Candida dubliniensis identification in Brazilian yeast stock collection

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42°C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42°C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.

Trypanosoma cruzi: genetic group with peculiar biochemical and biological behavior

Thirty-two Trypanosoma cruzi strains, isolated from chronic chagasic patients in the northwest of the state of Paraná (Brazil), were analyzed using molecular, biochemical and biological characteristics. Genotypic analysis using randomly amplified polymorphic DNA and simple sequence repeat-anchored polymerase chain reaction amplified profiles showed a large, genetically well-correlated group that contained the majority of the strains and a divergent group that included the PR-150 strain. For glycoconjugate composition, the PR-150 strain was different from the other strains considering the absence or presence of specific bands in aqueous or detergent phases. This strain was also totally different from the others in one out of the six parameters related to in vitro and in vivo biological behavior. We highlight the fact that the PR-150 was totally resistant to benznidazole. For the other biological parameters this strain was not totally distinct from the others, but it showed a peculiar behavior.

Biomphalaria tenagophila: dominant character of the resistance to Schistosoma mansoni in descendants of crossbreedings between resistant (Taim, RS) and susceptible (Joinville, SC) strains

The aim of the present work was to study parasitological, molecular, and genetic aspects in descendants of crossbreedings between a totally resistant Biomphalaria tenagophila strain (Taim, RS) and another one highly susceptible (Joinville, SC) to Schistosoma mansoni. Descendants F1 and F2 were submitted to S. mansoni infection (LE strain). The susceptibility rates for individuals from Group F1 were 0 to 0.6%, and from Group F2 was 7.2%. The susceptible individuals from Group F2 discharged a lower number of cercariae, when compared with the susceptible parental group, and in 2 out of 9 positive snails the cercarial elimination was discontinued. In order to identify genetic markers associated with resistance the genotype of parental snails and their offspring F1 and F2 were analyzed by means of the randomly amplified polymorphic DNA method. Nevertheless, it was not possible to detect any marker associated to resistance, but the results showed that in the mentioned species the resistance character is determined by two dominant genes.

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.

Molecular diversity of serial Cryptococcus neoformans isolates from AIDS patients in the city of São Paulo, Brazil

Despite highly active anti-retroviral therapy, cryptococcal meningoencephalitis is the second most prevalent neurological disease in Brazilian AIDS patients, being frequently a defining condition with several episodes. As knowledge of Cryptococcus neoformans isolates in the same episode is critical for understanding why some patients develop several episodes, we investigated the genotype characteristics of C. neoformans isolates in two different situations. By pulsed field gel electrophoresis and random amplifield polymorphic DNA analysis, 54 isolates from 12 patients with AIDS and cryptococcosis were analyzed. Group 1 comprised 39 isolates from nine patients with a single episode and hospitalization. Group 2 comprised 15 isolates from three patients with two episodes and hospitalizations. Except for three patients from group 1 probably infected with a single C. neoformans isolate, the other nine patients probably were infected with multiple isolates selected in different collection periods, or the infecting isolate might have underwent mutation to adapt and survive the host immune system and/or the antifungal therapy. However, the three patients from group 2 presented genetic diversity among isolates collected in both hospitalizations, possibly having hosted the initial isolate in both periods. These data, emphasize that Cryptococcus diversity in infection can contribute to strategies of treatment and prevention of cryptococcosis.

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.

Influence of anti-filarial chemotherapy strategies on the genetic structure of Wuchereria bancrofti populations

Lymphatic filarial (LF) parasites have been under anti-filarial drug pressure for more than half a century. Currently, annual mass drug administration (MDA) of diethylcarbamazine (DEC) or ivermectin in combination with albendazole (ALB) have been used globally to eliminate LF. Long-term chemotherapies exert significant pressure on the genetic structure of parasitic populations. We investigated the genetic variation among 210 Wuchereria bancrofti populations that were under three different chemotherapy strategies, namely MDA with DEC alone (group I, n = 74), MDA with DEC and ALB (group II, n = 60) and selective therapy (ST) with DEC (group III, n = 34) to understand the impact of these three drug regimens on the parasite genetic structure. Randomly amplified polymorphic DNA profiles were generated for the three groups of parasite populations; the gene diversity, gene flow and genetic distance values were determined and phylogenetic trees were constructed. Analysis of these parameters indicated that parasite populations under ST with a standard dose of DEC (group III) were genetically more diverse (0.2660) than parasite populations under MDA with DEC alone (group I, H = 0.2197) or with DEC + ALB (group II, H = 0.2317). These results indicate that the MDA may reduce the genetic diversity of W. bancrofti populations when compared to the genetic diversity of parasite populations under ST.

The relationship between genetic variability and the susceptibility of Biomphalaria alexandrina snails to Schistosoma mansoni infection

In the present study, Biomphalaria snails collected from five Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta), as well as reference control Biomphalaria alexandrina snails from the Schistosome Biological Supply Center (SBSC) (Theodor Bilharz Research Institute, Egypt), were subjected to species-specific polymerase chain reaction (PCR) assays to identify the collected species. All of the collected snails were found to be B. alexandrina and there was no evidence of the presence of Biomphalaria glabrata. Randomly amplified polymorphic DNA (RAPD)-PCR assays showed different fingerprints with varying numbers of bands for the first generation (F1) of B. alexandrina snail populations (SBSC, Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta). The primer OPA-1 produced the highest level of polymorphism and amplified the greatest number of specific bands. The estimated similarity coefficients among the B. alexandrina populations based on the RAPD-PCR profiles ranged from 0.56 (between SBSC and Ismailia snails) to 0.72 (between Ismailia and Kafr El-Sheikh snails). Experimental infection of the F1 of progeny from the collected snails with Schistosoma mansoni (SBSC strain) showed variable susceptibility rates ranging from 15% in the Fayoum snail group to 50.3% in SBSC snails. A negative correlation was observed between the infection rates in the different snail groups and the distances separating their corresponding governorates from the parasite source. The infection rates of the snail groups and their similarity coefficients with SBSC B. alexandrina snails were positively correlated. The variations in the rates of infection of different B. alexandrina groups with S. mansoni, as well as the differences in the similarity coefficients among these snails, are dependent not only on the geographical distribution of the snails and the parasite, but also on the genetic variability of the snails. Introduction of this variability into endemic areas may reduce the ability of the parasite to infect local hosts and consequently reduce schistosomiasis epidemiology.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea.

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Genetic variability of the Brazilian hair sheep breeds

The objectives of this work were to investigate the genetic structure of the Brazilian hair sheep breeds and to determine the origin of the Santa Inês breed. Molecular similarity was determined using Randomly Amplified Polymorphic DNA - Polymerase Chain Reaction markers in 238 individuals from five naturalized sheep breeds: Santa Inês (48 animals), Rabo Largo (48), Somali (48), Morada Nova (48) and Bergamasca (46), collected in Goiás, Sergipe, Bahia, and Ceará States as well as in the Federal District. Fifty-four loci were selected from 19 primers, after a pilot test using 140 primers. Qualitative analyses indicate diagnostic markers for all breeds. All breeds were significantly different from each other. Interbreed differences were explained by 14.92% of the total variation. Santa Inês clustered with Bergamasca (97% bootstrap) and with Rabo Largo, composing the third member of the group (81% bootstrap) while Morada Nova and Somali breeds clustered separately. Each breed should be considered as a separate management and conservation unit, and special care should be taken with Rabo Largo, Morada Nova and Somali breeds, represented by small herds in Brazil.

Genetic structure and gene flow of Eugenia dysenterica natural populations

This study was carried out to assess the genetic variability of ten "cagaita" tree (Eugenia dysenterica) populations in Southeastern Goiás. Fifty-four randomly amplified polymorphic DNA (RAPD) loci were used to characterize the population genetic variability, using the analysis of molecular variance (AMOVA). A phiST value of 0.2703 was obtained, showing that 27.03% and 72.97% of the genetic variability is present among and within populations, respectively. The Pearson correlation coefficient (r) among the genetic distances matrix (1 - Jaccard similarity index) and the geographic distances were estimated, and a strong positive correlation was detected. Results suggest that these populations are differentiating through a stochastic process, with restricted and geographic distribution dependent gene flow.

Rhizobium strains competitiveness on bean nodulation in Cerrado soils

The objective of this work was to identify the most competitive and effective Rhizobium strains in order to increase common bean yield by nitrogen fixation as alternative or complementation to the nitrogen fertilization. Competitiveness tests were lead in axenic conditions, in Cerrado soil pots and in three field experiments, with native Rhizobium strains that were previously identified, according to their effectiveness and genetic variability. The identification of strains in nodules was performed using serological tests (axenic conditions) - agglutination and enzyme linked immunosorbent (Elisa) assays - and random amplified polymorfic DNA (RAPD) (Cerrado soil). Plant yield was determined using the dry weight (greenhouse conditions), total N and grain yield (field experiments). Among the analyzed Rhizobium strains, native strain SLA 2.2 and commercial strain CIAT 899 were the dominant nodules in plants of the most productive plots, presenting yield productivity similar or higher to those obtained in treatments where 20 kg ha-1 of N were applied.

Ecophysiology of nontuberculous Mycobacteria in marine aquaculture ponds

The world demand for fish and fishery products is increasing steadily and it is generally accepted that it will not be possible to meet the heavy demand with resources exploited from capture fishery alone. Now aquaculture is well established and fastdeveloping industry in many countries and is a major focus sector for development. During recent decades, aquaculture has gained momentum, throughout the world especially in developing countries. According to Food and Agricultural Oganisation (FAO, 2000), global aquaculture production was 26.38 tones in 1996 have reached 32.9 million tonnes during 1999. Only marine aquaculture sector has contributed 13.1 million tonnes during 1999.India is a major fish producing country. About one half of lndia’s brackish water lands are currently being utilized for farming in order to reduce the gap between supply and demand for fish. Aquaculture has become a major source of livelihood for people and its role in integrated rural development, generation of employment and earning foreign exchange, thereby alleviating poverty is being greatly appreciated around the world.Among the infectious agents, bacteria are becoming the prime causal organisms for diseases in food fishes and other marine animals. Sindermann, (1970) reported that bacterial fish pathogen most commonly found among marine fishes is species of Pseudomonas, Vibrio and Mycobacterium. These can be categorized into primary pathogens; secondary invaders that may cause systemic disease in immunocompromised hosts; and normal marine flora which are not pathogenic but may occur on body surfaces or even within the tissues of the host. I-Iigh density of animals in hatchery tanks and ponds is conducive to the spread of pathogen and the aquatic environment with regular application of protein rich feed, is ideal for culturing bacteria. Bacteria, which are normally present in seawater or on the surface of fish, can invade and cause pathological effects in fishes, which are injured or subjected to other environmental stresses.Mycobacteria except parasites are known as nontuberculosis mycobacteria (NTM), atypical mycobacteria or mycobacteria other than tuberculosis(MO'l'l"). This group of mycobacteria includes opportunistic pathogens and saprophytes. Environmental mycobacteria are ubiquitous in distribution and the sources may include soil, water, warm-blooded as well as cold-blooded animals. Disease caused by environmental mycobacterial strains in susceptible humans (Goslee & Wolinsky, 1976; Grange, 1987), animals and fishes are increasingly attracting attention. Greatest importance of environmental mycobacteria is believed to be their role in immunological priming of humans and animals, thereby modifying their immune responses to subsequent exposure to pathogenic species.

Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering

We characterized 28 new isolates of Trypanosoma cruzi IIc (TCIIc) of mammals and triatomines from Northern to Southern Brazil, confirming the widespread distribution of this lineage. Phylogenetic analyses using cytochrome b and SSU rDNA sequences clearly separated TCIIc from TCIIa according to terrestrial and arboreal ecotopes of their preferential mammalian hosts and vectors. TCIIc was more closely related to TCIId/e, followed by TCIIa, and separated by large distances from TCIIb and TCI. Despite being indistinguishable by traditional genotyping and generally being assigned to Z3, we provide evidence that TCIIa from South America and TCIIa from North America correspond to independent lineages that circulate in distinct hosts and ecological niches. Armadillos, terrestrial didelphids and rodents, and domestic dogs were found infected by TCIIc in Brazil. We believe that, in Brazil, this is the first description of TCIIc from rodents and domestic dogs. Terrestrial triatomines of genera Panstrongylus and Triatoma were confirmed as vectors of TCIIc. Together, habitat, mammalian host and vector association corroborated the link between TCIIc and terrestrial transmission cycles/ecological niches. Analysis of ITS1 rDNA sequences disclosed clusters of TCIIc isolates in accordance with their geographic origin, independent of their host species. (C) 2009 Elsevier B.V. All rights reserved.

Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosomo cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TO presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TO and TCIIa from wild primates (16 TO and five TCIIa), Rhodnius spp, (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TO and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region. (C) 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.