A new experimental in vitro culture medium for cultivation of Leishmania species.

A new liquid culture medium prepared with chemicals that can be obtained economically and commercially was tested in in vitro cultivation of Leishmania promastigotes to obtain a large number of organisms to use in serological studies. The number of Leishmania infantum and Leishmania tropica promastigotes taken from Novy-MacNeal-Nicolle (NNN) medium reached 1 x 10(7)/ml at the end of the 8th day in our new medium, though in NNN medium the number of organisms reached only 5 x 10(6)/ml. After 10 subsequent passages, the culture medium prepared was evaluated as being quite inexpensive, simple, and successful compared with other commercially available liquid culture media.     

Milk of cow (Bos taurus), buffalo (Bubalus bubalis), and goat (Capra hircus): a better alternative than fetal bovine serum in media for primary isolation, in vitro cultivation, and maintenance of Leishmania donovani promastigotes

Tyndalized milk of goat, cow, and buffalo was found to be a potential substitute for fetal bovine serum (FBS) in the medium for the cultivation of Leishmania donovani promastigotes. The numbers (means) of promastigotes reached 2.6 x 10(7), 2.3 x 10(7), and 2.1 x 10(7)/ml, respectively, in the medium supplemented with 10% milk of goat, cow, and buffalo, in comparison to 1.9 x 10(7)/ml in the control with 10% FBS. In primary isolation, the milk-supplemented medium showed that 22 out of 26 samples were positive for promastigotes (84.6%) and the cells were maintained successfully during the observed period of 6 months.     

A simple method for cloning Leishmania spp

A rapid and simple technique for isolation of clones of Leishmania spp. was developed. This method consists to adapt promastigote forms of Leishmania infantum to an easily realized medium: the Panmede solid medium. We obtained cloned-population of this parasite for epidemiologic and toxonomic studies.     

Interaction of Leishmania PTS2 receptor peroxin 7 with the glycosomal protein import machinery

Leishmania proteins containing a peroxisomal targeting signal sequence 2 (PTS2) are selectively trafficked to the glycosome by associating with the peroxin 7 receptor protein (PEX7). The L. major PEX7 (LmPEX7) encodes a approximately 41 kDa protein that exhibits limited sequence identity with PEX7 homologues from other eukaryotic organisms. Functional characterization of recombinant and native LmPEX7 revealed that this receptor bound the PTS2 protein fructose-1,6-bisphosphate aldolase. Moreover, LmPEX7 also formed a tight association with the Leishmania PEX5, the cytosolic PTS1 receptor, and PEX14, a glycosomal peripheral membrane protein required for protein import into the glycosome. Mapping studies revealed that the Leishmania PEX7 binds to a domain on LdPEX5 encompassing residues 111-148 and to a site on LdPEX14 spanning residues 120-148. Finally, subcellular localization studies revealed that Leishmania PEX7 has a dual distribution within the cytosolic compartment and glycosomal lumen.     

Leishmania RAB7: characterisation of terminal endocytic stages in an intracellular parasite

Leishmania species are intracellular parasites that inhabit a parasitophorous vacuole (PV) within host macrophages and engage with the host endo-membrane network to avoid clearance from the cell. Intracellular Leishmania amastigotes exhibit a high degree of proteolytic/lysosomal activity that may assist degradation of MHC class II molecules and subsequent interruption of antigen presentation. As an aid to further analysis of the endosomal/lysosomal events that could facilitate this process, we have characterised a Leishmania homologue of the late endosomal marker, Rab7, thought to be involved in the terminal steps of endocytosis and lysosomal delivery. The Leishmania major Rab7 (LmRAB7) protein is expressed throughout the life-cycle, shows 73 and 64% identity to Trypanosoma cruzi and Trypanosoma brucei Rab7s (TcRAB7 and TbRAB7), respectively, and includes a kinetoplastid-specific insertion. The recombinant protein binds GTP and polyclonal antibodies raised against this antigen recognise structures in the region of the cell between the nucleus and kinetoplast. By immunoelectron microscopy of axenic amastigotes, Leishmania mexicana Rab7 (LmexRAB7) is found juxtaposed to and overlapping membrane structures labelled for the megasomal marker, cysteine proteinase B, confirming a late-endosomal/lysosomal localisation.     

Epidemiologic and parasitologic data concerning sporadic cutaneous leishmaniasis in northern Tunisia

This study refers to 23 patients presenting with the sporadic forms of cutaneous leishmaniasis encountered in northern most humid parts of Tunisia. Culture inoculation for parasitic isolation was processed using two media: the classical NNN and a rabbit serum based medium (SLC). Cultures were positive in 17 cases with SLC medium and 13 cases with NNN medium. Eight isolates were typed using 15 isoenzymes systems. Six isolates were identified as Leishmania infantum MON-24 which confirms the crucial role of this zymodeme in causing this form of cutaneous leishmaniasis. The other two isolates were identified as Leishmania infantum MON-1, which is the principal agent of visceral leishmaniasis in the Mediterranean area.     

Leishmania (Viannia) naiffi sp. n., a parasite of the armadillo, Dasypus novemcinctus (L.) in Amazonian Brazil

A new leishmanial parasite, Leishmania (Viannia) naiffi sp. n., is described from the nine-banded armadillo, Dasypus novemcinctus (Edentata: Dasypodidae), from Para State, north Brazil. The parasite grows luxuriantly in Diffco blood-agar medium (B47), but poorly in the skin of intradermally inoculated hamsters. A comparison of isoenzyme profiles by starch gel electrophoresis separates the parasite from L. (V) braziliensis and L. (V.) guyanensis by the enzymes ASAT, ALAT, PGM, GPI, G6PD, PEP, MPI and GD, and from Leishmania (Leishmania) chagasi, L. (L.) amazonensis and L. (L.) deanei by ASAT, ALAT, PGM, GPI, MPI, G6PD, MDH, PEP and ACON. Finally, L. (V.) naiffi is serologically differentiated from L. (V.) braziliensis, L. (V.) guyanensis and L. (V.) panamensis on monoclonal antibodies specific for these parasites.     

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.     

Development of a defined medium for heterologous expression in Leishmania tarentolae

A defined medium is essential for studying nutritional requirements of microorganisms and for selective supplementation of necessary substances. Hemin is an essential ingredient for growth of Leishmania tarentolae, but tends to precipitate in aqueous solutions without further stabilization. These aggregates disturb the measurement of the optical density or the cell density and the following downstream processing. Therefore, we were looking for stabilizing substances and established a PEG-hemin-solution, which avoided flocculation and allowed the cultivation of L. tarentolae in a medium, which we termed SFP(II) medium. With addition of RNA from Saccharomyces cerevisiae to SFP(II) medium the SFP(III) medium was established. In this medium, the specific cell division rate was increased (0.103 h(-1)) and stable for longer periods of time. The evaluation of the SFP(III) medium was done in shaker flasks by successful expression and segregation of the SAG2 protein, one of the main surface antigens of Toxoplasma gondii. With establishment and evaluation of this defined medium, the status of the Leishmania tarentolae expression system as an alternative to commonly used cell cultures is supported.     

In vitro maintenance of Leishmania promastigote in an egg based biphasic culture medium

Leishmaniasis, a parasitic disease caused by the protozoan Leishmania is endemic in many parts of tropics an subtropics. In vitro cultivation of the parasite plays an important role in the study and treatment of the disease. In the present study a new, simple and cheap yet reliable egg based bi-phasic culture medium is described which is capable of supporting long term cultivation of Leishmania promastigote in vitro without inclusion of foetal bovine serum or blood.     

An experimental model system for leishmaniasis. An ultrastructural study on cultured macrophages exposed to Leishmania parasites and sodium stibogluconate

To facilitate studies on the effect of chemotherapeutic agents on the host-parasite interaction in leishmaniasis, we have developed an experimental model for infecting mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigotes. As the drug action is often dependent on concentration, the distribution of sodium stibogluconate, which is the commonly used drug for treatment of leishmaniasis, was studied in various parts of the macrophages by energy dispersive X-ray microanalysis. The drug was found to accumulate in secondary lysosomes. The ultrastructural examination, using TEM and SEM, of macrophages, whose secondary lysosomes had been preloaded with gold particles, showed that leishmania parasites are phagocytosed and finally located in secondary lysosomes. Using flameless atomic absorption spectrophotometry, the concentration of Mn, Fe and Cu in promastigotes of Leishmania donovani, Leishmania aethiopica, Leishmania crithidia, Leishmania major and their culture media was estimated. Of the three transition metals, the parasites accumulated only Mn from the medium, which they may use in a primitive defense mechanism against reactive oxygen metabolites produced by macrophages during the respiratory burst associated with phagocytosis.     

Molecular karyotype and chromosomal localization of genes encoding two major surface glycoproteins, gp63 and gp46/M2, hsp70, and beta-tubulin in cloned strains of several Leishmania species.

The molecular karyotypes of several Leishmania isolates (Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 major surface glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and beta-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The beta-tubulin gene probe hybridized predominantly to large (1600-1750 kb) chromosome-size DNA and to 1-5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica. Gp46/M2 genes were located on 1 band in L. donovani, L. major, and L. aethiopica or 2 bands in L. tropica and L. amazonensis, but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii. Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.     

Evaluation of three new culture media for the cultivation and isolation of Leishmania parasites

The aim of this study was to establish novel culture media for Leishmania parasites with a potential of obtaining high amounts of promastigotes with long-term viability, and consisting of ingredients that were available in microbiology or parasitology laboratories. Other features of these media included no requirement for blood, FCS (Fetal calf serum) or erythrocyte lysate, inexpensiveness and easiness in preparation. In addition, aspiration samples obtained from cutaneous leishmaniasis (CL) suspected patients were cultivated in these media. Three culture media were prepared; trypticase beef extract hemoglobine (TBH) medium, including trypticase, beef extract and yeast extract as the protein source, glucose as the carbohydrate source, FeNH4 and bovine hemoglobine; Peptone-Yeast extract medium (PY), found to be effective in our previous studies for cultuvation of on Leishmania parasites, with bovine hemoglobine (PYH) and Brain Heart medium, containing bovine hemoglobine (BKH). The number of promastigotes were the highest on day 8 and 13 in RPMI 1640 and BKH medium, respectively. In TBH and PYH, the peak level of reproduction was between day 16 and 19, and it was found to be higher in TBH medium after the day 20. The number of promastigotes were found to be close in BKH, TBH and RPMI-1640 media and lower in PYH medium. Examination of the cultivation of the aqueous lesion specimens of the 10 CL-suspected cases in media revealed reproduction in 9 flasks of RPMI-1640 containing 10% FCS, 7 TBH, 6 BKH and 4 PYH. The differences between the culture media were not found to be statistically significant. These results suggested that, three liquid culture media, assessed in this study, with no requirement for FCS or erythrocyte lysate, were effective in the reproduction of promastigotes, and could be used effectively in the patient isolation and field studies, as well.     

Ecology of leishmaniasis in Southern France. 18. Enzymatic identification of Leishmania infantum Nicolle, 1908, isolated from Phlebotomus ariasi Tonnoir, 1921, spontaneously infected in the Cévennes

Out of 187 female Phlebotomus ariasi caught in the Cévennes focus of leishmaniasis 3 were found naturally infected with Leishmania. The infection in one of the three had spread from the midgut to the pharynx and proboscis. Stocks of Leishmania were isolated from two of the flies and 8 isoenzymes were examined. The newly isolated parasites were found to be indistinguishable from zymodeme 1 of Leishmania infantum s. st.     

Differential inhibition of macrophage microbicidal activity by liposomes.

In vitro culture of murine resident peritoneal macrophages with lymphokine (LK)-rich leukocyte culture fluids induces enhanced microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica. Macrophages infected with Leishmania and treated with LKs after infection acquire the capacity to kill the intracellular parasite within 72 h. When compared with control macrophage cultures treated with medium lacking LKs, 80 to 90% fewer macrophages treated with LKs contained amastigotes. In experiments designed to test liposome delivery of LKs to infected macrophages, addition of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine (molar ratio, 7:3) completely abrogated LK-induced microbicidal activity. Liposomes containing only phosphatidylcholine were not inhibitory. Inhibition of LK activity by the liposomes occurred regardless of whether the liposomes contained LKs. Liposomal inhibition of activated macrophage effector activity was limited to intracellular killing; LK-induced macrophage extracellular cytolysis (i.e., tumor cytotoxicity) was not affected by liposome treatment. These data indicate that elucidation of the effects of liposome composition on acquired host defense mechanisms may be useful for the design of drug delivery systems that allow expression or augmentation of immunologically induced mechanisms for the intracellular destruction of infectious agents.     

Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples

The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.     

Leishmania mexicana promastigotes secrete a protein tyrosine phosphatase

Leishmania mexicana is an intracellular protozoan parasite that infects macrophages and dendritic cells and causes a chronic cutaneous disease. Although many enzymatic activities have been reported in this parasite, the presence of kinases and phosphatases has been poorly studied. These enzymes control the phosphorylation and dephosphorylation of proteins. Specifically, protein tyrosine kinases phosphorylate tyrosine residues and protein tyrosine phosphatases (PTPases) dephosphorylate tyrosine residues. PTPase activities have been reported as pathogenic factors in various infectious microorganisms such as viruses, bacteria, and parasites. Also, it has been shown that the induction of one or more PTPase activities in macrophages represents an important pathogenicity factor in Leishmania. Recently, we reported a membrane-bound PTPase activity in promastigotes of Leishmania major. In the present work, we give evidence that promastigotes of L. mexicana are able to secrete a PTPase into the culture medium. Two antibodies: one monoclonal against the catalytic domains of the human placental PTPase 1B and a polyclonal rabbit anti-recombinant protein Petase7 from Trypanosoma brucei cross-reacted with a 50-kDa molecule. The anti-human PTPase 1B antibody depleted the enzymatic activity present in the conditioned medium. The pattern of sensitivity and resistance to specific PTPase and serine/threonine inhibitors showed that this enzyme is a protein tyrosine phosphatase.     

Heterogeneity of the genes encoding the major surface glycoprotein of Leishmania donovani.

The major surface glycoprotein of Leishmania (GP63) is present on all known species of Leishmania and likely plays an integral role during the infection of macrophages in the mammalian host. To identify regions of GP63 which may be of functional significance, the nucleotide sequence of a gene encoding GP63 of Leishmania donovani was determined and compared to the sequences reported for GP63 genes of Leishmania major and Leishmania chagasi. The GP63 nucleotide and predicted protein sequence was highly conserved among the 3 species despite their diverse geographical distribution. L. donovani GP63 is encoded by a multigene family and the gene locus contains at least 7 tandemly repeated genes and at least 3 genes which are dispersed from the tandem array. In addition, polymerase chain reaction and Southern blot analyses demonstrated that there was size heterogeneity within the pro-peptide coding regions of the multiple GP63 genes of L. donovani and that such genes were expressed concurrently in the promastigote life stage.     

杜氏利什曼原虫LACK基因真核表达重组质粒及其在真核细胞中的表达

利什曼原虫的 L ACK抗原是利什曼原虫活性蛋白激酶 C受体同源物 ,是一种新发现的抗原蛋白。我们以本实验室的重组质粒 T- L ACK为模板 ,PCR扩增获得 L ACK基因 ,与真核表达载体 pc DNA3.1( )定向重组 ,重组质粒经酶切和 PCR分析 ,含有约 95 0 bp的 L ACK基因 ,成功构建含有 L ACK基因的真核表达重组质粒 pc D-NA3- L ACK。将此重组质粒转染 COS- 7细胞 ,通过 RT- PCR及免疫荧光检测 L ACK基因在真核细胞中的表达。实验结果显示转染了重组质粒的 COS- 7细胞 ,其 RT- PCR及免疫荧光检测均呈阳性反应 ,证实重组质粒 pc DNA3-L ACK能在 COS- 7细胞中有效表达 L ACK蛋白。     

Leishmanial Excreted Factor: Protein-Bound and Free Forms from Promastigote Cultures of Leishmania tropica and Leishmania donovani

Leishmania spp. growing in culture produce an immunologically active substance called excreted factor (EF), which precipitates antibodies raised against intact cells and has been implicated as the conditioning agent for parasite infection of host macrophages. An improved method for isolation of the material is described, based on Sephadex column chromatography of growth medium which had been boiled at pH 5.0. This procedure allows the detection of differences among the EF molecules of different species, and it overcomes previous shortcomings through the monitoring of immunological activity throughout. Analysis of the products of this procedure revealed that EFs from Leishmania tropica and Leishmania donovani share a common carrier protein, identified as rabbit serum albumin, and are chemically quite similar. Growth medium from L. tropica boiled at acidic pH contains primarily an EF-albumin complex of 75,000 molecular weight. Treated growth medium from L. donovani, on the other hand, contains both the albumin complex and a smaller molecule (less than 27,000 molecular weight) that is not associated with rabbit protein. This material accounts for nearly 20% of the EF of one L. donovani strain, but constitutes only a minute fraction of L. tropica EF. Treatment of the EF-albumin complex with trichloroacetic acid separates the molecule into two major subunits, one having a molecular weight of about 61,000 (without anti-Leishmania activity) and the other having a molecular weight of about 18,000 (with no anti-rabbit activity). The protein-free EF of L. tropica differs from that released by trichloroacetic acid extraction in that it is capable of precipitating antisera of nonhomologous serotypes, whereas the albumin complex and the trichloroacetic acid-treated EF fragment are not. EFs from both species display pH-dependent affinity for certain lectins.