Molecular aspects of phylogenetic relationships among trypanosoma (Nannomonas) congolense.

At least four different genotypic groups of Trypanosoma (Nannomonas) congolense have been described based upon profiles of iso-enzymes, nucleotide composition of the highly repetitive satellite DNA, restriction fragment length polymorphisms (RFLPs) of conserved nuclear and kinetoplast DNA sequences and, more recently, randomly amplified polymorphic DNA (RAPD) patterns. The exact phylogenetic relationships of these morphologically identical but genetically heterogeneous parasites is obscure. I have designed specific oligonucleotide primers based on conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum, and used these in the polymerase chain reaction (PCR) to amplify genomic DNA from four different clones each representing a different genotypic group of T. congolense. PCR products of approximately 1 kb were generated using as template DNA from each of the trypanosomes. The nucleotide sequences of a segment of the PCR products were determined by direct sequencing to provide partial nucleotide sequence of the 18S rRNA gene in each of the trypanosomes. Using these sequences and those that have been published for the other Trypanosomatids, sequence similarity scores were determined with the help of appropriate computer software. Additionally, primers of arbitrary nucleotide sequence have been employed in the PCR amplification of genomic DNA from the four trypanosome clones representing the different T. congolense types. The patterns of randomly amplified polymorphic DNA (RAPD) have been used in the calculation of pairwise genetic similarity indices among these trypanosomes without making assumptions regarding relative band intensities. In a parallel study I have demonstrated that the PCR products of the 18S rRNA gene are sensitive probes for specific identification of trypanosomes. I have used heterologous PCR products as probes in hybridization analyses of genomic DNA and PCR products from the different types of T. congolense and from other protozoa. The RFLPs observed confirmed heterogeneity of the 18S rRNA gene sequences among the trypanosomes comprising the subgenus Nannomonas

Use of Restriction Fragment Length Polymorphism of 18S rRNA Gene for Identification of Trypanosome and Other Protozoan Species

Oligonucleotide primers based on conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of Trypanosoma brucei, Leishmania donovani, Triponema aequale and Legenidium gigantum were designed, synthesized and used to generate PCR products from different trypanosome species, subspecies and Leishmania donovani. The PCR products were digested with restriction enzyme Sau3A1, electrophoresed, transferred to nylon membrane and hybridized to a DNA probe (18pNS-1), which is specific to Trypanosoma congolense 18S rRNA gene. The restriction fragment length polymorphism (RFLP) pattern of PCR products obtained was the same for T. brucei subspecies: T.b. brucei and T.b. gambiense but different for other trypanosome species and L. donovani. RFLP analysis was also done with genomic DNA from different trypanosome species, subspecies and other protozoan species digested with Sau3A1 and other restriction enzymes. The genomic DNA RFLP pattern was the same for trypanosomes of the subgenus Trypanozoon: T. evansi, T.b. gambiense and T.b. brucei but different for L. donovani, Theilera parva and T. simiae. The genomic DNA RFLP pattern for T. Congolese genotypes: Savanna-type, Kilifi-type and West African riverine/forest type was also different. The results indicate intra- and inter-species genetic heterogeneity of 18S rRNA gene among the protozoans tested. The results further indicate that 18S rRNA gene of protozoans can potentially be used to identify different protozoan species and subspecies. The Kenya Veterinarian Vol. 29 2005: pp. 119-12

RAPD analysis of genotypic groups of Trypanosoma congolense and other African trypanosome species

Oligonucleotide primers of arbitrary nucleotide sequences were used in PCR reaction to amplify genomic DNA from the four types of T. congolense: Tsavo-type, Savannah-type, Kilifi-type and West African riverine/ forest-type. Several primers produced significantly different RAPD patterns for the four types of T. congolense. Similarity indices imply that the Kilifi-type and Savannahtype T. congolense (SI; 0.453) are the most closely related evolutionarily among T. congolense whereas the West African riverine/forest-type and Savannah type T. congolense (SI; 0.405) are the most distantly related. RAPD patterns of the four types of T. congolense were also compared with those of other trypanosome species. Similarity indices of this comparison implies that T. evansi and T.b. gambiense (SI; 0.667) are the most closely related whereas Kilifi-type T. congolense and T. b. brucei (SI; 0.315) are the most distantly related. The results further indicate that the T. congolense genotypic groups may be as different as they are from any other trypanosome species studied in this work. Bulletin of Animal Health and Production in Africa Vol. 54(1) 2006: 7-1

Additional file 1: Figure S1. of Toll-like receptor 2 (TLR2) plays a role in controlling cutaneous leishmaniasis in vivo, but does not require activation by parasite lipophosphoglycan

Lesion development in WT and TLR−/− mice upon infection with L. major after 18 weeks. WT, TLR2−/−, TLR1−/−, TLR6−/− and TLR4−/− mice (n = 4–5) were infected with 105 L. major promastigotes subcutaneously. Mice were monitored every week for the appearance and size of lesions. The mean lesion size (mm2) + SEM for each genotype is shown at each weekly time point post-infection up to the end of the experiment at 18 weeks. Knockout stains were compared to WT mice using the Mann-Whitney U test, where P < 0.05 was considered to indicate significant (*) differences. (TIF 28 kb