Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification.

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.

Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World.

INTRODUCTION: Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. METHODS: Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. RESULTS: Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. CONCLUSION: Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.

In vitro activity of an essential oil against Leishmania donovani.

The in vitro antileishmanial effect of the essential oil from Chenopodium ambrosioides against Leishmania donovani was investigated. The product showed significant activity against promastigotes and amastigotes, with a 50% effective concentration of 4.45 and 5.1 microg/mL, respectively. The essential oil caused an irreversible inhibition of the growth of promastigotes after a treatment with 100 or 10 microg/mL for 1 or 24 h, respectively. The phagocytic activity of the macrophages was preserved at a concentration toxic to the parasite. The essential oil from C. ambrosioides may be a potential candidate drug to development a new agent to combat this parasitic disease.

Nonlocal transplantation and outbreeding depression in the subshrub Lotus scoparius (Fabaceae).

The genetic background of transplants used to create or augment wild populations may affect the long-term success of restored populations. If seed sources are from differently adapted populations, then the relative performance of progeny from crosses among populations may decrease with an increase in genetic differences of parents and in the differences of parental environments to the transplant location. We evaluated the potential for such outbreeding depression by hybridizing individuals from six different populations of Lotus scoparius var. scoparius and L. s. var. brevialatus. We used allozyme data to calculate genetic distances between source populations, and compiled climatic data and measured soil traits to estimate environmental distances between source populations. We found significant outbreeding depression following controlled crosses. In the greenhouse, the success of crosses (seeds/flower × seedlings/seed) decreased with increasing genetic distance between populations revealing genetically based outbreeding depression unrelated to local adaptation. After outplanting to one native site (in situ common garden), field cumulative fitness of progeny (survival × fruit production) decreased significantly with mean environmental distance of the parental populations to the transplant site, but not with genetic distance between the crossed populations. This result is consistent with a disruption of local adaptation. At the second, ecologically contrasting common garden, where low survival reduced statistical power, field cumulative fitness (survival × progeny height) did not decrease significantly with either environmental distance or genetic distance. Overall, intervariety crosses were 40 and 50% as fit (seeds/flower × seedlings/seed × survival × fruits at the first garden or × height at the second) as intravariety crosses. These results suggest that the cumulative outbreeding depression was caused by a combination of genetically based ecological differences among populations and other genomic coadaptation. We conclude that mixing genetically differentiated seed sources of Lotus scoparius may significantly lower the fitness of augmented or restored populations. Genetic and environmental similarities of source populations relative to the transplant site should be considered when choosing source materials, a practice recommended by recent seed transfer policies. Geographic separation was not a good surrogate for either of these measures.

Association of mitochondria with the plasma membrane of the acinar cells of actively secreting and nonsecreting rat lacrimal and parotid glands.

The distance between mitochondria and the nearest plasma membrane was measured in actively secreting and nonsecreting exocrine glands in order to determine the effect of the secretory mechanism upon mitochondrial distribution. The greatest accumulation of mitochondria was at 0.6 micron from the plasma membrane. No difference was noted in mitochondrial distribution of actively secreting, untreated and atropine-treated animals. There was also no difference in mitochondrial distribution of lacrimal and parotid acinar cells. The possible physiological significance of this observation is discussed.

[The need for the use of chromogens for quantifying Leishmania promastigotes on plates from 96 wells]. / Necesidad del uso de cromógenos para cuantificar promastigotes de Leishmania en placas de 96 pozos.

The existing difficulties in the treatment of leishmaniasis justify the testing of the effect of new products on parasite forms in the search of a therapeutic alternative for the parasitosis. Given the need of establishing a method to evaluate the activity of natural synthetic products in vitro under the Cuban conditions, this paper was aimed at defining the usefulness of p-nitrophenilphosphate as a chromogenic substance for quantification of parasites in plaques from 96 wells. To standardize this colorimetric method the stages of the parasite growth curve were set. The study of linearity and selection of the sample size, which was optional for these assays, showed that it was possible to obtain maximum linear determination coefficient with 20 mm. Likewise, the variation coefficient was compared with and without the chromogen and the effect of changes in culture medium on the reading of absorbances was analyzed. The set limit of quantification proved the need of using chromogen for the purposes of this paper and the general results allow to recommend this less subjective, simpler and quicker methodology to test products of interest in this field.

[Study of the seroprevalence of toxocariasis in an infantile population in the City of Havana]. / Estudio de seroprevalencia de toxocariasis en una población infantil de Ciudad de La Habana.

A serological study to detect antibodies to Toxocara canis in a group of 156 healthy children from City of Havana is reported for the first time in Cuba. An ELISA method was employed using excretion/secretion antigens obtained in our laboratory. Data on epidemiological factors surveyed in this group are presented. Positivity percentage was of 5.2%. Results are discussed.

The hippocampus and stress induced 17-OHCS elevations.

The physiologic contribution of the limbic brain to emotionally induced stress is still poorly understood. The present study is designed to more specifically evaluate the role of the hippocampus in stress induced plasma 17-OHCS elevations. The conditional reflex to a sequential presentation of tone and shock was used as the stress agent in adult mongrel dogs. Plasma 17-OHCS levels were determined by the Porter-Silber method. Control and stress levels of 17-OHCS were determined before and after unilateral (left) hippocampectomy, and subsequent contralateral (right) hippocampectomy. A unilateral posterior hippocampal lesion partially attenuated (20%) the normal 17-OHCS stress response. In contrast to unilateral lesions, equivalent bilateral posterior hippocampal lesions abolished the normal 17-OHCS stress response. These observations support the thesis that the elevated 17-OHCS levels in response to the conditioning paradigm is dependent on the hippocampus. Furthermore, it is dependent upon the continuity of the hippocampal circuit and not upon the volumetric steroid binding capacity of the hippocampus. These studies also suggest that a unilaterally functioning hippocampus may be adequate to meet the physiologic needs of stress, as reflected by the 17-OHCS response.

[Cysteine-dependent protease in Trypanosoma cruzi useful for the diagnosis of Chagas disease]. / Proteasa dependiente de cisteína en Trypanosoma cruzi útil en el diagnóstico de la enfermedad de Chagas.

An immunoenzymatic system on solid phase for the detection of IgG antibodies in serum from chronic Chagasic patients was standardized. A protease from Trypanosoma cruzi (GP57/51KDa) was used as an antigen. The sensitivity, specificity and predictive values of the procedure were calculated taking into account the results obtained from conventional serology (CS), indirect immunofluorescence (IIF), indirect hemagglutination (IHA) and complement-mediated lysis test (CML) for the detection of anti-Trypanosoma cruzi antibodies in serum. Except one, the positive samples obtained by those techniques were also positive by our method. These results show that GP 57/51 is useful in the serodiagnosis of Chagas disease.