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Sleeping Sickness-causing Parasites Recombine, Affecting Virulence, Study Says

NEW YORK (GenomeWeb News) – Mixing between two protozoan parasite subspecies is associated with differences in virulence, as well as the presentation of the sleeping sickness they cause, according to a new study in mBio this week from the University of Liverpool's Neil Hall and his colleagues.

Two subspecies of the parasite Trypanosoma brucei can infect people, via a tsetse fly vector, causing more than 10,000 cases of sleeping sickness each year in sub-Saharan Africa. T. b. rhodesiense and T. b. gambiense are associated with acute and chronic disease in humans, respectively.

However, the researchers noted that those clinical demarcations between the subspecies are becoming fuzzier, as reports of both subspecies causing acute and chronic disease have been collected. A third subspecies, T. b. brucei, infects wildlife and livestock.

Further, isolates from a 1989 trypanosomiasis outbreak in Uganda indicated that genetic differences might affect virulence. Certain isoenzyme parasite groups in that outbreak were linked to different virulence levels.

In this study, the researchers sequenced isolates from those two Ugandan zymodemes — Busoga 17 and Zambezi 310 — to try to pinpoint loci behind such differences in virulence. The B17 isolate was linked to more severe disease, while Z310 was associated with a chronic course.

The researchers also compared the newly generated parasite genomes to a number of previously studied isolates and developed a SNP panel to quickly screen field isolates for differences linked to virulence.

"Our analysis demonstrates that the T. b. brucei subspecies causing [Human African trypanosomiasis] have undergone genetic exchange in natural populations, since the East African B17 and Z310 parasites share alleles with West African type 1 T. b. gambiense," Hall and his colleagues wrote. "The associated differences in disease progression in isolates with differentially derived haplotypes have clear implications for parasite control and diagnosis, as other important traits, such as human serum resistance or drug resistance, may move between parasite groups."

Initially, Hall and his colleagues characterized samples from the 1989 Ugandan outbreak using microsatellite analysis, but found that it could not differentiate between the Busoga and Zambezi zymodeme groups. Instead, they sequenced an isolate from each group using the SOLiD platform and aligned the reads they generated against the T. b. brucei reference genome, and compared them to publicly available trypanosome sequences.

While the B17 and Z310 isolates are broadly similar, sharing more than 99.8 percent of their genomes, at SNP loci, B17 was much more like T. b. gambiense — the parasite linked to "chronic" disease — than Z310 was.

Additionally, a Jukes-Cantor neighbor-joining tree indicated that West African T. b. gambiense and T. b. brucei cluster separately from East African T. b. brucei and T. b rhodesiense.

However, chromosome 8 from Z310 is more similar to chromosome 8s found in T. b. brucei while B17 appears to be halfway between that of an East African T. b. brucei strain and West African T. b. gambiense/T. b. brucei.

To bring more isolates into their analysis, the researchers genotyped an additional 31 T. b rhodesiense isolates from Uganda and a Zambian sample as well as 31 West African isolates, including five type 1 and 12 type 2 T. b. gambiense isolates, 11 T. b. brucei isolates, and three isolates from an unknown T. brucei subspecies using KASPar competitive allele-specific-PCR-based genotyping.

A phylogenetic tree incorporating that additional data indicated that West African T. b. brucei and type 1 and type 2 T. b. gambiense clustered together, which indicated to the researchers that West African T. b. brucei is more closely related to T. b. gambiense than to members of its same subspecies from East Africa.

Meanwhile, both the B17 and Z310 isolates clustered near the T. b. rhodesiense isolates and were distinct from East African T. b. brucei and West African T. b. gambiense and T. b. brucei.

As in their previous analysis, chromosome 8 SNPs showed that B17 strains are more similar to the T. b. gambiense genotype than the Z310 strains are, and the tree places the B17 zymodeme equidistant between the East African and West African parasites, consistent, the researchers noted, with it being heterozygous for the two genotypes.

Chromosome 8, Hall and his team noted, is a "strong candidate for loci that could underlie the differences in virulence between B1 and Z310 parasites." They added that only the isocitrate dehydrogenase isoenzyme differentiates the two groups.

"Our analysis showed that these strains from the Ugandan epidemic were intermediate between the reference genome sequences of T. b. gambiense and T. b. brucei and contained haplotypes that were present in both subspecies," Hall and his team noted. "This suggests that the human-infective subspecies of T. brucei are not genetically isolated, and our data are consistent with genomic introgression between East African and West African T. b. brucei subspecies."

They noted that such introgression has important ramifications for parasite control, adding that the parasites need to be monitored for recombination, adding that the panel of KASPar SNP markers they developed could be used to identify shared alleles and candidate loci.