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

Abstract

Direct PCR analysis of trypanosome infected blood samples in the quantities required for large scale epidemiological study has always been problematic. Current methods for identifying and differentiating trypanosomes typically require several species-specific reactions, many of which rely on mouse passaged samples to obtain quality concentrated genomic DNA. As a consequence important epidemiological information may be lost during the sample preparation stage. Here, we report a PCR methodology that reduces processing and improves on the sensitivity of present screening methods. The PCR technique targets the gene encoding the small ribosomal subunit in order to identify and differentiate 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, thereby making it a viable tool for large-scale epidemiological studies.

Introduction

The African trypanosomes comprise a group of important and complex pathogens, affecting animal and human health in much of sub Saharan Africa. The causative organisms are represented by a variety of species and subspecies of a heteroxenous parasite of the genus Trypanosoma, some of which are zoonotic, causing disease in man and animals (domestic and wild). Animal trypanosomiasis, or nagana, costs livestock producers and consumers an estimated $1340 million annually, this figure excludes indirect livestock benefits such as manure and traction (Kristjanson et al., 1999). Human African trypanosomiasis occurs in endemic foci across East and West Africa. The human infective Trypansoma brucei rhodesiense subspecies is maintained in wild animals (van Hoeve et al., 1967) and domestic livestock (Hide et al., 1996, Onyango et al., 1966) from where it may play a significant role in the generation of acute sleeping sickness epidemics in east Africa (Fevre, 2001, Hide et al., 1996, Welburn et al., 2001). Effective disease control and management depends heavily upon knowledge of the epidemiology of the disease, which in turn relies upon methods that incorporate screening of both animal and human populations (Hutchinson et al., 2003).

Methods of epidemiological screening include direct parasite examination using traditional dark ground microscopy, examination of buffy coat and more recently molecular methodologies based on the polymerase chain reaction (PCR). Microscopy is labour intensive and can lack sensitivity under field conditions due to routinely low peripheral parasitaemia in infected livestock (Picozzi et al., 2002). PCR based diagnostic methods have largely overcome difficulties associated with sensitivity and specificity. A number of methods have been developed for the following species and subspecies of Trypansoma–Trypanozoon (Artama et al., 1992, Kabiri et al., 1999), Trypanosoma congolense (Riverine/Forest) (Masiga et al., 1992), T. congolense (Kilifi) (Masiga et al., 1992), T. congolense (Savannah) (Masiga et al., 1992), Trypanosoma vivax (Masake et al., 1994, Masake et al., 1997), Trypanosoma simiae (Masiga et al., 1992), Trypanosoma evansi (Artama et al., 1992), T. congolense (Kenya Coast) (Masiga et al., 1992), and Trypanosoma theileri (Rodrigues et al., 2003). Using these approaches accurate species/subspecies differentiation requires up to eight different PCRs per sample, which increases the costs and impacts on the practical application of the technique for large-scale epidemiological studies. Furthermore, many of the PCR techniques developed in recent years are based on complex protocols requiring samples to be mouse passaged, and therefore mouse adapted, a process which some trypanosome isolates do not survive (Hoare, 1972, Masiga et al., 1992) resulting in loss of species or strains and selection and sampling bias (Coleman and Welburn, 2004).

Recent developments in matrices for sample collection and archive, which permit direct PCR identification from tissue/fluids may overcome such bias. Simplified protocols incorporating these improved sample collection techniques, together with rapid PCR-based screening methodologies for the direct analysis of field samples are therefore required. The internal transcribed spacers (ITS) located within the ribosomal RNA genes have been used to establish relationships and differentiate species in an extremely wide range of organisms (Mai and Coleman, 1997, Samuel, 1998, Schlotterer et al., 1994, Wesson et al., 1992). A high copy number combined with inter-species length variation makes the ITS region a useful marker for species differentiation in trypanosomes, as has been recently demonstrated (Desquesnes et al., 2001, McLaughlin et al., 1996, Njiru et al., 2004). However, this technique was shown to be relatively insensitive and in some cases was problematic for detection of T. vivax (the principal pathogenic species in cattle) in either concentrated genomic DNA or DNA extracted from field samples. Here, we report the development of a simple nested PCR method, which detects the inter-specific length variation of the ITS regions of ribosomal genes and thereby produces a unique size of PCR product for each species of trypanosome. The technique is able to detect the following African trypanosome species. (Trypanozoon, T. congolense (River/Forest), T. congolense (Kilifi), T. congolense (Savannah), T. vivax , T. simiae, T. evansi, T. congolense (Kenya Coast), and T. theileri). It is able to detect a single trypanosome and has been optimised for PCR amplification of blood applied to filter paper (Whatman FTA) permitting direct PCR analysis of field material.