Phylogenic analysis of Chinese Leishmania isolates based on small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA).

The leishmaniases are zoonotic diseases caused by protozoan parasites of the genus Leishmania. Leishmaniases are still endemic in China, especially in the west and northwest froniter regions. To revalue the preliminary phylogenetic results of Chinese Leishmania isolates, we amplified partial fragment of small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA), then tested the phylogenetic relationships among Chinese Leishmania isolates and their relatives by analyzing SSU rRNA gene sequences and 7SL RNA gene sequences. 19 SSU RNA sequences and 9 7SL RNA sequences were obtained in our study, then analyzed with 42 SSU RNA sequences and 32 7SL RNA sequences retrieved from Genbank, respectively. In the Bayesian analysis of the SSU RNA gene, the isolate MHOM/CN/93/GS7 and the isolate IPHL/CN/77/XJ771 are members of Leishmania donovani complex, while the isolate MHOM/CN/84/JS1 clustered with Leishmania tropica. The other 11 Chinese Leishmania isolates (MHOM/CN/90/WC, MCAN/CN/90/SC11, MHOM/CN/80/XJ801, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/ 89/GS5) form an unclassified group, defined as Leishmania sp., and the most relative species to this group is L. tarentolae. In the Bayesian analysis of the 7SL RNA gene, 9 Chinese Leishmania isolates also formed an unclassified group with L. tarentolae, including canine isolate 10, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/ CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/89/GS5. We concluded that: (1) Chinese Leishmania isolates are non-monophyly group; (2) an unclassified group may exist in China, and the most relative species to this group is L. tarentolae; (3) MHOM/CN/84/JS1, which was previously assigned as L. donovani, was most genetically related to L. tropica strain MHOM/SU/74/K27.

Axenic culture and identification of amastigotes from Sichuan human strain of Chinese Leishmania isolates.

This work describes a simple method to yield large amounts of the isolate MHOM/CN/90/SC10H2 amastigotes-like forms in axenic cultures using promastigotes as the starting population. The isolate MHOM/CN/90/SC10H2, used in this study, belongs to an undescribed species of Leishmania endemic to hill foci in China. The method describes induced extracellular amastigote transformation of this isolate. The rounded parasite obtained in axenic culture was morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as verified by the stage-specific genes (gp46 and p4 genes) with RT-PCR. A 70-80 kDa protein was recognized by polyclonal antibody HRP-IgG only in axenic-derived amastigotes and not in promastigotes.

Evaluation of the protective immunity of the Legionella pneumophila recombinant protein FlaA/MompS/PilE in an A/J mouse model.

To investigate the protect effects of the recombinant protein FlaA/MompS/PilE against Legionella pneumophila (L. pneumophila), the coding sequences of the three proteins were optimized by DNA Star software firstly, cloned, expressed by Escherichia coli BL21, and purified. To give an enhanced the immunological response, the proteins were linked together with (Linker) or without a linker insert (NLinker) and were purified from E. coli BL21. The A/J mouse model was used to determine the level of the induction of protective immunity from the purified proteins. Our results showed that the IgG titer, which was measured by ELISA, was increased after the administration of the five proteins. Compared to the administration of the individual proteins, the chimeric Linker and NLinker proteins displayed lasting immunity to a lethal dose of L. pneumophila challenge. The Linker protein protected the A/J mouse against a higher dose of L. pneumonia compared to the other proteins used in this study, as it contained a more effective immunogen. The work presented here demonstrates that the bioinformatics software, DNA Star, is a valid tool to analyse the epitopes of proteins and was useful in the optimization of proteins that could induce the protective immune response to L. pneumophila. The cross-immunity of recombinant proteins, such as the Linker and the NLinker chimera, have higher generates a greater immune than the single proteins.

Species delimitation and phylogenetic relationships of Chinese Leishmania isolates reexamined using kinetoplast cytochrome oxidase II gene sequences.

Leishmaniasis is a geographically widespread disease caused by protozoan parasites belonging to the genus Leishmania and transmitted by certain species of sand fly. This disease still remains endemic in China, especially in the west and northwest frontier regions. A recent ITS1 phylogeny of Chinese Leishmania isolates has challenged some aspects for their traditional taxonomy and cladistic hypotheses of their phylogeny. However, disagreement with respect to relationships within Chinese Leishmania isolates highlights the need for additional data and analyses. Here, we test the phylogenetic relationships among Chinese isolates and their relatives by analyzing kinetoplast cytochrome oxidase II (COII) gene sequences, including 14 Chinese isolates and three isolates from other countries plus 17 sequences retrieved from GenBank. The COII gene might have experienced little substitution saturation, and its evolutionary process was likely to have been stationary, reversible, and homogeneous. Both neighbor-joining and Bayesian analyses reveal a moderately supported group comprising ten newly determined isolates, which is closely related to Leishmania tarentolae and Endotrypanum monterogeii. In combination with genetic distance analysis as well as Bayesian hypothesis testing, this further corroborates the occurrence of an undescribed species of Leishmania. Our results also suggest that (1) isolate MHOM/CN/93/GS7 and isolate IPHL/CN/77/XJ771 are Leishmania donovani; (2) isolate MHOM/CN/84/JS1 is Leishmania tropica; (3) the status referring to an isolate MRHO/CN/62/GS-GER20 from a great gerbil in Gansu, China, as Leishmania gerbilli, formerly based on multilocus enzyme electrophoresis, is recognized; and (4) E. monterogeii is nested within the genus Leishmania, resulting in a paraphyletic Leishmania. In addition, the results of this study enrich our understanding of the heterogeneity and relationships of Chinese Leishmania isolates.