Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi

Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.

Malaria entomological risk factors in relation to land cover in the Lower Caura River Basin, Venezuela

To explore the effects of deforestation and resulting differences in vegetation and land cover on entomological parameters, such as anopheline species composition, abundance, biting rate, parity and entomological inoculation rate (EIR), three villages were selected in the Lower Caura River Basin, state of Bolívar, Venezuela. All-night mosquito collections were conducted between March 2008-January 2009 using CDC light traps and Mosquito Magnet(r) Liberty Plus. Human landing catches were performed between 06:00 pm-10:00 pm, when anophelines were most active. Four types of vegetation were identified. The Annual Parasite Index was not correlated with the type of vegetation. The least abundantly forested village had the highest anopheline abundance, biting rate and species diversity. Anopheles darlingi and Anopheles nuneztovari were the most abundant species and were collected in all three villages. Both species showed unique biting cycles. The more abundantly forested village of El Palmar reported the highest EIR. The results confirmed previous observations that the impacts of deforestation and resulting changes in vegetation cover on malaria transmission are complex and vary locally.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

Distinct population structure for co-occurring Anopheles goeldii and Anopheles triannulatus in Amazonian Brazil

To evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in Anopheles triannulatus, larval habitat characteristics across the Brazilian states of Roraima and Pará and genetic sequences were examined. A comparison with Anopheles goeldii was utilised to determine whether high genetic diversity was unique to An. triannulatus. Student t test and analysis of variance found no differences in habitat characteristics between the species. Analysis of population structure of An. triannulatus and An. goeldii revealed distinct demographic histories in a largely overlapping geographic range. Cytochrome oxidase I sequence parsimony networks found geographic clustering for both species; however nuclear marker networks depicted An. triannulatus with a more complex history of fragmentation, secondary contact and recent divergence. Evidence of Pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography. We hypothesise that niche partitioning is a driving force for diversity, particularly in An. triannulatus.

Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania) infantum chagasi-infected BALB/c mice

Pentavalent antimonials such as meglumine antimoniate (MA) are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania) infantum chagasi-infected mice. MA (Glucantime(r)) was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae)

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.

A new survey of the serology of human Trypanosoma cruziinfection in the Rio Negro microregion, Brazilian Amazon: a critical analysis

The serology of human Trypanosoma cruziinfection in the Rio Negro microregion is very complex because of the large numbers of false-positive cases that result from low antibody titres and cross-reactions with other infections. In the present study, we collected 4,880 blood samples on filter paper; of these, indirect immunofluorescence (IIF) was strongly reactive in 221 (4.5%), which were considered to be positive (IIF strongly reactive; high intensity of fluorescence) and weakly reactive in 302 (6.2%), which were considered to be doubtful (IIF weakly reactive; low intensity of fluorescence). The confirmatory test on the serum using at least two of three techniques (IIF, conventional ELISA and recombinant ELISA) on 137 samples that were positive in the screening test only confirmed 33 cases (24.1%). Of the 178 samples that were considered doubtful in the screening test, only 10 (5.6%) were considered to be positive in the confirmatory test. Finally, we recommend that the serological diagnosis of T. cruziinfection in the Amazon region be made using at least two different techniques, for example immunofluorescence and ELISA and confirmed by Western blot analysis when possible.

Molecular identification of Sporothrix species involved in the first familial outbreak of sporotrichosis in the state of Espírito Santo, southeastern Brazil

Sporotrichosis is a widespread subcutaneous mycosis caused by the dimorphic fungi now known as the Sporothrix schenckii complex. This complex is comprised of at least six species, including Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. Cases of sporotrichosis have significantly increased in Brazil over the past decade, especially in the state of Rio de Janeiro (RJ), where an epidemic among cat owners has been observed. The zoonotic transmission from cats to humans suggests a common source of infection and indicates that animals can act as vectors. We performed a molecular characterisation of samples collected during the first outbreak of familial sporotrichosis caused by S. brasiliensis in the state of Espírito Santo, Brazil. These results represent the first description of such an outbreak outside the endemic area of zoonotic sporotrichosis in RJ.

Genetic basis of triatomine behavior: lessons from available insect genomes

Triatomines have been important model organisms for behavioural research. Diverse reports about triatomine host search, pheromone communication in the sexual, shelter and alarm contexts, daily cycles of activity, refuge choice and behavioural plasticity have been published in the last two decades. In recent times, a variety of molecular genetics techniques has allowed researchers to investigate elaborate and complex questions about the genetic bases of the physiology of insects. This, together with the current characterisation of the genome sequence of Rhodnius prolixus allows the resurgence of this excellent insect physiology model in the omics era. In the present revision, we suggest that studying the molecular basis of behaviour and sensory ecology in triatomines will promote a deeper understanding of fundamental aspects of insect and, particularly, vector biology. This will allow uncovering unknown features of essential insect physiology questions for a hemimetabolous model organism, promoting more robust comparative studies of insect sensory function and cognition.

Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL) and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 ) gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia

Rhodnius barretti , a new triatomine species, is described based on adult specimens collected in rainforest environments within the Napo ecoregion of western Amazonia (Colombia and Ecuador). R. barretti resembles Rhodnius robustus s.l. , but mitochondrial cytochrome b gene sequences reveal that it is a strongly divergent member of the “robustus lineage”, i.e., basal to the clade encompassing Rhodnius nasutus , Rhodnius neglectus , Rhodnius prolixus and five members of the R. robustus species complex. Morphometric analyses also reveal consistent divergence from R. robustus s.l. , including head and, as previously shown, wing shape and the length ratios of some anatomical structures. R. barretti occurs, often at high densities, in Attalea butyracea and Oenocarpus bataua palms. It is strikingly aggressive and adults may invade houses flying from peridomestic palms. R. barretti must therefore be regarded as a potential Trypanosoma cruzi vector in the Napo ecoregion, where Chagas disease is endemic.