Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species

BackgroundDipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites’ growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species.MethodologyOrthologous DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples.FindingsSequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS) (-0.164, SE = 0.013) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex.A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples.ConclusionThe study demonstrates that DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance.     

Development and validation of a new real-time assay for the quantification of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope?=??3.405, r²?=?0.976, E?=?96.64 %) and the positive recombinant plasmid (y?=??3.36, r²?=?0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10² copies/μL (CV%?≈?1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g^?1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r²?=?0.944) and with the soil plating method (r²?=?0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.     

Photosynthetic Behaviour and Mineral Nutrition of <i>Tamarix gallica</i> Cultivated Under Aluminum and NaCl Combined Stress

The lack of knowledge of plant tolerance and differential response to aluminum (Al) encouraged many researchers, in the last decade, to elucidate Al toxicity and tolerance mechanisms. The current study reported the impact of Al, a toxic element with negative effects on plant growth and development, in halophytic plant Tamarix gallica. Plants were subjected to different Al concentrations (0, 200, 500 and 800 μM) with or without NaCl (200 mM) supplementation. Growth, photosynthesis and mineral content were assessed. Al stress had a significant decrease on shoots’ biomass production between 19 to 41%, and a little variation on chlorophyll content and photosynthetic efficiency (Fo, Fm, Fv fluorescence’s and Fv/Fm). Furthermore, the Al-treatments did not affect significantly the content of potassium, calcium, and magnesium in different plant parts, whereas NaCl addition to the medium induced a decrease in these elements’ concentrations. Our results have shown that T. gallica is able to accumulate the high levels of Al in shoots and roots, 6288 μg.g^-1 DW and 7834 μg.g^-1 DW respectively. It is considered as a hyperaccumulator plant of Al. In addition, Na⁺ contents in shoots and roots exceed 23000 μg.g^-1 DW. Therefore, T. gallica presents a high tolerance at the same time to Al and NaCl phytotoxicity, so it is interesting to use in phytoremediation programs.     

Effects of sewage disposal into the White Nile on the plankton community

Twenty water samples and 30 plankton samples were taken from the sewage disposal site (El Lamab, south of Khartoum), from 250 m upstream and 250 m downstream during January–March, 1987 and Februar–April, 1988. The water samples were examined for physical and chemical characteristics. Plankton samples were examined for species composition and relative abundance of phytoplankton and zooplankton.At the disposal site, the sewage effluent increased conductivity, nutrients and suspended solids, elevated BOD, and depleted oxygen. The pattern of abundance in the phytoplankton was almost reversed, due to the disappearance of many diatoms, replaced by blue-green algae, and the appearance of Euglenophyceae, found only at the disposal site. Copepods dominated over cladocerans, which are usually dominant under natural conditions. Rotifers, normally less abundant, also flourished at the disposal site. However, these effects were localized: physical, chemical and biological conditions returned to normal 250 m downstream.Dept. of Zoology, Hydrobiological Research Unit, University of Khartoum     

Inhibition of RANTES/CCR1-mediated chemotaxis by cosalane and related compounds

The anti-HIV agent cosalane and several of its analogues inhibited RANTES-induced migration of human monocytes, but they did not inhibit migration induced by MIP1alpha or MIP1beta. RANTES-induced migration of single receptor CCRI-HEK transfectants was also inhibited by the cosalanes. Acetylation of the reactive amino groups of RANTES abrogated the inhibitory activity of cosalane. The data suggest that cosalane and its structural analogues may interfere with the RANTES/CCR1 interaction by binding to RANTES.