The neuraminidase gene is present in the non-toxigenic Vibrio cholerae Amazonia strain: a different allele in comparison to the pandemic strains

The neuraminidase gene, nanH, is present in the O1, non-toxigenic Vibrio cholerae Amazonia strain. Its location has been assigned to a 150 kb NotI DNA fragment, with the use of pulsed-field gel electrophoresis and DNA hybridization. This NotI fragment is positioned inside 630 kb SfiI and 1900 kb I-CeuI fragments of chromosome 1. Association of the pathogenicity island VPI-2, carrying nanH and other genes, with toxigenic strains has been described by other authors. The presence of nanH in a non-toxigenic strain is an exception to this rule. The Amazonia strain nanH was sequenced (Genbank accession No. AY825932) and compared to available V. cholerae sequences. The sequence is different from those of pandemic strains, with 72 nucleotide substitutions. This is the first description of an O1 strain with a different nanH allele. The most variable domain of the Amazonia NanH is the second lectin wing, comprising 13 out of 17 amino acid substitutions. Based on the presence of nanH in the same region of the genome, and similarity of the adjacent sequences to VPI-2 sequences, it is proposed that the pathogenicity island VPI-2 is present in this strain.

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinanting B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.

Intermediate and definitive hosts of Schistosoma mansoni in Corrientes province, Argentina

Corrientes province is located in the humid subtropical region of Argentina northeast on the left riverbank of Paraná River in the border with the South of Brazil. This is a region without schistosomiasis but planorbid and rodents that would serve as host of the life cycle of Schistosoma mansoni inhabit here. The objective of this work is to know the role of rodent as definitive host of schistosomiasis. Biomphalaria tenagophila (4 to 8 mm Ø) from Maloyas, exposed each to 10 miracidia of SJ2 strain of S. mansoni natives from Brazil were susceptible (5%). The degree of compatibility was Class II of Frandsen. Five wild rodents captured in the same ecological niche were exposed transcutaneously to infection with 40 cercariae for animal: two Olygoryzomys flavescens, two Holochilus braziliensis, and one Scapteromys tuncidus. Only one H. braziliensis eliminated eggs in feces. Prepatent period was of 83 days. With these feces, two of six (33.3%) B. tenagophila from Maloyas were infected with miracidium. It was demonstrated, in an area free of schistosomiasis, that life cycle S. mansoni is closed with planorbid and rodents that live in the same ecological niche.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Autochthonous horizontal transmission of a CRF02_AG strain revealed by a human immunodeficiency virus type 1 diversity survey in a small city in inner state of Rio de Janeiro, Southeast Brazil

As part of an ongoing study on the features of AIDS spread towards small cities and rural areas, we present a molecular survey of human immunodeficiency virus type 1 (HIV-1) polymerase sequences recovered between 2004 and 2006 from 71 patients receiving care in the city of Saquarema, inner state of Rio de Janeiro. Phylogenetic reconstructions found the two prevalent lineages in the state (subtypes B [59 strains, 83.1%], F1 [6 strains; 8.4%], and BF1 recombinants [four strains; 5.6%]), as well as two (2.8%) CRF02_AG strains, which seems to be an emerging lineage in the capital. These CRF02_AG sequences were recovered from a married heterosexual couple who never traveled abroad, thus providing the first molecular evidence of autochthonous horizontal transmission of this lineage of major global importance. Also, three phylogenetic clusters of strains recovered from a total of 18.3% of the cohort were uncovered. Their close genetic relatedness suggests they were recovered from patients who probably took part in the same chain of viral spread. In conjunction with our previous surveys from inner Rio de Janeiro, these results suggest that although small cities harbor unique molecular features of HIV-1 infection, they also clearly reflect and may rapidly absorb the diversity recorded in large urban centers.

Life cycle, feeding and defecation patterns of Rhodnius ecuadoriensis (Lent & León 1958) (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Rhodnius ecuadoriensis is the second most important vector of Chagas Disease (CD) in Ecuador. The objective of this study was to describe (and compare) the life cycle, the feeding and defecation patterns under laboratory conditions of two populations of this specie [from the provinces of Manabí (Coastal region) and Loja (Andean region)]. Egg-to-adult (n = 57) development took an average of 189.9 ± 20 (Manabí) and 181.3 ± 6.4 days (Loja). Mortality rates were high among Lojan nymphs. Pre-feeding time (from contact with host to feeding initiation) ranged from 4 min 42 s [nymph I (NI)] to 8 min 30 s (male); feeding time ranged from 14 min 45 s (NI)-28 min 25 s (male) (Manabí) and from 15 min 25 s (NI)-28 min 57 s (nymph V) (Loja). The amount of blood ingested increased significantly with instar and was larger for Manabí specimens (p < 0.001). Defecation while feeding was observed in Manabí specimens from stage nymph III and in Lojan bugs from stage nymph IV. There was a gradual, age-related increase in the frequency of this behaviour in both populations. Our results suggest that R. ecuadoriensis has the bionomic traits of an efficient vector of Trypanosoma cruzi. Together with previous data on the capacity of this species to infest rural households, these results indicate that control of synanthropic R. ecuadoriensis populations in the coastal and Andean regions may have a significant impact for CD control in Ecuador and Northern Peru.

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

The bacterium Rickettsia rickettsii is the etiological agent of an acute, severe disease called Rocky Mountain spotted fever in the United States or Brazilian spotted fever (BSF) in Brazil. In addition to these two countries, the disease has also been reported to affect humans in Mexico, Costa Rica, Panama, Colombia and Argentina. Like humans, dogs are also susceptible to R. rickettsii infection. However, despite the wide distribution of R. rickettsii in the Western Hemisphere, reports of R. rickettsii-induced illness in dogs has been restricted to the United States. The present study evaluated the pathogenicity for dogs of a South American strain of R. rickettsii. Three groups of dogs were evaluated: group 1 (G1) was inoculated ip with R. rickettsii; group 2 (G2) was infested by R. rickettsii-infected ticks; and the control group (G3) was infested by uninfected ticks. During the study, no clinical abnormalities, Rickettsia DNA or R. rickettsii-reactive antibodies were detected in G3. In contrast, all G1 and G2 dogs developed signs of rickettsial infection, i.e., fever, lethargy, anorexia, ocular lesions, thrombocytopenia, anemia and detectable levels of Rickettsia DNA and R. rickettsii-reactive antibodies in their blood. Rickettsemia started 3-8 days after inoculation or tick infestation and lasted for 3-13 days. Our results indicate that a Brazilian strain of R. rickettsii is pathogenic for dogs, suggesting that canine clinical illness due to R. rickettsii has been unreported in Brazil and possibly in the other South American countries where BSF has been reported among humans.

The life history of Pygidiopsis macrostomum Travassos, 1928 (Digenea: Heterophyidae)

The life history of the trematode Pygidiopsis macrostomum Travassos, 1928 is described for the first time. Rediae and cercariae were obtained from naturally infected snails Heleobia australis (d´Orbigny), a new first intermediate host. Metacercariae were found encysted in the mesenteries of three naturally infected guppies, Phalloptychus januarius (Hensel), Jenynsia multidentata (Jenyns) (new host records) and Poecilia vivipara Bloch and Schneider. Experimental infections were successfully completed in the intermediate hosts H. australis and Poe. vivipara reared in the laboratory and hamsters Mesocricetus auratus Waterhouse were utilised as a definitive host.

The Pneumocystis life cycle

First recognised as "schizonts" of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.

Failure to demonstrate experimental vertical transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La Réunion Island, Indian Ocean

Aedes albopictus was responsible for transmission in the first outbreak of chikungunya (CHIK) on La Réunion Island, Indian Ocean, in 2005-2006. The magnitude of the outbreak on this island, which had been free of arboviral diseases for over 30 years, as well as the efficiency of Ae. albopictus as the main vector, raises questions about the maintenance of the CHIK virus (CHIKV) through vertical transmission mechanisms. Few specimens collected from the field as larvae were found to be infected. In this study, Ae. albopictus originating from La Réunion were orally infected with a blood-meal containing 10(8) pfu/mL of the CHIKV epidemic strain (CHIKV 06.21). Eggs from the first and second gonotrophic cycles were collected and raised to the adult stage. The infectious status of the progeny was checked (i) by immunofluorescence on head squashes of individual mosquitoes to detect the presence of viral particles or (ii) by quantitative RT-PCR on mosquito pools to detect viral RNA. We analysed a total of 1,675 specimens from the first gonotrophic cycle and 1,709 from the second gonotrophic cycle without detecting any viral particles or viral RNA. These laboratory results are compared to field records.

α-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an α-N-acetylglucosamine (α-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Life cycle of Triatoma ryckmani (Hemiptera: Reduviidae) in the laboratory, feeding patterns in nature and experimental infection with Trypanosoma cruzi

A cohort initiated with 121 eggs, yielding 105 first instar nymphs (eclosion rate: 86.78%), allowed us to observe the entire life cycle of Triatoma ryckmani under laboratory conditions (24ºC and 62% relative humidity), by feeding them on anesthetized hamsters. It was possible to obtain 62 adults and the cycle from egg to adult took a mean of 359.69 days with a range of 176-529 days (mortality rate of nymphs: 40.95%). Mean life span of adults was of 81 days for females and 148 days for males. The developmental periods of 4th and 5th nymphs were longer than those of the other instars. This suggests that young siblings have a better chance of taking a hemolymph meal from older ones, in order to survive during fasting periods during prolonged absences of vertebrate hosts from natural ecotopes. The stomach contents of 37 insects showed blood from rodents (15 cases), lizards (7 cases), birds (6 cases) and insect hemolymph (7 cases). Out of 10 insects fed by xenodiagnosis on a Trypanosoma cruzi infected mouse, all but one became infected with the parasite.