A comparative evaluation of PCR- based methods for species- specific determination of African animal trypanosomes in Ugandan cattle

Heba A Ahmed,Susan C Welburn,Ewan T Macleod,Kim Picozzi

doi:10.1186/1756-3305-6-316

Heba A Ahmed, Susan C Welburn + Show 2 more

Open Access

https://doi.org/10.1186/1756-3305-6-316

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Abstract

BackgroundIn recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. Individual species-specific multi-copy trypanosome DNA sequences can be targeted to identify parasites. Highly conserved ribosomal RNA (rRNA) genes are also useful for comparisons between closely related species. The internal transcribed spacer regions (ITS) in particular are relatively small, show variability among related species and are flanked by highly conserved segments to which PCR primers can be designed. Individual variations in inter-species length makes the ITS region a useful marker for identification of multiple trypanosome species within a sample.MethodsSix hundred blood samples from cattle collected in Uganda on FTA cards were screened using individual species-specific primers for Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax and compared to a modified (using eluate extracted using chelex) ITS-PCR reaction.ResultsThe comparative analysis showed that the species-specific primer sets showed poor agreement with the ITS primer set. Using species-specific PCR for Trypanozoon, a prevalence of 10.5% was observed as compared to 0.2% using ITS PCR (Kappa = 0.03). For Trypanosoma congolense, the species-specific PCR reaction indicated a prevalence of 0% compared to 2.2% using ITS PCR (Kappa = 0). For T. vivax, species-specific PCR detected prevalence of 5.7% compared to 2.8% for ITS PCR (Kappa = 0.29).ConclusionsWhen selecting PCR based tools to apply to epidemiological surveys for generation of prevalence data for animal trypanosomiasis, it is recommended that species-specific primers are used, being the most sensitive diagnostic tool for screening samples to identify members of Trypanozoon (T. b. brucei s.l). While ITS primers are useful for studying the prevalence of trypanosomes causing nagana (in this study the species-specific primers did not detect the presence of T. congolense) there were discrepancies between both the species-specific primers and ITS for the detection of T. vivax.

Highlights

In recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection
Species-specific DNA targets have been identified for the most important pathogenic trypanosome species that occur in cattle (Trypanosoma brucei s.l., Trypanosoma congolense and Trypanosoma vivax) and PCR based methods for their amplification developed
For detection of Trypanozoon (Trypanosoma brucei brucei, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi and Trypanosoma equiperdum), the most common target is the 177 bp DNA satellite repeat sequence originally described by Sloof et al [8] that exists in high copy number in the parasite genome (10,000)

Summary

Introduction

PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. The TBR universal primers developed to amplify the 177 bp sequences from Trypanozoon genomic DNA, were able to detect 0.1 pg of parasite DNA, i.e. the amount of DNA equated to that present in a single trypanosome [9]. To permit discrimination between samples carrying human serum resistant parasites and non human infective parasites a multiplex PCR that simultaneously detects the SRA gene as well as the gene for phospholipase C (GPI-PLC) was developed [12]. Both of these single copy genes are present in T. b. Both of these single copy genes are present in T. b. rhodesiense, but only GPI-PLC is present in T. b. brucei, so if a sample shows positive amplification for GPI-PLC this indicates that sufficient T. brucei s.l. genomic material is present to detect a single copy gene while the presence or absence of SRA determines whether T. b. rhodesiense is present

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