Polymorphism in the HASPB Repeat Region of East African Leishmania donovani Strains

Abstract

Background/ObjectivesVisceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission.Methodology/Principal FindingsSixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient's geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions.Conclusions/SignificanceHASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.

Differential diagnosis of the honey bee trypanosomatids Crithidia mellificae and Lotmaria passim

“…HRM proved to be a simple technique to classify genotypes of Leishmania donovani and Trypanosoma cruzi (Higuera et al, 2013;Zackay et al, 2013) and was also used to assess the strain diversity of Deformed wing virus (Martin et al, 2012). While HRM analysis of the type strains of C. mellificae, C. bombi, C. expoeki and L. passim appeared successful, we were not able to genotype infected honey bee samples.…”

“…Several loci, including cytochrome b and gGAPDH, showed enough interspecific identity to discriminate between both species, but sequencing remains necessary. Techniques like fragment length polymorphism and high resolution melting (HRM) were previously used to identify trypanosomatids in other host species (Higuera et al, 2013;Schmid-Hempel and Tognazzo, 2010;Zackay et al, 2013). We investigated both methods using infected honey bee samples from different geographic locations and trypanosomatid cell types from honey bees and bumble bees.…”

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

“…Leishmania k26 (also known as HASPB) protein, as well as SHERP that share the same locus on chromosome 23 [49], are stage-regulated proteins, expressed only in the mammalian host infective forms (metacyclic promastigotes and amastigotes) [50]. k26 has a central core composed of a 10–11 amino acid repeats (PKEDGHTQKND/PKEDGRTQKN in Additional file 3: Figure S2) which present high inter and intra-specific variability [49,51,52], a feature that makes it an interesting tool for molecular typing of different Leishmania species and strains [21]. Other than this, k26 is a highly immunogenic antigen [49] with proven efficacy as a vaccine in murine models of visceral leishmaniasis by L. donovani [51,53] and partial efficacy in canine leishmaniasis by L. infantum [54].…”

Leishmaniasis in Northern Syria during Civil War

“…Results of cpb-EF PCR ( 14 , 15 ) on patient samples identified as belonging to the Leishmania infantum/donovani complex in study of leishmaniasis control programs in northern Syria. Lane 1, step marker; lanes 2 and 3, L. donovani strain 275 with 3 and 6 μL of DNA, respectively; lane 4, negative control; lanes 5 and 6, L. infantum strain 282 with 3 and 6 μL of DNA, respectively.…”

“…To further discriminate species within the L. donovani/infantum complex, we ran 3 more PCR protocols for species differentiation: K26-PCR ( 13 ), cpbE/F-PCR ( 14 , 15 ), and HSP70-PCR ( 16 ) (Technical Appendix). …”