Molecular karyotype analysis and mapping of housekeeping genes to chromosomes of selected species complexes of Leishmania

The molecular karyotypes for 20 reference strais of species complexes of Leishmania were determined by contour-clamped homogeneous eletric field (CHEF) electrosphoresis. Determination of number/position of chromosome-sized bands and chromosomal DNA locations of house-keeping genes were the two criteria used for differentiating and classifying the Leishmania species. We have established two gel running conditions of optimal separation of chromosomes, wich resolved DNA molecules as large as 2,500 kilobase pairs (kb). Chromosomes were polymorphic in number (22-30) and size (200-2,500 kb) of bands among members of five complexes of Leishmania. Although each stock had a distinct karyotype, in general the differences found between strains and/or species within each complex were not clear enough for parasite identification. However, each group showed a specific number of size-concordant DNA molecules, wich allowed distinction among the Leishmania complex parasites. Clear differences between the Old and New world groups of parasites or among some New World Leishmania species were also apparent in relation to the chromosome locations of beta-tubulin genes. Based on these results as well as data from other published studies the potencial of using DNA karyotype for identifying and classifying leishmanial field isolates is discussed.

Life history studies of heterophyid Trematodes in the neotropical region: Ascocotyle (Leighia) hadra sp.n

The life cycle of Ascocotyle (Leighia) hadra n.sp. was experimentally reproduced, starting from cercariae from naturally infected Littoridina parchappei, collected from Los Ranchos stream, near Mercedes city, Buenos Aires Province, Argentina. Metacercariae were found encysted in the liver and mesentery of experimentally and naturally infected fishes Cnesterodon decemmaculatus and Jenynsia lineata. Adults were obtained experimentally in chicks and mice. The natural host is unknown. The new species is compared with Ascocotyle (Leighia) mcintoshi Price 1936 as described by Leigh, 1974, differing in behavior and morphology of cercarial, metacercarial and adult stages

Life history of Pygidiopsis crassus n. sp. (Trematoda, Digenea, Heterophyidae) in the neotropical region

The life cycle of Pygidiopsis crassus n. sp. was experimentally reproduced, starting from cercariae from naturally infected Littoridina parchappei collected from Lujan River and different ponds in Buenos Aires Province, Argentina. Metacercariae were found encysted in the body cavity of experimentally and naturally infected fishes Cnesterodon decemmaculatus and naturally infected Jenynsia lineata. Adults were obtained experimentally in chicks and mice. The natural host is unknown. The new species is compared with Pygidiopsis macrostomum Travassos 1928, from Rattus norvegicus and from Noctilio leporinus mastivus, differing in body and egg sizes, in the size relation of oral and ventral sucker and the shape of excretory vesicle.

Life history correlates of adult size in the malaria vector Anopheles darlingi

Adult dry weights of laboratory-reared Anopheles darlingi were highly correlated with wing lengths, which were used to estimate size variation in natural populations of this species. Significant differences in mean wing lengths of females trapped at baits were detected among collections in the same week at one site, but not between three sites in Brazil and Boliva. Relatively higher variability of wing lengths, compared to collections of other Anopheles (Nyssorhynchus), and platykurtic size distributions in large, single-night collections suggested that An. darlingi females caught at baits emerged from heterogenous larval habitats. No relationship was detected between parous state and the body size of wild-caught females. Adult males and females of laboratory-reared An. darlingi did not differ in body size. This absence of sexual size dimorphism is rare among mosquitoes and has not been noted previously in the genus Anopheles.

Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

Separation and Mapping of Chromosomes of Parasitic Protozoa

Many protozoan parasites represent an important group of human pathogens. Pulsed Field Gradient Gel Electrophoresis (PFGE) analysis has been an important tool for fundamental genetic studies of parasites like Trypanosoma, Leishmania, Giardia or the human malaria parasite Plasmodium falciparum. We present PFGE conditions allowing a high resolution separation of chromosomes ranging from 500 to 4000 kb within a two day electrophoresis run. In addition, we present conditions for separating large chromosomes (2000-6000 kb) within 36 hr. We demontrate that the application of two dimentional PFGE (2D-PFGE) technique to parasite karyotypes is a very useful method for the analysis of dispersed gene families and comparative studies of the intrachomosomal genome organization

The Trypanosoma cruzi Genome Project: Nuclear Karyotype and Gene Mapping of Clone CL Brener

By using improved pulsed field gel electrophoresis conditions, the molecular karyotype of the reference clone CL Brener selected for Trypanosoma cruzi genome project was established. A total of 20 uniform chromosomal bands ranging in size from 0.45 to 3.5 Megabase pairs (Mbp) were resolved in a single run. The weighted sum of the chromosomal bands was approximately 87 Mbp. Chromoblots were hybridized with 39 different homologous probes, 13 of which identified single chromosomes. Several markers showed linkage and four different linkage groups were identified, each comprising two markers. Densitometric analysis suggests that most of the chromosomal bands contain two or more chromosomes representing either homologous chromosomes and/or heterologous chromosomes with similar sizes

Selection, Recombination and History in a Parasitic Flatworm (Echinococcus) Inferred from Nucleotide Sequences

Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection) drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1). The phylogenetic analyses revealed several ambiguities, indicating that the taxonomic status of the E. granulosus horse strain should be revised

Life-history traits of Fossaria cubensis (Gastropoda: Lymnaeidae) under experimental exposure to Fasciola hepatica (Trematoda: Digenea)

The effect of exposing the lymnaeid snail Fossaria cubensis to the trematode Fasciola hepatica on the snail population's life-history traits was studied under laboratory conditions. Exposed individuals showed a lower survival rate than control snails, although from week 7 onward a slower decrease of this parameter in relation to the control group was observed. There were higher values of fecundity rate for the controls compared to the exposed group except during weeks 9, 10, 11 and 12, which was the time that followed the period when almost all of the infected snails died. Both the intrinsic and finite rates of natural increase were significantly higher for the control group, but exposed snails still attained a lower mean generation time. Age-specific trade-offs were found, mainly for the weekly increase in size versus the number of eggs per mass, the weekly increase in size versus the number of viable eggs per mass, the number of masses versus the hatching probability and the number of eggs versus the hatching probability. All these negative associations were significant for juveniles of both control and exposed snails and not for adults; however, exposed young individuals exhibited much higher values of the correlation coefficient than control animals.

The effect of isolation on the life-history traits of Pseudosuccinea columella (Pulmonata: Lymnaeidae)

A population of Pseudosuccinea columella was raised under laboratory conditions and its life tables were determined in isolated and paired snails. Isolated snails were significantly larger in shell size than paired snails from five weeks of age onward. Also, statistically significant differences were found for the number of eggs per mass per individual from week 5 to 9, isolated snails exhibiting the highest values. The intrinsic and finite rates of increase were greater in isolated than in paired snails. Either an inhibition of the reproductive output between individuals or the advantage of selfing may be the cause of the differences in this species, acting as a possible mechanism that increase the fitness of isolated snails.

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avançado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.

Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.