Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

IntroductionMicrosporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization.MethodsLarvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles.ResultsThe molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon.ConclusionsSamples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.

Phenotypic and molecular characterization of CTX-M extended-spectrum beta-lactamase-producing Escherichia coli isolates in Shiraz, Iran

AbstractINTRODUCTIONThe aim of this study was to detect the prevalence of the extended-spectrum beta-lactamase (ESBL)-encoding CTX-M gene in Escherichia coliisolates.METHODS:Phenotypic screening of 376 E. coli isolates for ESBL was conducted using disk diffusion. ESBL-producing isolates were tested using PCR and specific primers. The blaCTX-M cluster was identified using the RFLP method, and its genotype was sequenced.RESULTS:From 202 ESBL-producing E. coli , 185 (91.5%) possessed CTX-M genes. CTX-M-1 subtypes were found in 98% of the isolates. The blaCTX-M gene was identical to CTX-M-15.CONCLUSIONS:A high prevalence of CTX-M-1-producing E. coli apparently exists in Shiraz, Iran.

Structural and successional variability of periphytic algal community in a Amazonian lake during the dry and rainy season (Rio Branco, Acre)

The colonization process and successional patterns of a periphytic algal community were evaluated in a Amazonian Viveiro Lake (Rio Branco, Acre, Brazil). Sampling was performed over a period of 35 days; at four-day intervals for 20 days, and then at five-day intervals. Water sampling for physical, chemical and biological analyses was done during the dry and rainy season. Glass slides were used as artificial substrates for periphyton colonization. The structural community was evaluated through population density, algae class, diversity indices and descriptive species. Species richness, diversity and evenness increased as succession progressed. While density of Bacillariophyceae, Euglenophyceae and Zygnemaphyceae increased with succession, Cyanobacteria remained dominant. Synechocystis aquatilis, Synechocystis diplococcus and Navicula pseudolanceolata were the main descriptive species in both the dry and rainy season. Cymbela tumida, Frustulia rhomboides, Trachelomonas lacustris and Closterium acicularis was correlated with an increase in hydrologic level during the rainy season. Conversely, the density of Chlamydomonas sp., Chroomonas nordstedtii, Trachelomonas volvocinopsis, Trachelomonas volvocina and Synechococcus linearis was correlated with an increase in water transparency during the dry season. In general, the periphytic algal community showed high diversity and species richness independent of season. Season also had little influence on representation of algae class and main descriptive species. However, successional patterns varied by season, and changes in hydrologic levels acted directly on the succession path of periphytic algae. More research on periphyton dynamics is needed to improve our understanding of tropical lake ecosystems, especially in Amazonian.

Structural and functional characteristics of rat hearts with and without myocardial infarct. Initial experience with doppler echocardiography

OBJECTIVE: To assess by Doppler echocardiography the structural and functional alterations of rat heart with surgical induced extensive myocardial infarction. METHODS: Five weeks after surgical ligature of the left coronary artery, 38 Wistar-EPM rats of both sexes, 10 of them with extensive infarction, undergone anatomical and functional evaluation by Doppler echocardiography and then euthanized for anatomopathological analysis. RESULTS: Echocardiography was 100% sensible and specific to anatomopathological confirmed extensive miocardial infarction. Extensive infarction lead to dilatation of left ventricle (diastolic diameter: 0.89cm vs.0.64cm; systolic: 0.72cm vs. 0.33cm) and left atrium (0.55cm vs. 0.33cm); thinning of left ventricular anterior wall (systolic: 0.14cm vs. 0.23cm, diastolic: 0.11cm vs. 0.14cm); increased mitral E/ A wave relation (6.45 vs. 1.95). Signals of increased end diastolic ventricle pressure, B point in mitral valve tracing in 62.5% and signs of pulmonary hypertension straightening of pulmonary valve (90%) and notching of pulmonary systolic flow (60%) were observed in animals with extensive infarction. CONCLUSION: Doppler echocardiography has a high sensitivity and specificity for detection of chronic extensive infarction. Extensive infarction caused dilatation of left cardiac chambers and showed in Doppler signals of increased end diastolic left ventricular pressure and pulmonary artery pressure.

Chronic murine myocarditis due to Trypanosoma cruzi: an ultrastructural study and immunochemical characterization of cardiac interstitial matrix

In an attempt to define the mouse-model for chronic Chagas' disease, a serological, histopathological and ultrastructural study as well as immunotyping of myocardium collagenic matrix were performed on Swiss mice, chronically infected with Trypanosoma cruzi strains: 21 SF and mambaí (Type II); PMN and Bolivia (Type III), spontaneously surviving after 154 to 468 days of infection. Haemagglutination and indirect immunofluorescence tests showed high titres of specific antibodies. The ultrastructural study disclosed the cellular constitution of the inflammatory infiltrate showing the predominance of monocytes, macrophages with intense phagocytic activity, fibroblasts, myofibroblasts and abundant collagen matrix suggesting the association of the inflammatory process with fibrogenesis in chronic chagasic cardiomyopathy. Artertolar and blood capillary alterations together with dissociation of cardiac cells from the capillary wall by edema and inflammation were related to ultrastructural lesions of myocardial cells. Rupture of parasitized cardiac myocells contribute to intensify the inflammatory process in focal areas. Collagen immunotyping showed the predominance of Types III and IV collagen. Collagen degradation and phagocytosis were present suggesting a reversibility of the fibrous process. The mouse model seems to be valuable in the study of the pathogenetic mechanisms in Chagas cardiomyopathy, providing that T. cruzi strains of low virulence and high pathogenecity are used.

Role of divalent cations, pH, cytoskeleton componentes and surface charge on the adhesion of Trichomonas vaginalis to a polystyrene substrate

The process of adhesion of three different strains of Trichomonas vaginalis to a polystyrene substrate was analysed. The process of adhesion was dependent on the time of incubation and the pH of the phosphate-buffered solution (PBS) in which the parasites were suspended. The highest indices of adhesion were observed after an incubation time of 60 min at pH 6.6. The adhesion index increased when the parasites were incubated in the presence of culture media or when Ca++ or Mg++ was added to the PBS solution, whereas cytochalasin B, trypsin or neuraminidase reduced adhesion. Incubation of the parasites in the presence of poly-L-lysine facilitated the process of adhesion. Incubation of the parasites or polystyrene beads in the presence of poly-L-lysine led to important changes in their surface charge.

Isolation and antigenic characterization of human immunodeficiency virus (HIV) in Brazil

A retrovirus infecting a Brazilian AIDS patient was isolated and characterized in terms of its reactivity with sera from individuals infected with human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2). The Western blot analysis revealed that the Brazilian isolate is very similar to the well characterized HIV-1 strain. The serum of the patient from whom the virus was isolated did not react with the 140 kDa envelope glycoprotein specific for HIV-2.

Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Molecular and Antigenic Characterization of the Leishmania (Viannia) panamensis Kinetoplastid Membrane Protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (V) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

Zymodeme and Serodeme Characterization of Leishmania Isolates Obtained from Costa Rican Patients

Thirty-four Leishmania isolates obtained from Costa Rican patients, from different geographical areas, were characterized by isoenzyme electrophoresis and indirect immunofluorescense with monoclonal antibodies. Thirty-two were characterized as L. panamensis strains and two were L. braziliensis variants. We confirm the evident predominance of L. panamensis as the main etiological agent of leishmaniasis in Costa Rica and the existence of L. braziliensis in the country.

Antigenic and Genetic Characterization of Twenty-six Strains of Human Respiratory Syncytial Virus (Subgroup A) Isolated During Three Consecutive Outbreaks in Havana City, Cuba

Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100%) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus.

Studies on Cercariae from Kuwait Bay. XI. Description and surface topography of Cercaria kuwaitae XI sp.n. (Digenea: Echinostomatidae)

A new echinostome cercaria, Cercaria kuwaitae XI sp.n., from the prosobranch gastropod Cerithidea cingulata (Gmelin) from Kuwait Bay is described. The new cercaria is characterized by 23 collar spines and primary excretory tubules with distinct diverticula. The cercaria encysts in the snail host and is similar to those of Acanthoparyphium sp. The surface topography of the redia, cercaria and metacercarial cyst wall is studied by scanning electron microscopy. This is the first echinostome cercaria to be recorded in a gastropod from the Arabian Gulf region.

Ultrastructural, Antigenic and Physicochemical Characterization of the Mojuí dos Campos (Bunyavirus) Isolated from Bat in the Brazilian Amazon Region

The Mojuí dos Campos virus (MDCV) was isolated from the blood of an unidentified bat (Chiroptera) captured in Mojuí dos Campos, Santarém, State of Pará, Brazil, in 1975 and considerated to be antigenically different from other 102 arboviruses belonging to several antigenic groups isolated in the Amazon region or another region by complement fixation tests. The objective of this work was to develop a morphologic, an antigenic and physicochemical characterization of this virus. MDCV produces cytopathic effect in Vero cells, 24 h post-infection (p.i), and the degree of cellular destruction increases after a few hours. Negative staining electron microscopy of the supernatant of Vero cell cultures showed the presence of coated viral particles with a diameter of around 98 nm. Ultrathin sections of Vero cells, and brain and liver of newborn mice infected with MDCV showed an assembly of the viral particles into the Golgi vesicles. The synthesis kinetics of the proteins for MDCV were similar to that observed for other bunyaviruses, and viral proteins could be detected as early as 6 h p.i. Our results reinforce the original studies which had classified MDCV in the family Bunyaviridae, genus Bunyavirus as an ungrouped virus, and it may represent the prototype of a new serogroup.