Experimental Parasitology 111 (2005) 24–29

www.elsevier.com/locate/yexpr

A PCR based assay for detection and diVerentiation of African trypanosome species in blood

Andrew Cox a,b, Aimee Tilley a, Francis McOdimba a, Jenna Fyfe a, Mark Eisler a, GeoV Hide b, Susan Welburn a,¤

a Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin, Midlothian EH25 9RG, Scotland, UK

b Centre for Parasitology, Molecular Epidemiology and Ecology, Bioscience Research Institute, University of Salford, The Crescent, Salford, Manchester M5 4WT, UK

Received 26 January 2005; received in revised form 11 March 2005; accepted 13 March 2005Available online 29 April 2005

Abstract

Direct PCR analysis of trypanosome infected blood samples in the quantities required for large scale epidemiological study hasalways been problematic. Current methods for identifying and diVerentiating trypanosomes typically require several species-speciWcreactions, many of which rely on mouse passaged samples to obtain quality concentrated genomic DNA. As a consequence impor-tant epidemiological information may be lost during the sample preparation stage. Here, we report a PCR methodology that reducesprocessing and improves on the sensitivity of present screening methods. The PCR technique targets the gene encoding the smallribosomal subunit in order to identify and diVerentiate all clinically important African trypanosome species and some subspecies.The method is more economical, simple, and sensitive than current screening methods, and yields more detailed information, therebymaking it a viable tool for large-scale epidemiological studies. 2005 Elsevier Inc. All rights reserved.

Index Description and Abbreviations: Trypanosomiasis; Epidemiology; PCR; Diagnosis; Human African sleeping sickness; Ribosomal DNA;Whatman FTA; DNA, deoxyribonucleic acid; ITS, internal transcribed spacer; PCR, polymerase chain reaction; RNA, ribonucleic acid

Keywords: Trypanosomiasis; Epidemiology; PCR; Diagnosis; Human African sleeping sickness; Ribosomal DNA; Whatman FTA

1. Introduction

The African trypanosomes comprise a group ofimportant and complex pathogens, aVecting animal andhuman health in much of sub Saharan Africa. The caus-ative organisms are represented by a variety of speciesand subspecies of a heteroxenous parasite of the genusTrypanosoma, some of which are zoonotic, causing dis-ease in man and animals (domestic and wild). Animaltrypanosomiasis, or nagana, costs livestock producers

* Corresponding author.E-mail address: Sue.Welburn@ed.ac.uk (S. Welburn).

0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2005.03.014

and consumers an estimated $1340 million annually, thisWgure excludes indirect livestock beneWts such as manureand traction (Kristjanson et al., 1999). Human Africantrypanosomiasis occurs in endemic foci across East andWest Africa. The human infective Trypansoma bruceirhodesiense subspecies is maintained in wild animals(van Hoeve et al., 1967) and domestic livestock (Hide etal., 1996; Onyango et al., 1966) from where it may play asigniWcant role in the generation of acute sleeping sick-ness epidemics in east Africa (Fevre, 2001; Hide et al.,1996; Welburn et al., 2001). EVective disease control andmanagement depends heavily upon knowledge of theepidemiology of the disease, which in turn relies upon

A. Cox et al. / Experimental Parasitology 111 (2005) 24–29 25

methods that incorporate screening of both animal andhuman populations (Hutchinson et al., 2003).

Methods of epidemiological screening include directparasite examination using traditional dark groundmicroscopy, examination of buVy coat and morerecently molecular methodologies based on the polymer-ase chain reaction (PCR). Microscopy is labour intensiveand can lack sensitivity under Weld conditions due toroutinely low peripheral parasitaemia in infected live-stock (Picozzi et al., 2002). PCR based diagnostic meth-ods have largely overcome diYculties associated withsensitivity and speciWcity. A number of methods havebeen developed for the following species and subspeciesof Trypansoma–Trypanozoon (Artama et al., 1992; Kab-iri et al., 1999), Trypanosoma congolense (Riverine/For-est) (Masiga et al., 1992), T. congolense (KiliW) (Masigaet al., 1992), T. congolense (Savannah) (Masiga et al.,1992), Trypanosoma vivax (Masake et al., 1994, 1997),Trypanosoma simiae (Masiga et al., 1992), Trypanosomaevansi (Artama et al., 1992), T. congolense (Kenya Coast)(Masiga et al., 1992), and Trypanosoma theileri (Rodri-gues et al., 2003). Using these approaches accurate spe-cies/subspecies diVerentiation requires up to eightdiVerent PCRs per sample, which increases the costs andimpacts on the practical application of the technique forlarge-scale epidemiological studies. Furthermore, manyof the PCR techniques developed in recent years arebased on complex protocols requiring samples to bemouse passaged, and therefore mouse adapted, a processwhich some trypanosome isolates do not survive (Hoare,1972; Masiga et al., 1992) resulting in loss of species orstrains and selection and sampling bias (Coleman andWelburn, 2004).

Recent developments in matrices for sample collec-tion and archive, which permit direct PCR identiWcationfrom tissue/Xuids may overcome such bias. SimpliWedprotocols incorporating these improved sample collec-tion techniques, together with rapid PCR-based screen-ing methodologies for the direct analysis of Weld samplesare therefore required. The internal transcribed spacers(ITS) located within the ribosomal RNA genes havebeen used to establish relationships and diVerentiate spe-cies in an extremely wide range of organisms (Mai andColeman, 1997; Samuel, 1998; Schlotterer et al., 1994;Wesson et al., 1992). A high copy number combined withinter-species length variation makes the ITS region auseful marker for species diVerentiation in trypano-somes, as has been recently demonstrated (Desquesnes etal., 2001; McLaughlin et al., 1996; Njiru et al., 2004).However, this technique was shown to be relativelyinsensitive and in some cases was problematic for detec-tion of T. vivax (the principal pathogenic species in cat-tle) in either concentrated genomic DNA or DNAextracted from Weld samples. Here, we report the devel-opment of a simple nested PCR method, which detectsthe inter-speciWc length variation of the ITS regions of

ribosomal genes and thereby produces a unique size ofPCR product for each species of trypanosome. The tech-nique is able to detect the following African trypano-some species. (Trypanozoon, T. congolense (River/Forest), T. congolense (KiliW), T. congolense (Savannah),T. vivax , T. simiae, T. evansi, T. congolense (KenyaCoast), and T. theileri). It is able to detect a single try-panosome and has been optimised for PCR ampliWca-tion of blood applied to Wlter paper (Whatman FTA)permitting direct PCR analysis of Weld material.

2. Materials and methods

2.1. Samples

Field samples consisted of 245 samples of bovineblood taken from two villages in the Soroti and Tororodistricts of Uganda and collected on Whatman FTAcards. Genomic DNA stocks are as detailed in Table 1.

2.2. Primer design

Sixteen trypanosome ribosomal DNA sequences wereselected from the NCBI database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db D Nucleotide). T. bru-cei AF306771, AF306772, AF306773, AF306774, AF306775, AF306776, AF306777, and X05862; T. congolenseU22315; T. congolense (KiliW) U22316; T. congolense(River/Forest) U22317; T. congolense (Tsavo) U22318;T. vivax U22319, T. simiae U22320. Two additionalsequences (Trypanosoma cruzi AY362826 and Trypano-soma rangeli AY230240) were selected for comparison asout-groups to ensure optimal speciWcity of the primers.Sequences were aligned with CLUSTALX software(ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX) (Thompsonet al., 1997) and viewed using the Bioedit programme(Hall, 1999). A set of nested primers targeting the ribo-somal gene locus was selected using PRIMER3 web

Table 1Details and origin of trypanosome genomic DNA used in the develop-ment of the ITS-PCR protocol

Species Stock code Origin

T. brucei brucei BUTEBA135 Tororo, SE Uganda, Cow, 1990

T. brucei rhodesiense BUG H2 Kamuli, Uganda, Human, 2000

T. brucei rhodesiense DO Katerema, Uganda Human, 1990

T. congolense (Savannah) IL1180 (ILNat3.1) Serengeti, TanzaniaT. congolense (Forest) IL3900 Burkina FasoT. congolense (KaliW) IL45.1 KiliW, KenyaT. vivax ILDatt1.2 KenyaT. brucei OBUR C19 Soroti, Uganda,

Cow, 2000T. congolense (Forest) TSW103 Liberia, PigT. simiae TV008 Unknown

26 A. Cox et al. / Experimental Parasitology 111 (2005) 24–29

primer selection software (http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi). Primers were evalu-ated using NETPRIMER software available at (http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html). The speciWcity of the primers was evaluated usinga BLAST search against human and mouse genomes(http://www.ncbi.nlm.nih.gov/BLAST). The outer primersequences were ITS1 (5�-GAT TAC GTC CCT GCCATT TG-3�), and ITS2 (5�-TTG TTC GCT ATC GGTCTT CC-3�) (MWG Biotech), and inner primersequences ITS3 (5�-GGA AGC AAA AGT CGT AACAAG G-3�), and ITS4 (5�-TGT TTT CTT TTC CTCCGC TG-3�) (MWG Biotech). All PCR conditions wereoptimised using modiWed ‘Taguchi’ methods (Cobb andClarkson, 1994). Expected band sizes were calculatedfrom the distance between the primer locations as deter-mined from the sequences for each trypanosome speciespresent in bioinformatic databases. The expected bandsizes are shown in Table 2.

2.3. AmpliWcation of DNA

Blood and genomic DNA samples were applied toWhatman FTA cards and allowed to dry for a mini-mum of 24 h at room temperature. A 2 mm diameterpunch was cut from the cards and was washed accord-ing to the following protocol; three washes of 10 min inWhatman FTA reagent followed by two washes of5 min in 1 mM TE buVer. The punches were dried atroom temperature for a minimum of 90 min then placeddirectly in the PCR tubes for the Wrst round of PCR.The reaction volume of 25 �l contained the followingcomponents. Super-Taq PCR buVer from HT Biotech-nologies, Cambridge (Wnal concentrations of 10 mMTris–HCl, pH 9.0, 1.5 mM MgCl2, 50 mM KCl, 0.1%Triton X-100, and 0.01%(w/v) stabilizer), 2 �M of eachouter primer ITS1 and ITS2, 1 mM total dNTP’s and1.25 U of Biotaq (Bioline, London). The reaction condi-tions were as follows: 1 cycle of 95 °C for 7 min followedby 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and72 °C for 2 min, the thermal cycling was carried out on aStratagene Robocycler. For the second round reaction

Table 2Expected and obtained band sizes for ampliWcation using nested ITSprimers as calculated from the sequences present in bioinformatic dat-abases

Species Expected band size from NCBI database (bp)

Band sizes obtained (bp)

T. congolense (Forest) 1513 1501T. congolense (KiliW) 1422 1430T. congolense (Savannah) 1413 1408T. congolense (Tsavo) 954 951T. brucei 1207–1224 1215T. simiae 850 847T. vivax 611 620T. theileri 988 998

1 �l of the PCR product from the Wrst round reactionwas placed in a fresh tube and 24 �l of the reaction mix-ture was added as detailed for the outer primers, withthe exception of the substitution of the outer primers(ITS1 and 2) with the inner primers. (ITS3 and 4) Thereaction conditions were as detailed previously. Tenmicrolitres of the PCR product was run on a30 cm £ 20 cm 1.5% agarose gel run at 100 V. The gelwas stained with ethidium bromide and visualised usinga Flowgen Alpha 1220 gel imaging system.

3. Results

To diVerentiate important species (and some subspe-cies) of African trypanosome a nested PCR was devel-oped which ampliWed the variable ITS region of theribosomal gene locus, using primers designed to the con-served Xanking sequences (Fig. 1).

3.1. SpeciWcity

AmpliWcation of genomic DNA from trypanosomestocks (Table 1) resulted in a speciWc size band for eachspecies, which was within the bounds of measurementerror and was in complete agreement with the expectedband sizes (Table 2). Control DNA samples were notavailable for some trypanosome species (e.g., T. theileri),therefore when unexpected band sizes appeared in Weldsamples the bands were cut out, sequenced, and comparedwith database sequences to conWrm species identity (datanot shown). The speciWcity of the primers was furthertested by PCR ampliWcation with host DNA (human,cow, and mouse), which produced no visible bands.

3.2. Sensitivity

To investigate the sensitivity of the nested PCR, thetechnique was tested on a dilution series of whole try-

Fig. 1. The structure of part of the ribosomal RNA gene locus. Ribo-somal genes are present in tandem arrays of around 100–200 copiesper trypanosome. Each gene consists of a number of conserved codingregions and non-coding spacer regions. Large boxes represent con-served coding regions (SSU, small sub-unit; LSU, large subunit) andsmall boxes represent spacer regions. The two spacers, internal tran-scribed spacers (ITS) 1 and 2 are known to vary in size between speciesand occasionally subspecies. A set of nested primers designed to theconserved regions are represented by black arrows (outer primers)ITS1 and ITS2 and white arrows (inner primers) ITS3 and ITS4.

A. Cox et al. / Experimental Parasitology 111 (2005) 24–29 27

panosomes (diluted in phosphate-buVered saline) onWhatman FTA cards and a dilution series of genomicDNA (diluted in water) in liquid form and also appliedto Whatman FTA cards. In the two dilutions of genomicDNA positive ampliWcation was detected at a DNA con-centration of 49 pg ml¡1 (or less than a single trypano-some equivalent). To investigate the eYcacy of thetechnique on samples containing host material, trypano-somes were diluted in bovine blood (UK origin) andapplied to Whatman FTA cards to mimic Weld samples.Positive ampliWcation was detected at DNA a concentra-tion of 55 pg ml¡1, which is again equivalent to less thana single trypanosome.

3.3. Application to Weld samples

Application of the nested ITS primers to 245 samplesof bovine blood taken from the Tororo and Soroti Dis-tricts of Uganda and collected on Whatman FTA cardsresulted in successful ampliWcation of the target ITSregion as shown by species speciWc band sizes (Fig. 2).This technique was also able to show samples infected

Fig. 2. Representative gel showing bands obtained from PCR ampliW-

cation (using nested ITS primers) of 19 blood samples (on WhatmanFTA cards) taken from cattle in the Tororo district of Uganda. Sam-ples 2, 3, 8, 16, and 19 are all positive for T. brucei, Samples 2, 9, 16, 18,and 19 are positive for T. theileri. Sample 17 is positive for T. simiae,sample 5 is positive for T. vivax, and samples 1, 6, 7, 10, 11, 12, 13, and14 are negative. Lane M represents a marker graduated in 100 bpintervals (band sizes illustrated). Mixed species infections were foundin lanes 2, 3, 4, 5, 16, and 17.

1 2 3 4 5 6 7 8 M 9 10 11 12 13 14 15 16 17 18 19

1400bp1200bp1000bp800bp700bp600bp

with multiple species (e.g., Fig. 2; lanes 2, 16, and 19), asshown by the presence of multiple bands.

3.4. Technique evaluation

The eYcacy of the technique was tested against themost widely used screening method; individual speciesspeciWc PCR’s (Artama et al., 1992; Clausen et al., 1998;Majiwa et al., 1994; Masake et al., 1997; Masiga et al.,1992), using samples collected from two diVerent villagesin Uganda (Cow blood applied to Whatman FTAcards). Analysis of the 245 samples using the individualspecies-speciWc PCR screening method demonstrated alow prevalence of trypanosomes in cows from the Wrstvillage and a high prevalence of trypanosomes cowsfrom the second village. The new nested PCR analysismethod showed that a comparable prevalence andgreater number of species were detected in each case(Table 3).

4. Discussion

Existing methods for screening samples for detectionand diVerentiation of trypanosomes are not suited tolarge-scale epidemiological analysis. This studyaddressed the requirement for improved techniques thatsimplify the sample analysis process but maintain thesensitivity and speciWcity required for directly analysingWeld samples.

We developed, a new nested PCR targeted to includeboth internal transcribed spacers of the ribosomal RNAgenes (ITS PCR), that was capable of detecting trypano-somes in the presence of host DNA and the PCR inhibi-tors present in blood (Heme, Lactoferrin IgG and non-target DNA). This nested technique was found to be sen-sitive enough for detection of a single parasite in bloodsamples and has been shown to be able to diVerentiate allimportant African trypanosome species and some subspe-cies. To simplify the sample collection and processingmethodology, we investigated the storage of samples ontreated Wlter paper cards, which make possible the direct

Table 3Evaluation of detection of trypanosome DNA from Whatman FTA cards containing blood from low and high prevalence villages in the Tororo andSoroti districts of Uganda, using a nested PCR ampliWcation, compared with utilisation of individual species speciWc primers (Artama et al., 1992;Clausen et al., 1998; Majiwa et al., 1994; Masake et al., 1997; Masiga et al., 1992)

ND, not done.

Species Low prevalence village prevalence (%) High prevalence village prevalence (%)

Species speciWc PCR method ITS-PCR Species speciWc PCR method ITS-PCR

T. brucei 5 7 32 33T. theileri ND 3 ND 47T. congolense 0 1 1 5T. vivax 1 1 8 5T. simiae ND 0 ND 2

(N D 101) (N D 144)

28 A. Cox et al. / Experimental Parasitology 111 (2005) 24–29

analysis of biological samples, in addition to circumvent-ing the requirement for mouse passage. When the nestedtechnique was evaluated against the current single PCRper species screening method, using a complete sample setcontaining positive and negative samples, it was found tohave a similar level of detection, but was capable ofdetecting a greater number of species in both high and lowprevalence sample sets.

The epidemiology of African trypanosomiasis is com-plex and poorly understood and requires large-scale Weldbased investigation. This technique has greatly simpliWedepidemiological studies involving sample screening. As aresult the costs and time involved in screening samplesfor the eight major species/subspecies of trypanosomehave been reduced by a factor of four (conservative esti-mate). This nested PCR technique can be used to screenlarge numbers of biological samples directly, quickly,and accurately, making it a simple, cost eVective, robust,and reliable tool for investigating the complex epidemi-ology of African Trypanosomiasis.

Acknowledgments

This work was funded by the Animal Health Pro-gramme of the Department for International Develop-ment (DFID) of the United Kingdom and the Universityof Salford. Thanks are extended to Joseph Magona, theLivestock Research Institute and its Weld team in Tor-oro, Uganda. Charles Waiswa and Ian Anderson, thedistrict veterinary oYcers and their staV in TororoSoroti and Busia in Uganda. The views expressed arethose of the authors and not necessarily those of DFID.

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