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The detection of non-RoTat 1.2 Trypanosoma evansi
Experimental Parasitology 110 (2005) 30–38 www.elsevier.com/locate/yexpr
The detection of non-RoTat 1.2 Trypanosoma evansi J.M. Ngaira a b
a,b,¤
, N.K. Olembo c, E.N.M. Njagi b, J.J.N. Ngeranwa
b
Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 25530, Nairobi, Kenya Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844, Nairobi, Kenya c Department of Biochemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya Received 19 May 2004; received in revised form 23 December 2004; accepted 10 January 2005
Abstract The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR ampliWcation for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from diVerent trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR ampliWcation of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp ampliWcation product was present in all Wve non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is speciWc for non-RoTat 1.2 T. evansi. 2005 Elsevier Inc. All rights reserved. Keywords: Trypanosoma evansi; Variant surface glycoprotein; Diagnosis; Kenya
1. Introduction Three species comprise the subgenus Trypanozoon: Trypanosoma brucei, Trypanosoma evansi, and Trypanosoma equiperdum, with T. brucei further subdivided into three subspecies: T.b. brucei, T.b. rhodesiense, and T.b. gambiense (Hoare, 1972). Trypanosomes of the three species are morphologically indistinguishable save for short-stumpy forms that occur in T. brucei. A key distinguishing feature is that T. evansi and T. equiperdum lack functional mitochondria containing maxicircle kinetoplast DNA (kDNA) and are therefore unable to complete cyclical development in the tsetse Xy vector that transmits other African trypanosomes (Borst et al., 1987). Whereas there are no maxicircles in T. evansi, the
¤
Corresponding author. Fax: +254 568695. E-mail address: [email protected] (J.M. Ngaira).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.01.001
maxicircles in T. equiperdum are incomplete (Lun et al., 2004). Mechanical transmission by blood sucking insects has allowed T. evansi to spread in Africa, Asia, and South and Central America where it causes the disease “surra” in a wide range of animals including camels, horses, cattle, and buValo as well as wild animals. The variant surface glycoprotein (VSG) of T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 is a diagnostic antigen for T. evansi from diverse geographical regions. Serological and PCR tests based on the VSG of RoTat 1.2 have shown high speciWcity and sensitivity in studies in diVerent geographical regions of the world (Bajyana and Hamers, 1988; Urakawa et al., 2001; Verloo et al., 2000). It has been shown however, that a number of T. evansi in Kenya were not detected by tests based on RoTat1.2 VSG and four of the isolates lacked the RoTat1.2 VSG gene (Ngaira et al., 2004). A recent study in Kenya (Njiru et al., 2004) showed that RoTat 1.2-based CATT/T. evansi was 65.5% sensitive, agreeing
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
well with the 68.6% sensitivity observed earlier Ngaira et al. (2003) and emphasizing the need to address the problem of diagnosis of T. evansi in the region. The main objective of this study was to Wnd a diagnostic reagent for T. evansi infections that are not detected by RoTat 1.2 VSG-based tests. Our approach was to identify
Table 1 Trypanosoma evansi isolated from camels in Isiolo District, Kenya between 1996 and 1997 Stock
Date of isolation
Place of isolation
JN 2118Hu JN 4394Ha JN 5460Ke JN 3234Abd JN 3235Abd JN 3270Sa JN 4304Moh JN 4314Moh JN 4315Moh JN 4357Jum JN 4306Moh JN 5480Dai JN 6507Ru JN 6512Muh JN 6524Muh JN 4385Bo JN 3238Abd JN 3230Abd JN 4384Ha JN 4309Moh
30 October 1996 17 January 1997 7 June 1997 6 July 1997 6 July 1997 7 July 1997 7 July 1997 7 July 1997 7 July 1997 9 July 1997 10 July 1997 10 July 1997 10 July 1997 10 July 1997 10 July 1997 17 July 1997 6 July 1997 6 July 1997 17 January 1997 7 July 1997
Burat Burat Dahyal Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Burat Mlango
31
sequences of diagnostic potential in the VSG cDNA of a non-RoTat 1.2 T. evansi isolate.
2. Materials and methods Trypanosomes previously isolated from camels (Ngaira et al., 2003, 2004) were used in this study (Table 1). Reference trypanosomes from the KETRI cryobank and puriWed DNA samples obtained from ILRI are shown in Table 2. 2.1. PuriWcation of trypanosomes Trypanosomes were grown in rats and were separated from rat blood cells by anion-exchange chromatography with DEAE–cellulose (DE52, Whatman Biochemical) (Lanham and Godfery, 1970) as previously described (Ngaira et al., 2004). 2.2. Preparation of genomic DNA and total RNA Genomic DNA was extracted from the trypanosomes by standard methods (Van der Ploeg et al., 1982) and dissolved in 10mM Tris–HCl, pH 7.4 or water at 500g/ml. It was stored at 4°C or ¡20°C until needed. Total RNA was extracted from the trypanosomes using the single-step method described by Chomczynski and Sacchi (1987). The extracted RNA was kept frozen at ¡80°C until needed.
Table 2 Reference trypanosomes Designation
Species
Host
Locality, country where isolated
Year of isolation
KETRI 3094 KETRI 2440 KETRI 2454 KETRI 2455 KETRI 2448 KETRI 2479 KETRI 3261 KETRI 3572 KETRI 3117 RoTat 1.2 IL3960a IL3927a AnTat3.3a KETRI 2710 KETRI 3596 11E1a ILTat1.1a ILTat1.3a ILTat1.4a WaTat1.2a LiTat1.1a IL3707a IL2569a IL3662a IL3665a IL1180
T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. brucei T. brucei T. brucei T. brucei T. brucei T. brucei T.b. rhodesiense T.b. gambiense T.b. gambiense T. vivax T. congolense T. congolense T. congolense
Horse Camel Camel Camel Camel Camel Camel Camel Camel BuValo Camel Camel Capybara Not known Not known Cow Cow Cow Cow Man Man Man Tsetse Cow Cow Lion
South America Kulal, Kenya Kulal, Kenya Galana, Kenya Galana, Kenya Ngurunit Samburu, Kenya Nanyuki, Kenya Athi River Pekalongan, Indonesia Marsbit, Kenya Ipal, India South America Not known Apatit Teso, Kenya Uhembo, Kenya Uhembo, Kenya Uhembo, Kenya Uhembo, Kenya Nyanza, Kenya Cote d’Ivoire Bida, Nigeria Lugala, Uganda Transmara, Kenya Transmara, Kenya Serengeti, Tanzania
1973 1979 1954 1979 1979 1981 1990 1994 1988 1982 1980 Not known 1971 Not known 1994 1964 1964 1964 1964 1961 1952 1968 1969 1966 1966 1971
a
DNA samples obtained from ILRI.
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2.3. One-step RT-PCR
2.6. PCR ampliWcation of T. evansi JN 2118Hu VSG gene
To amplify only VSG-encoding transcripts, RT-PCR ampliWcation was performed using a forward primer designed to base pair with the reverse complement of the 3⬘ part of the mini-exon found in the 5⬘end of trypanosome mRNAs (Cornelissen et al., 1986) and a reverse primer designed to base pair with a conserved nucleotide sequence found in the 3⬘ untranslated region of VSG mRNAs of trypanosomes within the Trypanozoon (Majumder et al., 1981). The forward primer used was IL07725 (5⬘-CGG GTA CCT AGA ACA GTT TCT GTA CTA TAT TG-3⬘) designed to contain a KpnI site (Urakawa et al., 2001) and the reverse primer was IL07722 (CGG GAT CCA GGT GTT AAA ATA TA3⬘) designed to contain a BamHI site (Urakawa et al., 2001). The reaction was carried out using Qiagen OneStep RT-PCR kit according to the protocol provided by the supplier. BrieXy, a 50 l reaction mixture was prepared containing a Wnal concentration of 2.0 g total RNA, 0.6 M each primer, 400 M each dNTP, an optimal Qiagen OneStep RT-PCR Enzyme Mix and 1£ RT-PCR buVer. It was incubated at 50 °C for 30 min, followed by 95 °C for 15 min, followed by a three-step cycling of 34 cycles (denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min) and a Wnal extension at 72 °C for 10 min.
A segment of T. evansi JN 2118Hu VSG gene that lacked homology with sequences in the databanks was identiWed by BLAST homology searches. Two primers were designed to amplify the 273 bp fragment: forward primer (5⬘-TTCTACCAACTGACGGAGCG-3⬘) and reverse primer (5⬘-TAGCTCCGGATGCATCGGT-3⬘). PCR ampliWcation was carried out in 20 l Wnal reaction mixtures containing 100 ng genomic DNA, 10 mM Tris– HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 M each of the four dNTPs, 10 M each of the oligonucleotide primers, and 1.0 U Taq (Thermus aquaticus) DNA polymerase (Promega). The ampliWcation parameters were 94 °C for 1 min followed by 40 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min) followed by a Wnal extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis in a 1.5% agarose gel. The expected size of the ampliWed DNA fragment was 273 bp. The restriction enzyme sites in the 273 bp fragment of the sequenced cDNA were determined using the computer programme http://tools.neb.com/NEBcutter2/ index.php. To assess the sequence identity of the 273 bp PCR products, the DNA bands were puriWed from gel using QIAquick Gel Extraction kit (Qiagen) and digested with the restriction enzyme HaeIII using standard procedures (Maniatis et al., 1982).
2.4. Cloning and of PCR products The products of RT-PCR were resolved by electrophoreses in a 0.8% agarose gel and the expected band of approximately 1.6 kb eluted from the gel and cloned into the pGEM-T easy vector (Promega). The plasmids were used to transform Escherichia coli, which were subsequently plated on selective media for the isolation of recombinants. Plasmids were puriWed from individual bacterial colonies using the Wizard Plus SV Minipreps DNA PuriWcation System (Promega), and the sizes of inserts determined by PCR using vector primers. The nucleotide sequence of the inserts in the plasmids was determined commercially (Macrogene) by the procedure of Sanger (Sanger et al., 1977) using automatic DNA sequencing. Both strands of the VSG cDNA were completely sequenced. 2.5. Sequence similarity searches Similarity searches of GenBank/NCBI database were performed with the basic local alignment search program (BLAST) http://www.ncbi.nlm.nih.gov/BLAST/ (Altschul et al., 1997) using the default matrix. Alignment of two sequences was performed using the program FASTA (http://fasta.bioch.virginia.edu/ fasta_www/cgi/search_frm2.cgi).
3. Results 3.1. The general features of T. evansi JN 2118Hu VSG Two plasmids designated T. evansi JN 2118Hu VSG cDNA clone 1 and JN 2118Hu VSG cDNA clone 2 were sequenced completely on both strands. The predicted protein of the T. evansi JN 2118Hu VSG cDNA comprised 443 amino acids as shown in Figs. 1 and 2. The full-length VSG cDNA sequences of T. evansi JN 2118Hu clone 1 (1472 bp) and clone 2 (1490 bp) were highly similar to one another and had identical amino acids (99.55% similarity). A comparison of the predicted coding regions of T. evansi RoTat 1.2 VSG cDNA Accession No. AF317914 (Urakawa et al., 2001) and T. evansi JN 2118Hu VSG cDNA displayed 25.16% identity. Similarity searches of nucleotide and protein sequences in the GenBank/NCBI database found homology with trypanosome VSG sequences. The search on the predicted coding region displayed 75 protein sequences with signiWcant alignments, 32 of which were trypanosome VSG sequences. Identities (%) over the full-length of the protein were observed in six VSG sequences: T.b. rhodesiense MVAT4 VSG Accession No. AF068693.1 (20%), T. brucei VSG Accession No. U03710.1 (20%), T.b. rhodesiense metacyclic VSG sequence P02897 (20%),
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
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Fig. 1. The complete nucleotide and deduced amino acid sequence of T. evansi JN 2118Hu VSG cDNA clone 1. The full cDNA is 1472 bp. The ATG and TAA start and stop codons are underlined. The precursor VSG molecule constitutes 443 amino acid residues that include 17 residues of the Cterminal hydrophobic extension (underlined) and the signal peptide cleavage site between position 16 and 17 amino acid residues. The mini-exon and a 16-mer conserved among VSG mRNAs are underlined. Nucleotide sequences of the two internal primers used in the PCR ampliWcation of the T. evansi 2118 Hu VSG gene are underlined in bold. The EMBL accession number for this sequence is AJ870486.
T.b. brucei MITat 1.5 VSG P26333 (23%), T. evansi VSG Accession No. AY550977.1 (21%), T. evansi VSG Accession No. AF317926.1 (21%) and T. evansi VSG Accession No. AF317928.1 (20%). The C-terminal 100–150 amino acids of T. evansi JN 2118Hu VSG sequence displayed identities (%) with the corresponding regions in VSG sequences including T. brucei Accession No.
XM_340733.1 (35%), T.b. rhodesiense WRATatA P20946 (26%), T. evansi Accession No. AF317932.1 (41%), and T. brucei Accession No. AF294807.1 (36%). The similarity search on the full-length cDNA showed two blocks of sequences with high identities (96–100%): the spliced leader sequence (mini-exon) at the 5⬘ end and the conserved nucleotide sequence found in the 3⬘ untranslated
34
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Fig. 2. The complete nucleotide and deduced amino acid sequence of T. evansi JN 2118Hu VSG cDNA clone 2. The full cDNA is 1490 bp. The ATG and TAA start and stop codons are underlined. The precursor VSG molecule constitutes 443 amino acid residues that include 17 residues of the Cterminal hydrophobic extension (underlined) and the signal peptide cleavage site between position 16 and 17 amino acid residues. The mini-exon and a 16-mer conserved among VSG mRNAs are underlined. Nucleotide sequences of the two internal primers used in the PCR ampliWcation of the T. evansi 2118 Hu VSG gene are underlined in bold. The EMBL accession number for this sequence is AJ870487.
region of VSG mRNAs. When the nucleotide sequence (1332 bp) constituting the predicted coding region was used in similarity searches, no identities were present in the 5⬘ region of the sequence. High identities (84–95%) in the last 100 nucleotides of the 3⬘ end were present in the corresponding regions of trypanosome VSG cDNAs in the database.
The open reading frame of T. evansi JN 2118Hu VSG cDNA encoded a protein molecule with characteristics typical of VSGs, such as a signal peptide, a cysteine-rich C-terminal domain and a hydrophobic tail (Holder and Cross, 1981; Rice-Ficht et al., 1981). T. brucei VSGs are classiWed into three types of N-terminal domains and four types of C-terminal domains based on the primary struc-
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ture of the VSG (Carrington et al., 1991). The number and position of conserved cysteine residues and the potential N-glycosylation sites determine the domain type of the VSG. Our data showed that T. evansi JN 2118Hu VSG belongs to the type A of N-terminal domain and type 2 of C-terminal domain. The computer program (http:// www.cbs.dtu.dk, Nielsen and Anders Krogh, 1998; Nielsen et al., 1997) was used to determine the cleavage site of the signal peptide between amino acid residue 16 and 17 (Fig. 1). 3.2. The 273 bp diagnostic sequence Within the coding region of T. evansi JN 2118Hu, a 273 bp DNA segment was identiWed that lacked similarity with any other known trypanosome sequence in the databases. The restriction enzyme sites in the 273 bp sequence were determined using the computer programme http://tools.neb.com/NEBcutter2/index.php which displayed 27 single-cutter restriction enzymes (not shown). According to the computer analysis, digestion with HaeIII yielded two fragments, 124 and 149 bp. A pair of primers Xanking the 273 bp fragment was used in PCR to amplify the genomic DNA puriWed from various trypanosome populations including T. evansi, T. brucei, Trypanosoma vivax, Trypanosoma congolense. As control, a 488 bp fragment was ampliWed using primers derived from RoTat 1.2 VSG gene (Urakawa et al., 2001). PCR results showed that out of 32 T. evansi tested, Wve were positive for the expected 273 bp fragment while the rest of the T. evansi were negative. Fig. 3A shows the Wve positive samples and a representative 11 samples of the T. evansi samples that tested negative. Twenty-seven T. evansi were positive for the 488 bp fragment and Wve were negative. Fig. 3B shows a representative 11 of the positive samples. It shows all the Wve negative T. evansi. Fig. 3C shows that there were no products ampliWed from T. brucei samples. T. congolense and T. vivax samples were all negative (not shown). For convenience, T. evansi isolates that were not detected by RoTat 1.2-based PCR were referred to as non-RoTat 1.2 T. evansi. The sequence identity of the 273 bp PCR products was assessed by digestion using the restriction enzyme and HaeIII. Gel electrophoresis of the digest products displayed a common pattern in all the Wve non-RoTat 1.2 T. evansi, two of which are shown in Fig. 4. The restriction pattern was in agreement with the RE sites determined by computer (http://tools.neb.com/NEBcutter2/index.php). Due to lack of resolution however, the two bands 124 and 149 bp appeared as one band as shown in lanes 1 and 2 containing the digest products of samples JN 2118Hu and KETRI 2479, respectively. Lanes 3 contains the undigested 273 bp PCR product of sample JN 2118Hu.
35
4. Discussion The primary goal of this study was to determine the nucleotide sequence composition of T. evansi JN 2118Hu VSG and examine it for possible use in identiWcation of T. evansi populations that are undetectable by the currently available RoTat1.2-based tests. First, the structural features of the cloned molecule were examined to verify that the molecule was a trypanosome VSG. Mature polypeptides of trypanosome VSGs have an approximate molecular weight of 60,000 Mr (400–500 amino acid residues) and have at least one N-linked oligosaccharide (Cross, 1984). Other structural characteristics include a signal peptide, a cysteine-rich C-terminal domain, and a hydrophobic tail (Holder and Cross, 1981; Rice-Ficht et al., 1981). As shown in Figs. 1 and 2, the VSG cDNA from T. evansi JN 2118Hu encodes a protein with VSG C-terminal homologies (Carrington et al., 1991). The precursor VSG molecule is 443 amino acids that are processed to a mature protein by cleavage of the signal peptide and 17 amino acids of C-terminal extension. The 5⬘ untranslated region of the cDNA shows the mini-exon (Cornelissen et al., 1986) while the 3⬘ untranslated region shows the 16-mer oligonucleotide conserved at that position in all VSG mRNAs of trypanosomes of Trypanozoon (Majumder et al., 1981). These two sequence blocks indicate that our data represents a complete VSG gene. A search was made by BLAST to identify a nucleotide sequence in T. evansi JN 118Hu VSG cDNA that had no similarity with any other known trypanosome DNA sequence in the databases. As expected, sequences in the C-terminal region were unsuitable because of homologies with other trypanosome VSG sequences (Holder and Cross, 1981; Rice-Ficht et al., 1981). However, primary sequences within the N-terminal region of VSGs are extremely diverse, with little conservation (Rice-Ficht et al., 1981). It is in this region that a sequence was identiWed that had no similarity with other trypanosome sequences in the databases. The 273 bp fragment was ampliWed by PCR as described. Interestingly, fragments of identical size were ampliWed in four other T. evansi besides T. evansi JN 2118Hu. All the Wve positive samples were non-RoTat 1.2 T. evansi. Contamination can lead to artifacts in PCR ampliWcation, hence the need to assess the sequence identity of the PCR products. This was performed by restriction enzyme analysis of the 273 bp amplicons. The sites of single cutter restriction enzymes in the sequenced T. evansi JN 2118Hu VSG cDNA were determined by computer (http://tools.neb.com/NEBcutter2/index.php). To determine the restriction fragment length polymorphisms (RLFPs) in the 273 bp PCR products, digestion was carried out using the enzyme HaeIII which produced a common restriction pattern in all Wve non-RoTat 1.2 T. evansi DNA samples. The pattern agreed with that
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Fig. 3. PCR detection of non-RoTat 1.2 T. evansi. Gel electrophoresis (1.5% agarose, stained with ethidium bromide), showing PCR products from genomic DNA prepared from trypanosome populations. (A) Lane (M) 1 kb Plus DNA ladder (Invitrogen). The 273 bp PCR products ampliWed with T. evansi JN 2118Hu VSG-based primers are in lanes (1–16) as follows: (1) T. evansi JN 2118Hu; (2) T. evansi KETRI 2479; (3) T. evansi JN 4306Moh; (4) JN T. evansi JN 6512Muh; (5) T. evansi JN 6524Muh; (6) T. evansi RoTat 1.2; (7) T. evansi AnTat 3.3; (8) T. evansi KETRI 2455; (9) T. evansi KETRI 3117; (10) T. evansi KETRI 3261 (11) T. evansi JN 4314Moh; (12) T. evansi JN 4315Moh; (13) T. evansi JN 5480Dai; (14) T. evansi JN 6507Ru; (15) T. evansi JN 3235Abd; and (16) T. evansi JN 4394Ha. (B) Lane (M) 1 kb Plus DNA ladder (Invitrogen). The 488 bp PCR products ampliWed with RoTat 1.2 VSG-based primers are in lanes (1–16) as follows: (1) T. evansi RoTat 1.2; (2) T. evansi AnTat 3.3; (3) T. evansi KETRI 2455; (4) T. evansi KETRI 3117; (5) T. evansi KETRI 3261; (6) T. evansi JN 4314Moh; (7) T. evansi JN 4315Moh; (8) T. evansi JN 5480Dai; (9) T. evansi JN 6507Ru; (10) T. evansi JN 3235Abd; (11) T. evansi JN 4394Ha; (12) T. evansi JN 2118Hu; (13) T. evansi KETRI 2479; (14) T. evansi JN 4306Hu; (15) T. evansi JN 6512Muh; and (16) T. evansi JN 6524Muh. (C) Lane (M) 1 kb Plus DNA ladder (Invitrogen). The 273 bp PCR products ampliWed with T. evansi JN 2118Hu VSG-based primers are in lanes (1–7) as follows: (1) T. evansi JN 2118Hu; (2) T. brucei KETRI 3596; (3) T. brucei KETRI 2710; (4) T. brucei IIE1; (5) T. brucei ILTat1.1; (6) T.b. rhodesiense WaTat1.2; and (7) T.b. gambiense LiTat1.1.
produced from computer analysis of the cDNA sequence (http://tools.neb.com/NEBcutter2/index.php), inferring that the amplicons were speciWc and not produced by non-speciWc ampliWcation of DNA or by multimer formation of the PCR primers. The fact that the 273 bp fragment of the cDNA showed no similarities with trypanosome sequences in the database strongly suggested that this fragment was unique to the strain of T. evansi
described in this study. Whether Southern blots (Southern, 1975) would show similar results in the genomes of diVerent trypanosomes strains, remains to be investigated in future studies in this laboratory. Urakawa et al. (2001) using the DNA fragment that constitutes the RoTat 1.2 VSG open reading frame as hybridization probe observed that DNA sequences homologous to the RoTat 1.2 VSG gene were present in the genomes of
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
Fig. 4. Restriction enzyme digestion pattern of 273 bp amplicon gel electrophoresis (1.5% agarose, stained with ethidium bromide), showing 273 bp PCR products puriWed from gel and digested with restriction enzyme HaeIII. One kilo base Plus DNA ladder (lane M). T. evansi JN 2118Hu, 273 bp amplicon digested with HaeIII (lane 1); T. evansi KETRI 2479, 273 bp amplicon digested with HaeIII (lane 2); and T. evansi JN 2118Hu, 273 bp amplicon undigested (lane 3).
other trypanosomes, yet a 488 bp section of the fragment was speciWc for T. evansi by PCR ampliWcation. The sensitivity of the PCR test developed in this study can only be determined through the testing of a large number of isolates of diVerent geographical origin. For now, this test was 100% (Wve out of Wve) sensitive for non-RoTat 1.2 T. evansi examined in this study. The Wve samples that tested positive for the expected 273 bp fragment of the T. evansi JN 2118Hu VSG gene are the same that tested negative for the 488-bp fragment of the RoTat 1.2 VSG gene. The described test is an additional diagnostic tool for the detection of non RoTat 1.2 T. evansi, but may not necessarily detect all strains of nonRoTat 1.2 T. evansi as these are yet unknown. Regional genetic diVerences in T. evansi isolates may be present in Kenya, but this awaits conWrmation (Njiru et al., 2004). It has been argued that VSG sequences are not reliable diagnostic targets due to the large repertoire of VSG genes present in bloodstream trypanosomes (Radwanska et al., 2002). In T. evansi however, it is possible that the parasites’ potential for genetic variation is limited by the non-cyclical method of transmission (Boid, 1988), and this would make the use of VSG-based diagnosis more feasible in T. evansi than in cyclically transmitted trypanosomes. Studies have shown that the large majority of T. evansi isolates of diverse geographic origin belong to a major Type A group (Gibson et al., 1983). A minor group containing type B minicircles has been reported only in Kenya and the question now arises whether this group is the same as the non-RoTat 1.2 T. evansi described in this study. Claes et al. (2004) speculate that the non-RoTat 1.2 T. evansi reported by Ngaira et al. (2003) correspond to type B group and that all other T. evansi isolated elsewhere are the classical T. evansi type A group. Importantly, this would imply that
37
RoTat 1.2 VSG-based PCR tests (Claes et al., 2004; Urakawa et al., 2001) combined with the PCR test developed in this study could provide the sensitivity needed to detect the large majority of T. evansi. The potential of VSGs as diagnostic reagents is further demonstrated by the use of a VSG variant from the TEVA1 T. evansi Venezuelan isolate (Uzcanga et al., 2004). Moreover, dyskinetoplastic T. evansi strains that lack kDNA can be detected using VSG-based tests. In conclusion therefore, this study has shown that PCR ampliWcation of the internal 273 bp fragment of the T. evansi JN 2118Hu VSG gene can provide a convenient and reliable detection of T. evansi infections that would otherwise remain undetected by the current RoTat 1.2based tests.
Acknowledgments The authors funded this study. We are grateful for laboratory facilities at Kenya Trypanosomosis Research Institute (KETRI) and at International Livestock Research Institute (ILRI).
References Altschul, S.F., Madden, T.L., SchaVer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research 25 (17), 3389–3402. Bajyana, S.E., Hamers, R., 1988. A card agglutination test (CATT) for veterinary use based on an early VAT RoTat1/2 of Trypanosoma evansi. Annales de la Société Belge de Médecine Tropicale 68, 233–240. Boid, R., 1988. Isoenzyme characterisation of 15 stocks of Trypanosoma evansi isolated from camels in the Sudan. Tropical Medicine and Parasitology 39, 45–50. Borst, P., Fase-Fowler, F., Gibson, W.C., 1987. Kinetoplast DNA of Trypanosoma evansi. Molecular and Biochemical Parasitology 23, 31–38. Carrington, M., Miller, N., Blum, M., Roditi, I., Wiley, D., Turner, M., 1991. Variant speciWc glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. Journal of Molecular Biology 221, 823–835. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analtycal Biochemistry 162, 156–159. Claes, F., Radwanska, M., Urakawa, T., Majiwa, P.A., Goddeeris, B.M., Buscher, P., 2004. Variable surface glycoprotein RoTat1.2 PCR as a speciWc diagnostic tool for the detection of Trypanosoma evansi infections. Kinetoplastid Biology and Disease 3, 3. Cornelissen, A.W., Verspieren, M.P., Toulme, J.J., Swinkels, B.W., Borst, P., 1986. The common 5⬘ terminal sequence on trypanosome mRNAs: A target for anti-messenger oligodeoxynucleotides. Nucleic Acids Research 14, 5605–5614. Cross, G.A., 1984. Structure of the variant glycoproteins and surface coat of Trypanosoma brucei. Philosophical Transactions of the Royal Society of London 307, 3–12. Gibson, W.C., Wilson, A.J., Moloo, S.K., 1983. Characterisation of Trypanosoma (Trypanozoon) evansi from camels in Kenya using isoenzyme electrophoresis. Research in Veterinary Science 34, 114–118.
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Hoare, C.A., 1972. The Trypanosomes of Mammals: A Zoological Monograph, Blackwell ScientiWc Publication, Oxford and Edinburgh, UK. Holder, A.A., Cross, G.A., 1981. Glycopeptides from variant surface glycoproteins of Trypanosoma brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties. Molecular and Biochemical Parasitology 2, 135–150. Lanham, S.M., Godfrey, D.G., 1970. Isolation of salivarian trypanosomes from man and other mammals using DEAE–cellulose. Experimental Parasitology 28, 521–534. Lun, Z.R., Li, A.X., Chen, X.G., Lu, L.X., Zhu, X.Q., 2004. Molecular proWles of Trypanosoma brucei, T. evansi and T. equiperdum stocks revealed by the random ampliWed polymorphic DNA method. Parasitology Research 92, 335–340. Majumder, H.K., Boothroyd, J.C., Weber, H., 1981. Homologous 3⬘terminal regions of mRNAs for surface antigens of diVerent antigenic variants of Trypanosoma brucei. Nucleic Acids Research 9, 4745–4753. Maniatis, T., Fritsch, E.F., Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. N1 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Ngaira, J.M., Bett, B., Karanja, S.M., Njagi, E.N., 2003. Evaluation of antigen and antibody rapid detection tests for Trypanosoma evansi infection in camels in Kenya. Veterinary Parasitology 114, 131– 141. Ngaira, J.M., Njagi, E.N., Ngeranwa, J.J., Olembo, N.K., 2004. PCR ampliWcation of RoTat 1.2 VSG gene in Trypanosoma evansi isolates in Kenya. Veterinary Parasitology 120, 23–33. Nielsen, H., Anders Krogh, A., 1998. Prediction of signal peptides and signal anchors by a hidden Markov model. In: Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, California, pp. 122–130. Nielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. IdentiWcation of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10, 1–6.
Njiru, Z.K., Constantine, C.C., Ndung’u, J.M., Robertson, I., Okaye, S., Thompson, R.C., Reid, S.A., 2004. Detection of Trypanosoma evansi in camels using PCR andCATT/T. evansi tests in Kenya. Veterinary Parasitolgy 124, 187–199. Radwanska, M., Chamekh, M., Vanhamme, L., Claes, F., Magez, S., Magnus, E., de Baetselier, P., Buscher, P., Pays, E., 2002. The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense. American Journal of Tropical Medicine and Hygiene 67, 684–690. Rice-Ficht, A.C., Chen, K.K., Donelson, J.E., 1981. Sequence homologies near the C-termini of the variable surface glycoproteins of Trypanosoma brucei. Nature 294, 53–57. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, United States of America 74, 5463–5467. Southern, E.M., 1975. Detection of speciWc sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503–517. Urakawa, T., Verloo, D., Moens, L., Buscher, P., Majiwa, P.A., 2001. Trypanosoma evansi: Cloning and expression in Spodoptera frugiperda [correction of fugiperda] insect cells of the diagnostic antigen RoTat 1.2. Experimental Parasitology 99, 181–189. Uzcanga, G.L., Perrone, T., Noda, J.A., Perez-Pazos, J., Medina, R., Hoebeke, J., Bubis, J., 2004. Variant surface glycoprotein from Trypanosoma evansi is partially responsible for the cross-reaction between Trypanosoma evansi and Trypanosoma vivax. Biochemistry 43, 595–606. Van der Ploeg, L.H., Valerio, D., De Lange, T., Bernards, A., Borst, P., Grosveld, F.G., 1982. An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome. Nucleic Acids Research 10, 5905–5923. Verloo, D., Holland, W., My, L.N., Thanh, N.G., Tam, P.T., Goddeeris, B., Vercruysse, J., Buscher, P., 2000. Comparison of serological tests for Trypanosoma evansi natural infections in water buValoes from North Vietnam. Veterinary Parasitology 92, 87–96.
The detection of non-RoTat 1.2 Trypanosoma evansi J.M. Ngaira a b
a,b,¤
, N.K. Olembo c, E.N.M. Njagi b, J.J.N. Ngeranwa
b
Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 25530, Nairobi, Kenya Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844, Nairobi, Kenya c Department of Biochemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya Received 19 May 2004; received in revised form 23 December 2004; accepted 10 January 2005
Abstract The majority of Trypanosoma evansi can be detected using diagnostic tests based on the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2. Exceptions are a number of T. evansi isolated in Kenya. To characterize T. evansi that are undetected by RoTat 1.2, we cloned and sequenced the VSG cDNA from T. evansi JN 2118Hu, an isolate devoid of the RoTat 1.2 VSG gene. A 273 bp DNA segment of the VSG gene was targeted in PCR ampliWcation for the detection of non-RoTat 1.2 T. evansi. Genomic DNA samples from diVerent trypanosomes were tested including 32 T. evansi, 10 Trypanosoma brucei, three Trypanosoma congolense, and one Trypanosoma vivax. Comparison was by PCR ampliWcation of a 488 bp fragment of RoTat1.2 VSG gene. Results showed that the expected 273 bp ampliWcation product was present in all Wve non-RoTat 1.2 T. evansi tested and was absent in all 27 RoTat 1.2-positive T. evansi tested. It was also absent in all other trypanosomes tested. The PCR test developed in this study is speciWc for non-RoTat 1.2 T. evansi. 2005 Elsevier Inc. All rights reserved. Keywords: Trypanosoma evansi; Variant surface glycoprotein; Diagnosis; Kenya
1. Introduction Three species comprise the subgenus Trypanozoon: Trypanosoma brucei, Trypanosoma evansi, and Trypanosoma equiperdum, with T. brucei further subdivided into three subspecies: T.b. brucei, T.b. rhodesiense, and T.b. gambiense (Hoare, 1972). Trypanosomes of the three species are morphologically indistinguishable save for short-stumpy forms that occur in T. brucei. A key distinguishing feature is that T. evansi and T. equiperdum lack functional mitochondria containing maxicircle kinetoplast DNA (kDNA) and are therefore unable to complete cyclical development in the tsetse Xy vector that transmits other African trypanosomes (Borst et al., 1987). Whereas there are no maxicircles in T. evansi, the
¤
Corresponding author. Fax: +254 568695. E-mail address: [email protected] (J.M. Ngaira).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.01.001
maxicircles in T. equiperdum are incomplete (Lun et al., 2004). Mechanical transmission by blood sucking insects has allowed T. evansi to spread in Africa, Asia, and South and Central America where it causes the disease “surra” in a wide range of animals including camels, horses, cattle, and buValo as well as wild animals. The variant surface glycoprotein (VSG) of T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 is a diagnostic antigen for T. evansi from diverse geographical regions. Serological and PCR tests based on the VSG of RoTat 1.2 have shown high speciWcity and sensitivity in studies in diVerent geographical regions of the world (Bajyana and Hamers, 1988; Urakawa et al., 2001; Verloo et al., 2000). It has been shown however, that a number of T. evansi in Kenya were not detected by tests based on RoTat1.2 VSG and four of the isolates lacked the RoTat1.2 VSG gene (Ngaira et al., 2004). A recent study in Kenya (Njiru et al., 2004) showed that RoTat 1.2-based CATT/T. evansi was 65.5% sensitive, agreeing
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
well with the 68.6% sensitivity observed earlier Ngaira et al. (2003) and emphasizing the need to address the problem of diagnosis of T. evansi in the region. The main objective of this study was to Wnd a diagnostic reagent for T. evansi infections that are not detected by RoTat 1.2 VSG-based tests. Our approach was to identify
Table 1 Trypanosoma evansi isolated from camels in Isiolo District, Kenya between 1996 and 1997 Stock
Date of isolation
Place of isolation
JN 2118Hu JN 4394Ha JN 5460Ke JN 3234Abd JN 3235Abd JN 3270Sa JN 4304Moh JN 4314Moh JN 4315Moh JN 4357Jum JN 4306Moh JN 5480Dai JN 6507Ru JN 6512Muh JN 6524Muh JN 4385Bo JN 3238Abd JN 3230Abd JN 4384Ha JN 4309Moh
30 October 1996 17 January 1997 7 June 1997 6 July 1997 6 July 1997 7 July 1997 7 July 1997 7 July 1997 7 July 1997 9 July 1997 10 July 1997 10 July 1997 10 July 1997 10 July 1997 10 July 1997 17 July 1997 6 July 1997 6 July 1997 17 January 1997 7 July 1997
Burat Burat Dahyal Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Mlango Burat Mlango
31
sequences of diagnostic potential in the VSG cDNA of a non-RoTat 1.2 T. evansi isolate.
2. Materials and methods Trypanosomes previously isolated from camels (Ngaira et al., 2003, 2004) were used in this study (Table 1). Reference trypanosomes from the KETRI cryobank and puriWed DNA samples obtained from ILRI are shown in Table 2. 2.1. PuriWcation of trypanosomes Trypanosomes were grown in rats and were separated from rat blood cells by anion-exchange chromatography with DEAE–cellulose (DE52, Whatman Biochemical) (Lanham and Godfery, 1970) as previously described (Ngaira et al., 2004). 2.2. Preparation of genomic DNA and total RNA Genomic DNA was extracted from the trypanosomes by standard methods (Van der Ploeg et al., 1982) and dissolved in 10mM Tris–HCl, pH 7.4 or water at 500g/ml. It was stored at 4°C or ¡20°C until needed. Total RNA was extracted from the trypanosomes using the single-step method described by Chomczynski and Sacchi (1987). The extracted RNA was kept frozen at ¡80°C until needed.
Table 2 Reference trypanosomes Designation
Species
Host
Locality, country where isolated
Year of isolation
KETRI 3094 KETRI 2440 KETRI 2454 KETRI 2455 KETRI 2448 KETRI 2479 KETRI 3261 KETRI 3572 KETRI 3117 RoTat 1.2 IL3960a IL3927a AnTat3.3a KETRI 2710 KETRI 3596 11E1a ILTat1.1a ILTat1.3a ILTat1.4a WaTat1.2a LiTat1.1a IL3707a IL2569a IL3662a IL3665a IL1180
T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. evansi T. brucei T. brucei T. brucei T. brucei T. brucei T. brucei T.b. rhodesiense T.b. gambiense T.b. gambiense T. vivax T. congolense T. congolense T. congolense
Horse Camel Camel Camel Camel Camel Camel Camel Camel BuValo Camel Camel Capybara Not known Not known Cow Cow Cow Cow Man Man Man Tsetse Cow Cow Lion
South America Kulal, Kenya Kulal, Kenya Galana, Kenya Galana, Kenya Ngurunit Samburu, Kenya Nanyuki, Kenya Athi River Pekalongan, Indonesia Marsbit, Kenya Ipal, India South America Not known Apatit Teso, Kenya Uhembo, Kenya Uhembo, Kenya Uhembo, Kenya Uhembo, Kenya Nyanza, Kenya Cote d’Ivoire Bida, Nigeria Lugala, Uganda Transmara, Kenya Transmara, Kenya Serengeti, Tanzania
1973 1979 1954 1979 1979 1981 1990 1994 1988 1982 1980 Not known 1971 Not known 1994 1964 1964 1964 1964 1961 1952 1968 1969 1966 1966 1971
a
DNA samples obtained from ILRI.
32
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
2.3. One-step RT-PCR
2.6. PCR ampliWcation of T. evansi JN 2118Hu VSG gene
To amplify only VSG-encoding transcripts, RT-PCR ampliWcation was performed using a forward primer designed to base pair with the reverse complement of the 3⬘ part of the mini-exon found in the 5⬘end of trypanosome mRNAs (Cornelissen et al., 1986) and a reverse primer designed to base pair with a conserved nucleotide sequence found in the 3⬘ untranslated region of VSG mRNAs of trypanosomes within the Trypanozoon (Majumder et al., 1981). The forward primer used was IL07725 (5⬘-CGG GTA CCT AGA ACA GTT TCT GTA CTA TAT TG-3⬘) designed to contain a KpnI site (Urakawa et al., 2001) and the reverse primer was IL07722 (CGG GAT CCA GGT GTT AAA ATA TA3⬘) designed to contain a BamHI site (Urakawa et al., 2001). The reaction was carried out using Qiagen OneStep RT-PCR kit according to the protocol provided by the supplier. BrieXy, a 50 l reaction mixture was prepared containing a Wnal concentration of 2.0 g total RNA, 0.6 M each primer, 400 M each dNTP, an optimal Qiagen OneStep RT-PCR Enzyme Mix and 1£ RT-PCR buVer. It was incubated at 50 °C for 30 min, followed by 95 °C for 15 min, followed by a three-step cycling of 34 cycles (denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min) and a Wnal extension at 72 °C for 10 min.
A segment of T. evansi JN 2118Hu VSG gene that lacked homology with sequences in the databanks was identiWed by BLAST homology searches. Two primers were designed to amplify the 273 bp fragment: forward primer (5⬘-TTCTACCAACTGACGGAGCG-3⬘) and reverse primer (5⬘-TAGCTCCGGATGCATCGGT-3⬘). PCR ampliWcation was carried out in 20 l Wnal reaction mixtures containing 100 ng genomic DNA, 10 mM Tris– HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 M each of the four dNTPs, 10 M each of the oligonucleotide primers, and 1.0 U Taq (Thermus aquaticus) DNA polymerase (Promega). The ampliWcation parameters were 94 °C for 1 min followed by 40 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min) followed by a Wnal extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis in a 1.5% agarose gel. The expected size of the ampliWed DNA fragment was 273 bp. The restriction enzyme sites in the 273 bp fragment of the sequenced cDNA were determined using the computer programme http://tools.neb.com/NEBcutter2/ index.php. To assess the sequence identity of the 273 bp PCR products, the DNA bands were puriWed from gel using QIAquick Gel Extraction kit (Qiagen) and digested with the restriction enzyme HaeIII using standard procedures (Maniatis et al., 1982).
2.4. Cloning and of PCR products The products of RT-PCR were resolved by electrophoreses in a 0.8% agarose gel and the expected band of approximately 1.6 kb eluted from the gel and cloned into the pGEM-T easy vector (Promega). The plasmids were used to transform Escherichia coli, which were subsequently plated on selective media for the isolation of recombinants. Plasmids were puriWed from individual bacterial colonies using the Wizard Plus SV Minipreps DNA PuriWcation System (Promega), and the sizes of inserts determined by PCR using vector primers. The nucleotide sequence of the inserts in the plasmids was determined commercially (Macrogene) by the procedure of Sanger (Sanger et al., 1977) using automatic DNA sequencing. Both strands of the VSG cDNA were completely sequenced. 2.5. Sequence similarity searches Similarity searches of GenBank/NCBI database were performed with the basic local alignment search program (BLAST) http://www.ncbi.nlm.nih.gov/BLAST/ (Altschul et al., 1997) using the default matrix. Alignment of two sequences was performed using the program FASTA (http://fasta.bioch.virginia.edu/ fasta_www/cgi/search_frm2.cgi).
3. Results 3.1. The general features of T. evansi JN 2118Hu VSG Two plasmids designated T. evansi JN 2118Hu VSG cDNA clone 1 and JN 2118Hu VSG cDNA clone 2 were sequenced completely on both strands. The predicted protein of the T. evansi JN 2118Hu VSG cDNA comprised 443 amino acids as shown in Figs. 1 and 2. The full-length VSG cDNA sequences of T. evansi JN 2118Hu clone 1 (1472 bp) and clone 2 (1490 bp) were highly similar to one another and had identical amino acids (99.55% similarity). A comparison of the predicted coding regions of T. evansi RoTat 1.2 VSG cDNA Accession No. AF317914 (Urakawa et al., 2001) and T. evansi JN 2118Hu VSG cDNA displayed 25.16% identity. Similarity searches of nucleotide and protein sequences in the GenBank/NCBI database found homology with trypanosome VSG sequences. The search on the predicted coding region displayed 75 protein sequences with signiWcant alignments, 32 of which were trypanosome VSG sequences. Identities (%) over the full-length of the protein were observed in six VSG sequences: T.b. rhodesiense MVAT4 VSG Accession No. AF068693.1 (20%), T. brucei VSG Accession No. U03710.1 (20%), T.b. rhodesiense metacyclic VSG sequence P02897 (20%),
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
33
Fig. 1. The complete nucleotide and deduced amino acid sequence of T. evansi JN 2118Hu VSG cDNA clone 1. The full cDNA is 1472 bp. The ATG and TAA start and stop codons are underlined. The precursor VSG molecule constitutes 443 amino acid residues that include 17 residues of the Cterminal hydrophobic extension (underlined) and the signal peptide cleavage site between position 16 and 17 amino acid residues. The mini-exon and a 16-mer conserved among VSG mRNAs are underlined. Nucleotide sequences of the two internal primers used in the PCR ampliWcation of the T. evansi 2118 Hu VSG gene are underlined in bold. The EMBL accession number for this sequence is AJ870486.
T.b. brucei MITat 1.5 VSG P26333 (23%), T. evansi VSG Accession No. AY550977.1 (21%), T. evansi VSG Accession No. AF317926.1 (21%) and T. evansi VSG Accession No. AF317928.1 (20%). The C-terminal 100–150 amino acids of T. evansi JN 2118Hu VSG sequence displayed identities (%) with the corresponding regions in VSG sequences including T. brucei Accession No.
XM_340733.1 (35%), T.b. rhodesiense WRATatA P20946 (26%), T. evansi Accession No. AF317932.1 (41%), and T. brucei Accession No. AF294807.1 (36%). The similarity search on the full-length cDNA showed two blocks of sequences with high identities (96–100%): the spliced leader sequence (mini-exon) at the 5⬘ end and the conserved nucleotide sequence found in the 3⬘ untranslated
34
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
Fig. 2. The complete nucleotide and deduced amino acid sequence of T. evansi JN 2118Hu VSG cDNA clone 2. The full cDNA is 1490 bp. The ATG and TAA start and stop codons are underlined. The precursor VSG molecule constitutes 443 amino acid residues that include 17 residues of the Cterminal hydrophobic extension (underlined) and the signal peptide cleavage site between position 16 and 17 amino acid residues. The mini-exon and a 16-mer conserved among VSG mRNAs are underlined. Nucleotide sequences of the two internal primers used in the PCR ampliWcation of the T. evansi 2118 Hu VSG gene are underlined in bold. The EMBL accession number for this sequence is AJ870487.
region of VSG mRNAs. When the nucleotide sequence (1332 bp) constituting the predicted coding region was used in similarity searches, no identities were present in the 5⬘ region of the sequence. High identities (84–95%) in the last 100 nucleotides of the 3⬘ end were present in the corresponding regions of trypanosome VSG cDNAs in the database.
The open reading frame of T. evansi JN 2118Hu VSG cDNA encoded a protein molecule with characteristics typical of VSGs, such as a signal peptide, a cysteine-rich C-terminal domain and a hydrophobic tail (Holder and Cross, 1981; Rice-Ficht et al., 1981). T. brucei VSGs are classiWed into three types of N-terminal domains and four types of C-terminal domains based on the primary struc-
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
ture of the VSG (Carrington et al., 1991). The number and position of conserved cysteine residues and the potential N-glycosylation sites determine the domain type of the VSG. Our data showed that T. evansi JN 2118Hu VSG belongs to the type A of N-terminal domain and type 2 of C-terminal domain. The computer program (http:// www.cbs.dtu.dk, Nielsen and Anders Krogh, 1998; Nielsen et al., 1997) was used to determine the cleavage site of the signal peptide between amino acid residue 16 and 17 (Fig. 1). 3.2. The 273 bp diagnostic sequence Within the coding region of T. evansi JN 2118Hu, a 273 bp DNA segment was identiWed that lacked similarity with any other known trypanosome sequence in the databases. The restriction enzyme sites in the 273 bp sequence were determined using the computer programme http://tools.neb.com/NEBcutter2/index.php which displayed 27 single-cutter restriction enzymes (not shown). According to the computer analysis, digestion with HaeIII yielded two fragments, 124 and 149 bp. A pair of primers Xanking the 273 bp fragment was used in PCR to amplify the genomic DNA puriWed from various trypanosome populations including T. evansi, T. brucei, Trypanosoma vivax, Trypanosoma congolense. As control, a 488 bp fragment was ampliWed using primers derived from RoTat 1.2 VSG gene (Urakawa et al., 2001). PCR results showed that out of 32 T. evansi tested, Wve were positive for the expected 273 bp fragment while the rest of the T. evansi were negative. Fig. 3A shows the Wve positive samples and a representative 11 samples of the T. evansi samples that tested negative. Twenty-seven T. evansi were positive for the 488 bp fragment and Wve were negative. Fig. 3B shows a representative 11 of the positive samples. It shows all the Wve negative T. evansi. Fig. 3C shows that there were no products ampliWed from T. brucei samples. T. congolense and T. vivax samples were all negative (not shown). For convenience, T. evansi isolates that were not detected by RoTat 1.2-based PCR were referred to as non-RoTat 1.2 T. evansi. The sequence identity of the 273 bp PCR products was assessed by digestion using the restriction enzyme and HaeIII. Gel electrophoresis of the digest products displayed a common pattern in all the Wve non-RoTat 1.2 T. evansi, two of which are shown in Fig. 4. The restriction pattern was in agreement with the RE sites determined by computer (http://tools.neb.com/NEBcutter2/index.php). Due to lack of resolution however, the two bands 124 and 149 bp appeared as one band as shown in lanes 1 and 2 containing the digest products of samples JN 2118Hu and KETRI 2479, respectively. Lanes 3 contains the undigested 273 bp PCR product of sample JN 2118Hu.
35
4. Discussion The primary goal of this study was to determine the nucleotide sequence composition of T. evansi JN 2118Hu VSG and examine it for possible use in identiWcation of T. evansi populations that are undetectable by the currently available RoTat1.2-based tests. First, the structural features of the cloned molecule were examined to verify that the molecule was a trypanosome VSG. Mature polypeptides of trypanosome VSGs have an approximate molecular weight of 60,000 Mr (400–500 amino acid residues) and have at least one N-linked oligosaccharide (Cross, 1984). Other structural characteristics include a signal peptide, a cysteine-rich C-terminal domain, and a hydrophobic tail (Holder and Cross, 1981; Rice-Ficht et al., 1981). As shown in Figs. 1 and 2, the VSG cDNA from T. evansi JN 2118Hu encodes a protein with VSG C-terminal homologies (Carrington et al., 1991). The precursor VSG molecule is 443 amino acids that are processed to a mature protein by cleavage of the signal peptide and 17 amino acids of C-terminal extension. The 5⬘ untranslated region of the cDNA shows the mini-exon (Cornelissen et al., 1986) while the 3⬘ untranslated region shows the 16-mer oligonucleotide conserved at that position in all VSG mRNAs of trypanosomes of Trypanozoon (Majumder et al., 1981). These two sequence blocks indicate that our data represents a complete VSG gene. A search was made by BLAST to identify a nucleotide sequence in T. evansi JN 118Hu VSG cDNA that had no similarity with any other known trypanosome DNA sequence in the databases. As expected, sequences in the C-terminal region were unsuitable because of homologies with other trypanosome VSG sequences (Holder and Cross, 1981; Rice-Ficht et al., 1981). However, primary sequences within the N-terminal region of VSGs are extremely diverse, with little conservation (Rice-Ficht et al., 1981). It is in this region that a sequence was identiWed that had no similarity with other trypanosome sequences in the databases. The 273 bp fragment was ampliWed by PCR as described. Interestingly, fragments of identical size were ampliWed in four other T. evansi besides T. evansi JN 2118Hu. All the Wve positive samples were non-RoTat 1.2 T. evansi. Contamination can lead to artifacts in PCR ampliWcation, hence the need to assess the sequence identity of the PCR products. This was performed by restriction enzyme analysis of the 273 bp amplicons. The sites of single cutter restriction enzymes in the sequenced T. evansi JN 2118Hu VSG cDNA were determined by computer (http://tools.neb.com/NEBcutter2/index.php). To determine the restriction fragment length polymorphisms (RLFPs) in the 273 bp PCR products, digestion was carried out using the enzyme HaeIII which produced a common restriction pattern in all Wve non-RoTat 1.2 T. evansi DNA samples. The pattern agreed with that
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J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
Fig. 3. PCR detection of non-RoTat 1.2 T. evansi. Gel electrophoresis (1.5% agarose, stained with ethidium bromide), showing PCR products from genomic DNA prepared from trypanosome populations. (A) Lane (M) 1 kb Plus DNA ladder (Invitrogen). The 273 bp PCR products ampliWed with T. evansi JN 2118Hu VSG-based primers are in lanes (1–16) as follows: (1) T. evansi JN 2118Hu; (2) T. evansi KETRI 2479; (3) T. evansi JN 4306Moh; (4) JN T. evansi JN 6512Muh; (5) T. evansi JN 6524Muh; (6) T. evansi RoTat 1.2; (7) T. evansi AnTat 3.3; (8) T. evansi KETRI 2455; (9) T. evansi KETRI 3117; (10) T. evansi KETRI 3261 (11) T. evansi JN 4314Moh; (12) T. evansi JN 4315Moh; (13) T. evansi JN 5480Dai; (14) T. evansi JN 6507Ru; (15) T. evansi JN 3235Abd; and (16) T. evansi JN 4394Ha. (B) Lane (M) 1 kb Plus DNA ladder (Invitrogen). The 488 bp PCR products ampliWed with RoTat 1.2 VSG-based primers are in lanes (1–16) as follows: (1) T. evansi RoTat 1.2; (2) T. evansi AnTat 3.3; (3) T. evansi KETRI 2455; (4) T. evansi KETRI 3117; (5) T. evansi KETRI 3261; (6) T. evansi JN 4314Moh; (7) T. evansi JN 4315Moh; (8) T. evansi JN 5480Dai; (9) T. evansi JN 6507Ru; (10) T. evansi JN 3235Abd; (11) T. evansi JN 4394Ha; (12) T. evansi JN 2118Hu; (13) T. evansi KETRI 2479; (14) T. evansi JN 4306Hu; (15) T. evansi JN 6512Muh; and (16) T. evansi JN 6524Muh. (C) Lane (M) 1 kb Plus DNA ladder (Invitrogen). The 273 bp PCR products ampliWed with T. evansi JN 2118Hu VSG-based primers are in lanes (1–7) as follows: (1) T. evansi JN 2118Hu; (2) T. brucei KETRI 3596; (3) T. brucei KETRI 2710; (4) T. brucei IIE1; (5) T. brucei ILTat1.1; (6) T.b. rhodesiense WaTat1.2; and (7) T.b. gambiense LiTat1.1.
produced from computer analysis of the cDNA sequence (http://tools.neb.com/NEBcutter2/index.php), inferring that the amplicons were speciWc and not produced by non-speciWc ampliWcation of DNA or by multimer formation of the PCR primers. The fact that the 273 bp fragment of the cDNA showed no similarities with trypanosome sequences in the database strongly suggested that this fragment was unique to the strain of T. evansi
described in this study. Whether Southern blots (Southern, 1975) would show similar results in the genomes of diVerent trypanosomes strains, remains to be investigated in future studies in this laboratory. Urakawa et al. (2001) using the DNA fragment that constitutes the RoTat 1.2 VSG open reading frame as hybridization probe observed that DNA sequences homologous to the RoTat 1.2 VSG gene were present in the genomes of
J.M. Ngaira et al. / Experimental Parasitology 110 (2005) 30–38
Fig. 4. Restriction enzyme digestion pattern of 273 bp amplicon gel electrophoresis (1.5% agarose, stained with ethidium bromide), showing 273 bp PCR products puriWed from gel and digested with restriction enzyme HaeIII. One kilo base Plus DNA ladder (lane M). T. evansi JN 2118Hu, 273 bp amplicon digested with HaeIII (lane 1); T. evansi KETRI 2479, 273 bp amplicon digested with HaeIII (lane 2); and T. evansi JN 2118Hu, 273 bp amplicon undigested (lane 3).
other trypanosomes, yet a 488 bp section of the fragment was speciWc for T. evansi by PCR ampliWcation. The sensitivity of the PCR test developed in this study can only be determined through the testing of a large number of isolates of diVerent geographical origin. For now, this test was 100% (Wve out of Wve) sensitive for non-RoTat 1.2 T. evansi examined in this study. The Wve samples that tested positive for the expected 273 bp fragment of the T. evansi JN 2118Hu VSG gene are the same that tested negative for the 488-bp fragment of the RoTat 1.2 VSG gene. The described test is an additional diagnostic tool for the detection of non RoTat 1.2 T. evansi, but may not necessarily detect all strains of nonRoTat 1.2 T. evansi as these are yet unknown. Regional genetic diVerences in T. evansi isolates may be present in Kenya, but this awaits conWrmation (Njiru et al., 2004). It has been argued that VSG sequences are not reliable diagnostic targets due to the large repertoire of VSG genes present in bloodstream trypanosomes (Radwanska et al., 2002). In T. evansi however, it is possible that the parasites’ potential for genetic variation is limited by the non-cyclical method of transmission (Boid, 1988), and this would make the use of VSG-based diagnosis more feasible in T. evansi than in cyclically transmitted trypanosomes. Studies have shown that the large majority of T. evansi isolates of diverse geographic origin belong to a major Type A group (Gibson et al., 1983). A minor group containing type B minicircles has been reported only in Kenya and the question now arises whether this group is the same as the non-RoTat 1.2 T. evansi described in this study. Claes et al. (2004) speculate that the non-RoTat 1.2 T. evansi reported by Ngaira et al. (2003) correspond to type B group and that all other T. evansi isolated elsewhere are the classical T. evansi type A group. Importantly, this would imply that
37
RoTat 1.2 VSG-based PCR tests (Claes et al., 2004; Urakawa et al., 2001) combined with the PCR test developed in this study could provide the sensitivity needed to detect the large majority of T. evansi. The potential of VSGs as diagnostic reagents is further demonstrated by the use of a VSG variant from the TEVA1 T. evansi Venezuelan isolate (Uzcanga et al., 2004). Moreover, dyskinetoplastic T. evansi strains that lack kDNA can be detected using VSG-based tests. In conclusion therefore, this study has shown that PCR ampliWcation of the internal 273 bp fragment of the T. evansi JN 2118Hu VSG gene can provide a convenient and reliable detection of T. evansi infections that would otherwise remain undetected by the current RoTat 1.2based tests.
Acknowledgments The authors funded this study. We are grateful for laboratory facilities at Kenya Trypanosomosis Research Institute (KETRI) and at International Livestock Research Institute (ILRI).
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