Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice.

AIM: To assess the efficiency of a medium-pressure UV reactor under full-scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. METHODS AND RESULTS: Six/seven-day-old mice were administered orally 2-10x10(4)Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm(-2) UV irradiation of ingested oocysts resulted 7 days later in a 3.4-4.0 log10 reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. CONCLUSION: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. SIGNIFICANCE AND IMPACT OF THE STUDY: Present results suggest that in WTP conditions, a medium-pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro-organisms present in environmental waters.

Microplate method for obtaining Leishmania clonal populations.

The various clinical expressions observed in human leishmaniases result from complex host-parasite relationships in which the biodiversity of the parasite is a determining factor. Because Leishmania strains isolated from humans are composed of heterogeneous populations, it is crucial to use clonal lineages for studies on the characterization of these parasites. Presently, techniques used for cloning Leishmania spp. parasites are time-consuming and show poor efficiency. Here, a method developed in 96-well microplates is described, which allows one to rapidly obtain numerous clones of Leishmania in the most versatile and efficient way. The technique may be useful for cloning various protozoa as well as Leishmania spp.

Virulence of Leishmania infantum is expressed as a clonal and dominant phenotype in experimental infections.

Human Leishmania infantum infection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence in L. infantum visceral leishmaniasis is clonal and dominant in nature.

Assessment of Leishmania promastigote growth in vitro by means of nucleoside hydrolase activity determination.

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of protozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl-beta-D-ribofuranoside (NPR) substrate for the quantification of Leishmania infantum. The free promastigote stage of L. infantum contains high amounts of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide range of concentrations (5 x 10(4) 2 x 10(8) parasites ml(-1)) in a single assay. No difference in NH activity was observed between various strains at equivalent concentrations and growth curves determined with NH dosage were similar to optical counts. Samples can be stored at -20 degrees C for weeks before use in this unique assay without significant loss of NH activity. The method is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. infantum promastigotes in vitro.

Experimental pathogenicity of a presumed monoxenous trypanosomatid isolated from humans in a murine model.

Two strains of a presumed lower trypanosomatid isolated from immunocompetent and HIV-infected humans in French West Indies were investigated in vitro and in vivo in a murine experimental model. The ability of parasites to grow in vitro in bone marrow-derived macrophages and their virulence in vivo were assessed. For in vivo infection, two groups of BALB/c mice were inoculated either by the subcutaneous or intravenous route with 10(7) promastigotes at day 0. Infection was monitored by measuring parasite load in liver, spleen, foot pad, popliteal, and mesenteric lymph nodes and brain from day 7 to day 150 post-infection using a microtitration technique. Parasites multiplied in mouse macrophages in vitro. In vivo, both strains proved infective to mice and capable of visceralization and dissemination in the popliteal and mesenteric lymph nodes, liver, spleen, and even brain. Both strains elicited a strong humoral response against trypanosomatid antigen in mice, which cross-reacted with Leishmania antigen. Contrasting with the straightforward dissemination of parasites, the infection was strikingly well tolerated by the murine host with no clinical signs and minimal tissue changes around parasitized macrophage infiltrates.

Evidence for determining parasitic factors in addition to host genetics and immune status in the outcome of murine Leishmania infantum visceral leishmaniasis.

C.B-17 SCID and congenic BALB/C mice were used to examine Leishmania infantum strain pathogenicity independently of host genetic factors. While parasite loads were significantly higher in immunodeficient mice than in immunocompetent mice, the kinetics of infection during a long-term follow-up were similar, suggesting that intrinsic parasitic factors also influence the outcome of L. infantum infection.

Inhibition of Friend cells proliferation by spi-1 antisense oligodeoxynucleotides.

The spi-1 proto-oncogene encodes the transcription factor PU.1 which is normally expressed in all hematopoietic cell lineages except in T cell lines. During the murine acute erythroleukemia induced by the Friend retrovirus, SFFV, spi-1 deregulation by insertional mutagenesis results in the overexpression of Spi-1/PU.1 in the malignant proerythroblastic cell. To assess the Spi-1 role in the proliferation and the differentiation arrest of the Friend tumor cells we inhibited spi-1 gene expression in two Friend cell lines by using antisense oligodeoxyribonucleotides. Proliferation and cloning efficiency of both cell lines were significantly inhibited by spi1 antisense. This antiproliferative effect was not related to an apparent maturation of erythroleukemic cells demonstrating that repression of spi-1 expression is not sufficient per se to restore the ability of the proerythroblastic cells to spontaneously differentiate in mature erythroblasts. These data suggest that the spi-1 gene would be involved in the Friend leukemic process by promoting the proerythroblast to proliferate.

Protooncogene expression in normal, preleukemic, and leukemic murine erythroid cells and its relationship to differentiation and proliferation.

The expression of 18 protooncogenes was examined by Northern blot analysis in preleukemic and leukemic stages of murine erythroleukemias induced by Friend viruses. As controls, erythropoietically stimulated spleens from phenylhydrazine-treated mice were studied. Expression of 10 protooncogenes (c-erb-A, c-erb-B, c-ets, c-sis, c-mos, c-rel, c-src, c-fes, c-fms, N-myc [corrected] was not detectable in Friend erythroleukemias. One protooncogene (c-src) was found expressed in normal erythroid cells but not in erythroleukemias. Four protooncogenes (c-fos, c-abl, N-ras, and c-raf) were expressed at low levels in both steps of erythroleukemia. c-fos and c-abl RNAs were barely detectable in normal erythroid cells. High levels of four protooncogene transcripts (c-H-ras, c-K-ras, c-myc, and c-myb) were detected in preleukemic and leukemic tissues. While c-H-ras RNA was found at similar levels in normal and leukemic erythroid cells, c-myc, c-myb, and c-K-ras were not expressed in normal erythroid cells. To determine whether the elevated levels of c-myc, c-myb, and c-K-ras RNAs in erythroleukemic cells are related to the proliferative state or the undifferentiated state of the cells, the effect of dimethyl sulfoxide-induced differentiation on oncogene expression in two erythroleukemia cell lines was examined. Terminal differentiation was associated with lack of c-myb expression while c-myc and c-K-ras expression was essentially unaffected. These results suggest that the high levels of c-myb transcripts in erythroleukemias may reflect the undifferentiated state of the leukemic cells. In contrast, the elevated expression of c-myc and c-K-ras at both stages of the Friend diseases is probably not related to the stage of differentiation but rather to the uncontrolled proliferation of the cells. Finally among 18 protooncogenes surveyed, only the accumulation of c-myc and c-K-ras RNAs appears to be associated with the Friend erythroleukemic process before the late leukemic phase develops.

Presence of a c-myc transcript initiated in intron 1 in Friend erythroleukemia cells and in other murine cell types with no evidence of c-myc gene rearrangement.

In Friend murine erythroleukemia cells, although no detectable c-myc gene rearrangement was found, we observed, in addition to the normal 2.3-kilobase c-myc transcript, the presence of a 2.3-kilobase c-myc mRNA initiated in intron 1 at a promoter site called P3. The intron 1-initiated transcript has a longer half-life than the normal c-myc mRNA. This c-myc transcript initiated in intron 1 was also found in other murine cell types where no rearrangement of the c-myc locus has been reported.

Retroviral endogenous transcripts related to the envelope gene of Friend spleen focus-forming virus in normal mouse tissues.

Retroviral endogenous sequences related to the envelope (env) gene of Friend spleen focus forming virus (SFFV) and of mink cell focus forming viruses (MCF) are present in the genome of various mouse strains. We have examined the transcription of these SFFV/MCF-related sequences in normal tissues of two mouse strains, ICFW and DBA/2. Cytoplasmic Poly A+ RNAs of normal mouse tissues were analyzed by dot-blot and Northern blot hybridizations with a subcloned env SFFV DNA fragment (0.4 kbp BamH I-Sma I). In both mice, the level of SFFV/MCF env related transcripts was very low in bone marrows and spleens whereas it was high in kidneys. Intermediate levels of transcripts were observed in other tissues (thymus, liver and brain). In both mouse strains, the size of SFFV/MCF env related transcripts varied from one tissue to another. Some transcripts in DBA/2 mice were reminiscent of full-size viral message indicating an occasional expression of xenotropic/MCF endogenous virus in this low-leukemic strain. Sizes of the other SFFV/MCF related env transcripts were unusual, but were similar in both strains for each tissue studied. This last result suggests a tissue-specific transcription of endogenous sequences related to the SFFV/MCF env gene. A 1.8 kb SFFV/MCF env RNA was the major transcript in the tissues which expressed a high level of these env transcripts. Treatment of mice with phenylhydrazine which greatly stimulates erythroid differentiation in spleens increased the level of SFFV/MCF related env RNAs only in the spleens, suggesting a possible correlation between the SFFV/MCF env transcription and the stimulation of the erythroid spleen cells.