Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting

Abstract

Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98 %, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together.

Genetic Diversity of Trypanosoma evansi in Buffalo based on Internal Transcribed Spacer (ITS) Regions

“…Previously, the intra-species genetic variation of Trypanosoma have been reported in T. brucei [2], T. congolense [30], and T. evansi [33,46] in different hosts basically revealed by PCR based method. Herein, we investigated the diversity of T. evansi in a single host (buffalo) using the nucleotide data of 18S rDNA, 5.8S rDNA, and ITS regions.…”

Molecular Epidemiology of Parasitic Diseases: The Chagas Disease Model

“…Fingerprinting based on AFLPs has been useful in differentiating subspecies of T. brucei, which is the aetiological agent of sleeping sickness, a pathology that affects more than 8 million people in Africa. These techniques have shown great reproducibility in distinguishing T. b. rhodesiense and T. b. gambiense, two sympatric species that generate different symptomatologies, demonstrating how molecular epidemiology can assist in understanding disease outcomes in certain pathologies, thus aiding in developing proper treatment and management measures (Agbo et al, 2002;Masiga et al, 2006). RFLPs have been applied to parasites such as T. gondii and Trichinella spiralis, which are two species for which pigs play an important role in transmission dynamics.…”

Multiplex-endonuclease genotyping approach (mega): a tool for the fine-scale detection of unlinked polymorphic DNA markers

“…Trypanosoma brucei consists of three subspecies that are indistinguishable by conventional morphological and antigenic criteria, but differ in their geographical distribution and host specificity (Gibson et al 1980). For epidemiological identification and tracking of the subspecific strains, T. b. garnbiense can be distinguished from T. b. brucei and T. b. rhodesiense (Hide et al 1998;Agbo et al 2002). However, the distinction between T. b. brucei and T. b. rhodesiense has always been more difficult to determine (Godfrey et al 1990;Hide 1999;Agbo et al 2002).…”

“…For epidemiological identification and tracking of the subspecific strains, T. b. garnbiense can be distinguished from T. b. brucei and T. b. rhodesiense (Hide et al 1998;Agbo et al 2002). However, the distinction between T. b. brucei and T. b. rhodesiense has always been more difficult to determine (Godfrey et al 1990;Hide 1999;Agbo et al 2002). Resistance to normal human serum (equivalent to human infectivity) is an important trait that distinguishes T. b. gambiense and T. b. rhodesiense (which are resistant) from T. b. brucei strains (which are sensitive).…”

“…A review by Masiga et al (2000) highlighted the possible value of conventional AFLP in parasite genetic studies but was recently reported to offer low resolution in the genotyping of trypanosome subspecies (Agbo et al 2002), and previously in closely related strains in other taxa (Lindstedt et al 2000). Simons et al (1997) pioneered attempts at the use of additional endonucleases to increase the discriminatory power of AFLP but reported only slight improvement in fingerprint patterns compared with the standard protocol.…”