A Laboratory-based Approach to Biological Control of Snails

Development of Schistosoma mansoni in the intermediate host Biomphalaria glabrata is influenced by a number of parasite and snail genes. Understanding the genetics involved in this complex host/parasite relationship may lead to an often discussed approach of introducing resistant B. glabrata into the field as a means of biological control for the parasite. For the snail, juvenile susceptibility to the parasite is controlled by at least four genes, whereas one gene seems to be responsible for adult nonsusceptibility. Obtaining DNA from F2 progeny snails from crosses between parasite-resistant and-susceptible snails, we have searched for molecular markers that show linkage to either the resistant or susceptible phenotype. Both restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) approaches have been used. To date, using a variety of snail and heterologous species probes, no RFLP marker has been found that segregates with either the resistant or susceptible phenotype in F2 progeny snails. More promising results however have been found with the RAPD approach, where a 1.3 kb marker appears in nearly all resistant progeny, and a 1.1 kb marker appears in all susceptible progeny

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

Identification of Transcribed Sequences (ESTs) in the Trypanosoma cruzi Genome Project

Random single pass sequencing of cDNA fragments, also known as generation of Expressed Sequence Tags (ESTs), has been highly successful in the study of the gene content of higher organisms, and forms an integral part of most genome projects, with the objective to identify new genes and targets for disease control and prevention and to generate mapping probes. In the Trypanosoma cruzi genome project, EST sequencing has also been a starting point, and here we report data on the first 797 sequences obtained, partly from a CL Brener epimastigote non-normalized library, partly on a normalized library. Only around 30% of the sequences obtained showed similarity with Genbank and dbEST databases, half of which with sequences already reported for T. cruzi.

Evaluation of DNA Recombinant Methodologies for the Diagnosis of Plasmodium falciparum and their Comparison with the Microscopy Assay

Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolívar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.

Mexican Trypanosoma cruzi stocks: analysis of minicircle kDNA homologies by cross-hybridization

Homologies of minicircle kDNA of 27 Mexican stocks were studied by cross-hybridization with four kDNA probes derived from three reference stocks belonging to groups Trypanosoma cruzi I (SO34 cl4 and Silvio) and T. cruzi II (MN) and one Mexican stock. High homologies were only observed with Silvio (six stocks) and Mexican probes (11 stocks). After 30 min exposure (low homology) additional stocks were recognized with SO34 cl4 (three stocks) and Silvio (six stocks) probes; with the Mexican probe only five stocks remained non-reactive. All the stocks were typed by isoenzyme (16 loci) and Mexican stocks belonged to T. cruzi I. Hybridization patterns were not strictly correlated with the observed clustering and cross-hybridization of kDNA minicircles is not available to distinct Mexican stocks.

Selection of Trypanosoma cruzi clonal genotypes (clonet 20 and 39) isolated from Bolivian triatomines following subculture in liquid medium

Previous studies showed that two groups of Trypanosoma cruzi clonal genotypes named clonet 20 and clonet 39 were predominant in Triatoma infestans, the unique vector of Chagas disease in Bolivia. These groups of clones correspond to distinct genetic clusters. These clonets were detected in T. infestans and Rhodnius pictipes fecal samples before isolation and after culture by kDNA PCR (polymerase chain rreaction) and hybridization of the amplified products with clonet specific kDNA probes named 20 and 39 as previously reported. Forty eight T. infestans and three R. pictipes infected insects captured at random in different Bolivian departments were proceeded. As previously reported the direct identification of the two major clonets in fecal samples allowed the detection of abundant mixed infections: 41% in the original sample, however after culture, only 6% of mixed infections were detected. Among the 21 parasite stocks isolated from digestive tracts where mixed infections were initially detected (clonet 20 + 39) clonet 20 alone was detected in 81% of them. This result clearly showed that the culture step selected clonet 20 parasites over those belonging to clonet 39. The taxonomic status of the isolated stocks was also confirmed by isoenzyme typing, and correlation was observed between clustering topology and hybridization patterns with the probes 20 and 39.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

Cotton Rats (Sigmodon hispidus) and Black Rats (Rattus rattus) as Possible Reservoirs of Leishmania spp. in Lara State, Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.

Human papillomavirus prevalence among women with cervical intraepithelial neoplasia III and invasive cervical cancer from Goiânia, Brazil

This study estimated the prevalence and distribution of human papillomavirus (HPV) types among women with cervical intraepithelial neoplasia (CIN) grade III and invasive cervical cancer from Goiás (Brazil Central Region). Seventy-four cases were analyzed and consisted of 18 CIN III, 48 squamous cell carcinomas, 4 adenocarcinomas, 1 adenosquamous carcinoma and 3 undifferentiated carcinomas. HPV-DNA sequences were examined in formalin-fixed and paraffin-embedded tissues using primers from L1 region GP5+/GP6+. Polymerase chain reaction products were typed with dot blot hybridization using probes for HPV 16, 18, 31, 33, 45, 54, 6/11, 42/43/44, 51/52, 56/58. The prevalence of HPV was estimated to be 76% (56/74). HPV 16 was the most frequently found type, followed by HPV 33, 18 and 31. The prevalence of untyped HPV was 6%; 79% percent of the squamous cell carcinoma cases and 61% percent of the CIN III were positive for HPV and the prevalence rate of HPV types was the same for the total number of cases. According to other studies, HPV type 16 is the most prevalent virus in all Brazilian regions, but there is variation regarding to other types. Type 18 is the second most prevalent HPV in North, Southeast and South Brazil regions and types 31 and 33 are the second most prevalent HPV in Northeast and Central Brazil, respectively.

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera, Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20ºC) and old (20 to 42 h, 28ºC) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hibridization whit the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O?H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.