† Luc Moens,‡ Philippe Buscher,† andPhelix A. O. Majiwa*,2

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*International Livestock Research Institute, ILRI, P.O. Box 30709, Na†Department of Parasitology, Institute of Tropical Medicine, Nationale‡Department of Biochemistry, University of Antwerp, Universiteitsplei

Urakawa, T., Verloo, D., Moens, L., Buscher, P., and Majiwa, P. A.O. 2001. Trypanosoma evansi: Cloning and expression in Spodopterafugiperda insect cells of the diagnostic antigen RoTat1.2. ExperimentalParasitology 99, 181–189. A complementary DNA encoding the vari-ant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypano-zoon antigenic type (RoTat)1.2, currently used for experimental sero-logical diagnosis of T. evansi infection in livestock, was cloned asa recombinant plasmid and sequenced. A recombinant baculoviruscontaining the coding region of RoTat1.2 VSG was constructed toexpress the protein in Spodoptera fugiperda insect cells. From this,sufficient quantities of the recombinant protein are being producedfor empirical and wide-scale objective assessment of the diagnosticpotential of this antigen. The gene encoding the RoTat1.2 VSG wasshown by PCR to be present in the genomes of many different clonedisolates of T. evansi, but not T. brucei, from geographically separateregions of Africa, Asia, and South America. With the recombinantRoTat1.2 at hand, it is now possible to investigate the extent to which

epitopes on this VSG are conserved among different T. evansi iso-lates. q 2001 Elsevier Science (USA)

Index Descriptors and Abbreviations: Trypanosoma evansi; CATT,card agglutination test for trypanosomiasis; VSG, variant surface glyco-protein; Surra; ELISA, enzyme-linked immunosorbent assay; Ro-Tat1.2, Rode Trypanozoon antigenic type 1.2.

1Present address: London School of Hygiene & Tropical Medicine,Keppel Street, London WC1E 7HT, United Kingdom.

2To whom correspondence should be addressed. Fax: 254-2-631499.E-mail: p.majiwa@cgiar.org.

0014-4894/01 $35.00 181q 2001 Elsevier Science (USA)All rights reserved.

, Kenya;at 155, B-2000 Antwerp, Belgium; andB2610 Antwerp, Belgium

INTRODUCTION

Trypanosomosis remains an important disease in manydeveloping countries of the world, affecting both humansand livestock. In sub-Saharan Africa, tsetse-transmitted try-panosomosis is the most debilitating disease of humans andtheir livestock, resulting in economic losses estimated atover $1.3 billion annually (Kristjanson et al. 1999). How-ever, there are species of pathogenic trypanosomes whichare transmitted through other means than the tsetse fly. Tryp-anosoma evansi, which causes the disease generally knownas Surra in livestock, is transmitted by biting flies and isfound outside the tsetse belt in Africa and in South America,the Middle East, and Asia. The disease appears to be spread-ing to other countries, such as China, Indonesia, Thailand,Vietnam, and a number of countries in South America, someof which were previously free of Surra. There is evidencethat T. evansi is approaching Australia through Papua NewGuinea (Reid et al. 1999). All this makes T. evansi thesingle pathogenic salivarian trypanosome with the widest

worldwide distribution. However, very little information onthe precise distribution, incidence, and impact of Surra ina majority of the countries affected is available. The lackof accurate diagnostic tests and tools for characterization ofT. evansi forms a major constraint to treatment and deploy-ment of rational control strategies for Surra.

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From the analyses of nuclear DNA, kinetoplast DNA, andmultilocus isoenzymes (Gibson et al. 1980; Borst et al. 1987;Songa et al. 1990; Stevens and Godfrey, 1992; Zang andBaltz, 1994; Mathieu-Daude and Tibayrenc, 1994), it hasbeen suggested that T. evansi, in contrast to the trypanosomesthat develop cyclically in the tsetse fly, has a limited variantsurface glycoprotein (VSG) antigenic repertoire. A similarconclusion has been reached from serological analysis of T.evansi isolates from different countries including Indonesiaand China (Jones and Payne 1992; Lun et al. 1992). Serologi-cal data has been obtained from both immune-trypanolysisand ELISA analyses indicating that RoTat1.2 VSG is a pre-dominant variant antigen type (VAT) expressed in all T.evansi stocks examined so far (Verloo et al. 1997).

Consequently, several antibody detection assays havebeen developed based on the RoTat1.2. These include adirect agglutination test (CATT/T. evansi; Songa and Hamers1988), an indirect agglutination test (LATEX/T. evansi; Ver-loo et al. 2000), and an ELISA/T. evansi (Verloo et al. 2000)which have been used in many studies seeking to clarify theepidemiology of T. evansi in the affected countries (Payneet al. 1991; Monzon et al. 1995; Verloo et al. 2000; Davisonet al. 2000). The CATT/T. evansi uses whole, formaldehyde-fixed, Coomassie-stained, freeze-dried RoTat1.2 trypano-somes, while the antigen in LATEX/T. evansi and in ELISA/T. evansi consists of soluble-form RoTat1.2 VSG purifiedfrom the trypanosomes. With these tests, false positive re-sults may occur due to cross-reacting epitopes on the anti-gens; furthermore, each of the tests requires a large numberof laboratory rodents in which to grow the RoTat1.2 trypano-somes for preparation of the antigens.

An antigen detection test, Suratex (Nantulya 1994), hasalso been developed for diagnosis of T. evansi infections,but this is based on monoclonal antibodies that recognizeunknown antigen(s) linked to latex particles.

To improve the serological diagnostic tests for T. evansiinfections based on RoTat1.2 VSG, we have cloned andexpressed the VSG in a heterologous eukaryotic expressionsystem. This will facilitate standardization of the test, usinga recombinant antigen produced in a predictable manner

with minimum batch-to-batch variation. It is expected thatthe use of a recombinant RoTat1.2 antigen or peptides basedupon conserved epitopes of RoTat1.2 will eliminate a major-ity of the fortuitous false positives experienced with thecurrent version of tests based on this VSG. The availabilityof robust diagnostic tests for the screening of livestock herdsand diagnosis of Surra in individual animals is likely toincrease effective management of the disease.

URAKAWA ET AL.

MATERIALS AND METHODS

The trypanosomes. The trypanosomes expressing the RoTat1.2were obtained from the collection of trypanosome stabilates maintainedat the ITM-Antwerp, (Table I). The RoTaR1 was derived by cloningfrom an isolate of T. evansi found in a naturally infected buffalo atPekalongan, Indonesia (Songa and Hamers 1988). The trypanosomeswere grown in lethally irradiated rats, which were monitored for para-sitemia daily by microscopic examination of tail blood. At the firstpeak parasitemia, the rats were euthanatized and then bled by cardiacpuncture. The trypanosomes were separated from the blood elementsby chromatography in a column of DE-52 (Lanham and Godfrey 1970).The purified trypanosomes were washed with 15 ml of phosphate-buffered saline–glucose, pH 8.0, and then pelleted by centrifugation.The pellet was either used immediately or stored at 2708C for later use.

Preparation of RNA and DNA. Total RNA was extracted from thetrypanosomes using the single-step method described by Chomczynskiand Sacchi (1987) and kept frozen at 2808C until needed. DNA wasextracted from the trypanosomes following routine procedures anddissolved in 10 mM Tris–HCl, pH 7.4, at 500 mg/ml. This was keptat 48C until needed for use. The nucleic acids were analyzed followingestablished molecular biological procedures (Sambrook et al. 1989).

cDNA synthesis. The cDNA was synthesized from the total RNAusing MMLV reverse transcriptase (New England BioLabs, Beverly,MA). To amplify only VSG-encoding transcripts, an oligonucleotide,complementary to the conserved segment of the 38 end noncodingregion of the Trypanozoon VSGs mRNA (Rice-Ficht et al. 1981)was used in priming the first-strand cDNA synthesis. This primer,designated ILO7722 or sVATBam (CGG GAT CCA GGT GTT AAAATA TA), was designed to contain a BamHI site for ease of subsequentmanipulations. The synthesis reaction was performed for 2 h at 378C.

The PCR. To amplify from the sscDNA only the sequences encod-ing VSGs, a set of primers, one which is complementary to the 38 endportion of the miniexon, but containing a KpnI site, designated ILO7725(CGG GTA CCT AGA ACA GTT TCT GTA CTA TAT TG) andthe other designated the sVATBam, were used in PCR amplificationcontaining 2–4 mg of sscDNA in a final volume of 50 ml. The amplifica-tion parameters consisted of 20 cycles each at 948C for 30 s, 428C for30 s, and 728C for 1.5 min, followed by additional 20 cycles each at948C for 30 s, 468C for 30 s, and 728C for 1.5 min.

To amplify sequences encoding the RoTat1.2 VSG from total geno-mic DNA, the PCR was conducted using the set of primers ILO7957(GCC ACC ACG GCG AAA GAC) and ILO8091 (TAA TCA GTGTGG TGT GC) in a final reaction volume of 10 ml containing 100 pgof genomic DNA. The amplification parameters were 948C for 30 s,528C for 30 s, and 728C for 60 s, followed by a 10-min extension at728C for 5 min. The samples were subsequently cooled to 48C andthen kept frozen until use.

Cloning of PCR products. The PCR yielded a product of approxi-

mately 1.6 kb whenever RNA of any Trypanozoon was used. Theproducts were resolved by electrophoresis in a 0.8% agarose gel; the1.6-kb band was eluted from the gel and then subcloned into theplasmid TA-vector pCR2.1 (Invitrogen, San Diego, CA). The plasmidswere transformed into Escherichia coli, which were subsequently platedon selective media. Plasmids were purified from individual bacterialcolonies and the sizes of inserts in each of them determined by PCR,using vector primers.

Nucleotide sequence analysis. Nucleotide sequence composition

WaTat1.2 T. b. rhosesiense Nyanza, Kenya Man 196111E1 T. brucei Uhembo, Kenya Cow 1964

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ILTat1.1 T. brucei UhemILTat1.3 T. brucei UhemGuTat3.1 T. b. brucei BusoSTIB247 T. brucei SereLiTat1.1 T. b. gambiense CoteIL2343 T. b. gambiense? CoteIL3935 T. brucei Yim

of the inserts in the individual plasmids was determined by cyclesequencing, using an ABI 377 DNA automated sequencer (Perkin–Elmer, Foster, CA). To avoid errors that may be introduced by Taqpolymerase, three independent clones of each PCR product were se-quenced from each ligation event. Gaps in the sequence were filledby primer walking using internal oligonucleotide primers. Thesequences were assembled and analyzed using common computerprograms.

Cotransfection into SF21AE insect cells. The insert shown by se-quence analysis to contain the putative RoTat1.2 VSG cDNA wasamplified by PCR with a pair of primers ILO7791 (AAA CTG CAGTAT GCA AAC CAA GGC GCTC) and ILO7722, mentioned above.The product obtained was subcloned into the pCR-2.1 vector. The 1.6-kb DNA fragment was subsequently cut with Pst1 and BamH1 andthen ligated into the PstI/BamHI insertion site of a transfer plasmidpVL1392 (Invitrogen). The correct insertion of the insert into theplasmid was confirmed by sequence determination. The recombinantplasmid DNA and the linearized BacPaK6 DNA (CLONTECH, PaloAlto, CA) digested with Bsu36I were cotransfected into Spodopterafrugiperda SF21AE insect cells with Lipofectin (Cellfectin; GIBCO-BRL, Grand Island, NY). Plaque assays (Urakawa et al. 1989) wereperformed on the individual progeny viruses in insect cells; lysatesfrom the cells were resolved by SDS–PAGE, which were subsequentlyanalyzed by staining with Coomassie brilliant blue R-250 or immu-

noblotting (Burnette 1981).

Cells and media. S. frugiperda insect cells, SF21AE, were usedfor propagation of high-titer baculoviruses and for plaque assays. Thecells were grown in TC100 insect cell medium (GIBCO-BRL) supple-mented with 10% fetal calf serum. High Five (Tn5) cells derivedfrom Trichoplusiani (Invitrogen) were used for higher production ofrecombinant proteins. These were cultured in EX-CELL 401 serum-free medium (JRH Biosciences, Lenexa, KS) kept at 288C.

The recombinant protein in insect cells. After electrophoretic sepa-ration of the proteins expressed by baculovirus in insect cells by SDS–

, Kenya Cow 1964, Kenya Cow 1964Uganda Cow 1966

ti, Tanzania Kongoni 1971voire Man 1952voire Man 1978Sudan Man 1982

PAGE, the gel was soaked in 0.25 M KCl for 5 min at 48C. The mainprotein band corresponding to the size predicted to be produced fromthe insert was clearly visible in the gels. The protein bands were cutout of the gel and the gel pieces were homogenized. These wereused in the immunization of Balb/c mice, by repeated interperitonealinjection. After two boosters, the antisera were collected from the miceand pooled.

Immunoblot analyses. SDS–polyacrylamide gel electrophoresisand immunoblot analyses of proteins in the lysates of trypanosomesand insect cells harboring recombinant baculoviruses were performedas already described (Urakawa et al. 1995). The membranes onto whichhad been blotted the proteins in the lysates were washed intensivelywith phosphate-buffered saline containing 0.05% Tween 20; they werethen incubated with horseradish peroxidase-conjugate and subsequentlydetected by reaction with luminescence (ECL; Amersham, Little Chal-font, UK).

Sequencing of the RoTat1.2 VSG. The soluble form of RoTat1.2was purified from trypanosomes homogeneously expressing the VSG,following conventional procedures (Cross 1975). The purified protein,showing a single band by SDS–PAGE, was dialyzed against pure waterand was subjected to N-terminal amino acid sequencing in a ABI 471-B as recommended by the manufacturer.

PCR amplification of RoTat1.2 VSG gene in T. evansi and T. bruceiisolates. The PCR amplification was carried out using a pair of

Trypanosoma evansi DIAGNOSTIC ANTIGEN RoTat1.2 183

TABLE I

Trypanosomes Used in This Study

Designation Species Locale, country where isolated Host Year

RoTat1.2 T. evansi Pekalongan, Indonesia Buffalo 1982AnTat3.3 T. evansi South America Capybara 1971IL3927 T. evansi Ipal, India Camel N/AIL3928 T. evansi Ukunda, Kenya Camel 1978IL3960 T. evansi Marsabit, Kenya Camel 1980KETRI 394 T. evansi Isiolo, Kenya Camel 1997IL1695 T. evansi Ukunda, Kenya Camel 1978IL3354 T. evansi Adel, Mali Camel N/AIL3382 T. evansi Nara, Mali Camel N/A

internal primers, ILO7957 (GCC ACC ACG GCG AAA GAC) andILO8091 (TAA TCA GTG TGG TGT GC), both of which had beendetermined to be within the RoTat1.2 VSG coding region. GenomicDNA from nine different cloned isolates each of T. evansi and T. bruceiwere used as templates at the concentration of 100 ng/ml in a finalreaction of 10 ml. The amplification parameters consisted of 40 cycleseach at 948C for 30 s, 528C for 30 s, and 728C for 1 min. The PCRproducts were analyzed by electrophoresis in a 1.5% agarose gel. Theexpected size of the amplified DNA fragment was 488 bp.

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RESULTS

The trypanosomes used express RoTat1.2 VSG. Trypa-nosomes purified from the host blood elements were spreadon a glass slide and then stained by immunofluorescenceusing monovalent antisera to RoTat1.2. Greater than 99%of the organisms were stained. Furthermore, in the immune-trypanolysis, .99% of the trypanosomes were lysed whenmonovalent antisera to RoTat1.2 was used in the assay (datanot shown). Collectively, these data indicate that a vast ma-jority (.99%) of the trypanosomes used in the study expressthe RoTat1.2 VSG.

The cloned cDNA encodes RoTat1.2 VSG. The nucleo-tide sequence composition of the cloned cDNA was deter-mined completely on both strands, as already described. Thesequence obtained was translated into the predicted aminoacid sequence using conventional computer programs. Con-currently, the N-terminal amino acid sequence of the solubleform of the protein was determined directly from the VSGpurified from the trypanosomes expressing the RoTat1.2.The amino acid sequences from the two sources werealigned. The amino acid residues, underlined in Fig. 1, werefound to be identical in both the purified protein and thepredicted translation of the cloned cDNA. These amino acidresidues represent the N-terminal amino acid sequence ofthe mature, soluble form of RoTat1.2 VSG. Analysis ofthe predicted amino acid sequence using the SignalP-NNcomputer program (http://www.cbs.dtu.dk; Nielsen et al.1997) predicts that the signal peptide spans the amino acidresidues 225 to 21 and that the cleavage occurs betweenresidues 21 and 1 (Fig. 1). These data confirm that thecloned cDNA encodes the RoTat1.2 VSG.

RoTat1.2 VSG expressed in insect cells. A recombinantbaculovirus, designated AcRoTat1.2, was generated by co-transfection of S. frugiperda insect cells with baculovirusgenomic DNA and a transfer recombinant plasmid con-taining the coding region of the putative RoTat1.2 VSGcDNA. The proteins produced by the insect cells were ana-lyzed by SDS–PAGE. The insect cells infected with AcRo-Tat1.2 produced major protein bands of 62 and 60 kDa (Fig.2A, lane 2), which are similar in size to the native RoTat1.2

VSG (Fig. 2A, lane 4). When Tn5 cells were used for theexpression of these proteins, the amount of protein producedwas approximately 30% of the total cell proteins as judgedby visual inspection of the proteins in a gel (data not shown).Growth of the cells in medium containing tunicamycin at10 mg/ml resulted in a significant reduction in the productionof the 62-kDa band (Fig. 2A, lane 3), indicating that thisparticular protein is glycosylated.

URAKAWA ET AL.

Both the 60- and the 62-kDa proteins immunoreacted withthe rabbit serum raised against the purified native RoTat1.2VSG (Fig. 2B, lanes 2–4) and with the monospecific poly-clonal antibodies made against the recombinant protein (Fig.2C, lane 2–4). Therefore the S. frugiperda insect cells in-fected with AcRoTat1.2 recombinant baculovirus expressRoTat1.2 VSG.

General features of RoTat1.2 VSG. Carrington et al(1991) classified T. brucei VSGs into three types of the N-terminal domain and four types of the C-terminal domain,based upon the primary structure of each VSG. The numberand position of cysteine residues and potential N-glycosyla-tion sites on a VSG determine the domain type to which itbelongs. According to this classification scheme, the Ro-Tat1.2 VSG belongs to the type A of the N-terminal domainand the type 2 of the C-terminal domain. Upon alignmentof the predicted amino acid sequence of this VSG with theamino acid sequence of the other Trypanozoon VSGs alreadyin the public domain databases, we found no significantmatches.

RoTat1.2 VSG gene in the genomes of other Trypanozoon.While in the bloodstream of a mammalian host, the trypano-some expresses only one of several potential VSG genes.We sought to determine whether RoTat1.2 VSG gene existsin the genomes of the other Trypanozoon. For this, the DNAfragment constituting the RoTat1.2 VSG open reading framewas used as a probe in Southern blot (Southern 1975) hybrid-ization analysis of DNA from other trypanosome specieswithin the Trypanozoon. The fragment hybridized with DNAfrom all the trypanosomes whose DNA were examined inthis way (Fig. 3). There was evident restriction fragmentlength polymorphism, as would be expected of a VSG gene.Apparently, DNA sequences homologous to the RoTat1.2VSG gene are present in the genomes of the other trypano-somes. Whether the RoTat1.2 VSG is ever expressed in thesetrypanosomes was not determined.

Diverse isolates of T. evansi contain intact RoTat1.2 VSGgene. The data shown in Fig. 3 indicate that different iso-lates of trypanosome species within the Trypanozoon havesequences homologous to the RoTat1.2 VSG gene. To inves-tigate whether the whole segment of the gene encodingRoTat1.2 VSG is present in the genomes of these trypano-

somes, we designed a pair of primers flanking a 488-bpsegment of the RoTat1.2 VSG cDNA and used these in PCRto amplify the genomic DNA purified from the differentcloned isolates of the trypanosomes, including T. evansi andT. brucei. The data obtained are shown in Fig. 4. As expected,this pair of primers amplified a fragment of approximately500 bp in the DNA of RoTat1.2 trypanosomes (Fig. 4, lane1). Interestingly, a fragment of identical size was amplified

Trypanosoma

evansiD

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NO

STIC

AN

TIG

EN

RoTat1.2

185

acid sequence. The 24n 38 end are a portion ofth residues are numberedf d of the mature protein.N oTat1.2 VSG gene areu

FIG. 1. The nucleotide sequence of RoTat1.2 VSG cDNA Accession No AF317914 and the deduced aminoucleotides (nt) underlined at the 58 end are a portion of the miniexon/spliced leader, and the 18 nt underlined at thee VAT-primer. The amino acid residues at the N-terminal end of the RoTat1.2 VSG are underlined. The nucleotide

rom the start to the end of the insert; the amino acid residues are numbered starting with the one at the N-terminal enucleotide sequences of the two internal primers, ILO7957 and ILO8091, used in the PCR amplification of the Rnderlined and boldfaced.

FIG. 2. Serological identity between recombinant and native RoTat1.2. Cell lysates made in sample buffer were resolved by SDS–PAGE andg th). LoTatevae in3, p

the gel was stained with Coomassie blue (A) Western blots containinrecombinant RoTat1.2 (B) or rabbit antisera made to RoTat1.2 VSG (C2, SF21AE insect cells infected with the recombinant baculovirus AcRAcRoTat1.2, grown in the presence of tunicamycin at 10 mg/ml; 4, T.IL3962. M contains protein molecular weight markers, with sizes of thpoints to the p10, a nonstructural viral protein, apparent in lanes 2 and

186 URAKAWA ET AL.

in the DNA of all the other T. evansi isolates used (Fig. 4,lanes 2–9), irrespective of the VSG that they express. Incontrast, no fragment of a similar size was amplified in theDNA of any of the T. brucei clones used (Fig. 4, lanes10–18).

DISCUSSION

In earlier comparative studies of the serological tests fordetection of trypanosome infection in animals, it was ob-served that a more sensitive test for detection of T. evansiinfections could be developed based upon RoTat1.2 VSG,because this antigen is thought to be expressed during theearly part of infection with this trypanosome (Songa andHamers 1988). Data obtained from immune-trypanolysis andVAT specific immunofluorescence show that this VSG orVSGs with similar epitopes occur frequently in spontaneous

relapse populations of different strains and geographic iso-lates of T. evansi (Verloo et al. 1997). Therefore, the Ro-Tat1.2-based antibody detection tests can be used in de-tecting T. evansi infections in different breeds of livestockincluding swine, cattle, and buffalo (Verloo et al. 2000).

T. evansi is probably the single trypanosome species with

e proteins were probed with either mouse antisera made against theanes contain lysates as follows: 1, mock-infected SF21AE insect cells;1.2; 3, SF21AE insect cells infected with the recombinant baculovirusnsi RoTat1.2; 5, T. evansi AnTat3.3; 6, T. evansi IL3927; 7, T. evansidividual proteins indicated in kDa on the left side. An arrowhead in Aroduced by the baculovirus vector.

the widest distribution worldwide. It is thought to haveevolved by mutation from T. brucei in the desert margins,by adopting a lifestyle that dispensed with cyclical develop-ment in the tsetse fly but required only mechanical transmis-sion to the host (Hoare 1970). The adaptation is thought tohave occurred in two phases, as suggested by analysis ofthe kinetoplast minicircle sequences (Borst et al. 1987; Lunet al. 1992). As would be expected, therefore, T. evansishares a significant number of morphological and physiolog-ical features with the bloodstream forms of T. brucei. Thisis supported by data obtained from comparative molecularanalyses using various criteria, including molecular karyo-types and the predominant repetitive genomic DNA elements(Waitumbi and Young 1994; Kimmel et al. 1987). Sequenceanalyses of trypanosome ribosomal RNA genes indicate thatT. evansi can indeed be regarded a variant of T. brucei(Urakawa et al. 1998; Urakawa and Majiwa, 2001).

Unlike T. brucei, T. evansi thrives outside the tsetse belt,where it causes a serious disease in livestock. It is in these

areas where reliable reagents are needed for detection ofthis parasite and for diagnosis of the disease that it causesin different species of livestock. Where no other Africantrypanosomes exist, the problem of distinguishing T. evansifrom other trypanosomatids is less compounded. For thispurpose, serological tests are most desirable because of theirrelative simplicity. The CATT/T. evansi was developed for

nal 488-bp segment of the cDNA encoding RoTat1.2 was

trypanosomes. Two micrograms of DNA from different trypanosomeswas digested with the restriction enzymes indicated and the blots of

these were hybridized with the segment of the cDNA, which encodesRoTat1.2 VSG. In both panels, lanes contain DNA digests from thefollowing trypanosomes: 1, T. evansi IL3927; 2, T. evansi AnTat3.3;3, T. evansi IL3960; 4, T. evansi RoTat1.2; 5, T. brucei ILTat1.3. Thesizes of the main fragments of DNA are indicated in kb on the rightside of each panel.

use in such areas. The test has been used for a number ofstudies investigating the prevalence of T. evansi infectionsin different host species in different regions of the world(Gutierrez et al. 2000; Payne et al. 1991; Verloo et al. 2000).In a majority of the studies, the test has a very high sensitivityand specificity and shows a good correlation between posi-tivity, parasitemia, and low PCV in the infected animals. Incertain instances, the test detected as positive some animalsin which the parasites could not be demonstrated (Verloo etal. 2000). However, since the test in its present form employscrude antigen, there is often cross-reactivity; furthermore,the test may fail to detect some true positive animals becauseit lacks sensitivity. As a first step in an attempt to improvethe performance of this and related tests, we have cloned and

expressed the RoTat1.2 VSG upon which the tests are based.

The evidence that the gene cloned and expressed in thecurrent study encodes RoTat1.2 VSG are severalfold: first,in priming the first-strand cDNA synthesis, use was madeof an oligonucleotide known to be conserved only in VSGmRNA of the trypanosome species within the Trypanozoon(Rice-Ficht et al. 1981); second, the amino acid sequences

187

predicted from the nucleotide sequence of the cloned geneare identical to those found at the N-terminal end of thepurified soluble form of RoTat1.2 VSG (Fig. 1); third, theantisera raised to the purified RoTat1.2 VSG reacts with therecombinant protein expressed by the insect cells infectedwith the recombinant baculovirus containing the cloned tran-script (Fig. 2); finally, the antisera raised against the recombi-nant protein expressed by the recombinant baculovirus reactswith the purified RoTat1.2 VSG and the VSG on the trypano-somes expressing the RoTat1.2 VSG (Fig. 2 and data notshown).

Although the sequences homologous to RoTat1.2 VSGgene were detected in the genomes of other cloned isolatesof trypanosomes within the Trypanozoon (Fig. 3), we ob-tained no direct evidence that these trypanosomes do indeedexpress the RoTat1.2 VSG. However, the fact that the inter-

Trypanosoma evansi DIAGNOSTIC ANTIGEN RoTat1.2

FIG. 3. RoTat1.2 VSG gene homologue in the genomes of other

found in the DNA of different isolates of T. evansi (Fig. 4)provides strong indirect evidence that the trypanosomes havethe potential to express the RoTat1.2 VSG. Whether theyexpress this VSG remains to be proven empirically. Theoligonucleotide primers based upon the sequence of the Ro-Tat1.2 VSG gene can serve to distinguish T. evansi from

FIG. 4. Sequences encoding RoTat1.2 VSG in other trypanosomes.Oligonucleotide primers flanking ,500 bp internal segment of theRoTat1.2 open reading frame were used in PCR to amplify genomicDNA from different cloned isolates of Trypanozoon. An aliquot of thePCR products was resolved in a gel, a photograph of which is shown.Lanes contain the PCR products from DNA prepared from trypanosome

clones as follows; 1, T. evansi RoTat1.2; 2, T. evansi AnTat3.3; 3, T.evansi IL3927; 4, T. evansi IL3298; 5, T. evansi, IL3960; 6, T. evansiKETRI 394; 7, T. evansi IL1695; 8, T. evansi IL3354; 9, T. evansiIL3382; 10, T. brucei WaTat1.2; 11, T. brucei 11E1;12, T. bruceiILTat1.1; 13, T. brucei ILTat1.3; 14, T. brucei GuTat3.1; 15, T. bruceiSTIB247; 16, T. brucei gambiense LiTat1.1; 17, T. brucei IL2343; 18,T. brucei IL3935. Lane M contains the 100-bp DNA molecular weightladder, with sizes indicated in kb on the right side.

188

the other Trypanozoon. The availability of recombinant Ro-Tat1.2 now makes it possible to evaluate more objectivelythe potential of this antigen to serve as basis for the develop-ment of an improved serological diagnostic test for Surra.

ACKNOWLEDGMENTS

from Pantanal of Mato Grosso - Brazil. Veterinary Parasitology

This study received financial support from the ILRI Livestock HealthProgram and the Belgian Directorate General for International Coopera-tion. We gratefully acknowledge the grants to L.M. (Project Nos.G.2023.94 and G.0314.00) from the Fund for Scientific Research-Flanders (FWO).

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Received 20 February 2001; accepted 27 November 2001