Phylogeny, ultrastructure, histopathology and prevalence of Myxobolus oliveirai sp. nov., a parasite of Brycon hilarii (Characidae) in the Pantanal wetland, Brazil

This paper presents the morphological, histological and ultrastructural characteristics of Myxobolus oliveirai sp. nov., a parasite of the gill filaments in Brycon hilarii from the Brazilian Pantanal. Out of 216 B. hilariispecimens examined (126 wild and 90 cultivated), 38.1% of wild specimens (n = 48) were infected. The parasites form elongated plasmodia primarily in the tip of gill filaments, reaching about 3 mm in length. A thorough comparison with all the Myxobolus species described from South American hosts, as well as nearly all the Myxobolus species described so far is provided. Partial sequencing of the 18S rDNA gene revealed a total of 1,527 bp. The Myxobolus species parasite of B. hilarii did not match any of the Myxozoa available in GenBank. In the phylogenetic analysis, M. oliveirai sp. nov. composed a monophyletic group with eight other species: five species of Myxobolus parasites of mugilid fishes, two parasites of pangasiid and one of centrarchid. Infection prevalence values of the parasite revealed no significant differences between wet and dry seasons or between males and females. The importance of the infection to the farming of the host species is emphasized.

Phylogeny and revision of the bee genus Rhinocorynura Schrottky (Hymenoptera, Apidae, Augochlorini), with comments on its female cephalic polymorphism

Phylogeny and revision of the bee genus Rhinocorynura Schrottky (Hymenoptera, Apidae, Augochlorini), with comments on its female cephalic polymorphism. A taxonomic revision and a phylogeny for the species of Rhinocorynura are provided. Six species are recognized: R. briseis, R. crotonis, R. inflaticeps and R. vernoniae stat. nov., the latter removed from synonymy with R. inflaticeps, in addition to two newly described species, R. brunnea sp. nov. and R. viridis sp. nov. Lectotypes for Halictus crotonis Ducke, 1906 and Halictus inflaticeps Ducke, 1906 are hereby designated. Another available name included in Rhinocorynura, Corynuropsis ashmeadi Schrottky, 1909, is removed from the genus and treated as species inquerenda in Augochlorini. Rhinocorynura is monophyletic in the phylogenetic analysis and the following relationships were found among its species: (R. crotonis (R. briseis ((R. brunnea sp. nov. + R. viridis sp. nov.) (R. inflaticeps + R. vernoniae)))). Biogeographic relationships within the genus and comparisons with related taxa are presented. Females of all species exhibit pronounced variation in body size, in two of them, R. inflaticeps and R. vernoniae, with structural modifications possibly linked to division of labor. Identification key and illustrations for the species are provided.

Molecular analysis of the dengue virus type 1 and 2 in Brazil based on sequences of the genomic envelope-nonstructural protein 1 junction region

The genomic sequences of the Envelope-Non-Structural protein 1 junction region (E/NS1) of 84 DEN-1 and 22 DEN-2 isolates from Brazil were determined. Most of these strains were isolated in the period from 1995 to 2001 in endemic and regions of recent dengue transmission in São Paulo State. Sequence data for DEN-1 and DEN-2 utilized in phylogenetic and split decomposition analyses also include sequences deposited in GenBank from different regions of Brazil and of the world. Phylogenetic analyses were done using both maximum likelihood and Bayesian approaches. Results for both DEN-1 and DEN-2 data are ambiguous, and support for most tree bipartitions are generally poor, suggesting that E/NS1 region does not contain enough information for recovering phylogenetic relationships among DEN-1 and DEN-2 sequences used in this study. The network graph generated in the split decomposition analysis of DEN-1 does not show evidence of grouping sequences according to country, region and clades. While the network for DEN-2 also shows ambiguities among DEN-2 sequences, it suggests that Brazilian sequences may belong to distinct subtypes of genotype III.

THE INFLUENCE OF HIV-1 SUBTYPES C, CRF31_BC AND B ON DISEASE PROGRESSION AND INITIAL VIROLOGIC RESPONSE TO HAART IN A SOUTHERN BRAZILIAN COHORT

Background: Although most HIV-1 infections in Brazil are due to subtype B, Southern Brazil has a high prevalence of subtype C and recombinant forms, such as CRF31_BC. This study assessed the impact of viral diversity on clinical progression in a cohort of newly diagnosed HIV-positive patients. Methods: From July/2004 to December/2005, 135 HIV-infected patients were recruited. The partial pol region was subtyped by phylogeny. A generalized estimating equation (GEE) model was used to examine the relationship between viral subtype, CD4+ T cell count and viral load levels before antiretroviral therapy. Hazard ratio (Cox regression) was used to evaluate factors associated with viral suppression (viral load < 50 copies/mL at six months). Results: Main HIV-1 subtypes included B (29.4%), C (28.2%), and CRF31_BC (23.5%). Subtypes B and C showed a similar trend in CD4+ T cell decline. Comparison of non-B (C and CRF31_BC) and B subtypes revealed no significant difference in the proportion of patients with viral suppression at six months (week 24). Higher CD4+ T cell count and lower viral load were independently associated with viral suppression. Conclusion: No significant differences were found between subtypes; however, lower viral load and higher CD4+ T cell count before therapy were associated with better response.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Hepatitis C: sexual or intrafamilial transmission? Epidemiological and phylogenetic analysis of hepatitis C virus in 24 infected couples

The role of sexual or intrafamilial transmission of hepatitis C is controversial. A phylogenetic analysis was performed on the non-structural region 5B of the hepatitis C virus (NS5B-HCV). High percentages of homology (mean of 98.3%) were shown between the couples. Twenty (83.3%) of the 24 men but only two of the women (8.3%) reported having had sexually transmitted diseases during their lives. The risk factors for HCV acquisition were blood transfusion (10 couples), use of illegal injected drugs (17), use of inhalants (15), acupuncture (5) and tattoos (5). The shared use of personal hygiene items included toothbrushes between six couples (25%), razor blades between 16 (66.7%), nail clippers between 21 (87.5%) and manicure pliers between 14 (58.3%). The high degree of similarity of the hepatitis C virus genome supports the hypothesis of hepatitis C virus transmission between these couples. The shared use of personal hygiene items suggests the possibility of intrafamilial transmission of infection.

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.

Cladistic analysis of the Neotropical genera Cerqueirellum Py-Daniel, 1983, Coscaroniellum Py-Daniel, 1983 and Shelleyellum Py-Daniel & Pessoa, 2005 (Diptera: Simuliidae)

Females of simuliid black flies are haematophagous insects and vectors of several pathogenic agents of human diseases such as the filarial worms Mansonella ozzardi and Onchocerca volvulus. The genus Cerqueirellum is one of the most important groups of vectors of mansonellosis and onchocerciasis diseases in South America, and the genera Coscaroniellum and Shelleyellum are phylogenetically close to Cerqueirellum. There is not yet an agreement among authors about the generic classification of the species which compose these three genera, being all lumped by some taxonomists within Psaroniocompsa. A cladistic analysis of all species of Coscaroniellum, Cerqueirellum, and Shelleyellum, based on 41 morphological characters were done. Species closely related to Cerqueirellum were included in the analysis. The genera Cerqueirellum, Coscaroniellum and Shelleyellum were demonstrated as consistent basal entities and well-defined monophyletic clades.

A new filaria of a lizard transmitted by sandflies

A description is given of Madathamugadia wanjii n. sp., a Splendidofilariinae parasite of the gecko Ptyodactylus hasselquistii, on the west bank of the River Jordan and of its life cycle in Phlebotomus duboscqi. The new species is close to M. ivaschkini (Annaev, 1976) n. comb., of Turkmenistan, wich is also transmitted by sandflies (Reznik, 1982). The genus Madathamugadia is now comprised of four species, two from Madagascar and two from the Mediterranean sub-region; it differs from the genus Thamugadia by the presence of a double row of papillae anterior to the cloaca of the male. The larval characters of Splendidofilarinae of lizards confirm the affinity of these parasites to the Splendidofilarinae of birds (Chandlerella and Splendidofilaria); the first group could have arisen from the second by "captures" wich could have occurred in several places.

Neotropical Monogenoidea. 24. Rhinoxenus bulbovaginatus n. sp. (Dactylogyridae, Ancyrocephalinae) from the nasal cavity of Salminus maxillosus (Osteichthyes, Characidae) from the Rio Paraná, Paraná, Brazil

Rhinoxenus bulbovaginatus n. sp. is described from the nose of Salminus maxillosus (Characidae) collected in the basin of the rio Paraná, near the city of Porto Rico, state of Paraná, Brazil. The new species can be differentiated from the other three species in the genus by the morphology of the copulatory complex, vagina, and ventral anchor. The sister group relationship of the known species of Rhinoxenus was determined using techniques of Phylogenetic Systematics (Cladism). The resulting cladogram (C.I.=100%) indicates that the new species is most closely related to R. piranhus Kritsky, Boeger and Thatcher, 1988. The other two species of the genus, R. arietinus Kritsky, Boeger and Thatcher, 1988 and R. nyttus Kritsky, Boeger and Thatcher, 1988, both parasites of Anostomidae fishes, have a paraphyletic position in the cladogram, suggesting that the origin of at least one of them can not be associated to cospeciation.

Study of the Weight of Eggs from Six Ixodid Species from Brazil

Eggs from engorged females of six ixodidae species (Ixodes loricatus, Amblyomma rotundatum, A. cajennense, Haemaphysalis leporis-palustris, Boophilus microplus, Dermacentor nitens) maintained in laboratory were counted to calculate the number and mean weight in 1g of eggs from each species. Phylogenetic considerations are discussed based on the results

Molecular Epidemiology and Emergence of Rift Valley Fever

Rift Valley fever (RVF) is a mosquito-borne viral disease which manifested itself during recent epidemics and revealed its significant potential of emergence. Studies on molecular epidemiology undertaken to better understand the factors leading to RVF emergence, have confirmed the mode of circulation of the virus and highlithted probable risks and obstacles for prevention and control. As for several other viral agents, molecular epidemiology is becoming a useful tool in the study of the emergence of RVF as a serious infectious disease.

Systematics and Population Level Analysis of Anopheles darlingi

A new phylogenetic analysis of the Nyssorhynchus subgenus (Danoff-Burg and Conn, unpub. data) using six data sets {morphological (all life stages); scanning electron micrographs of eggs; nuclear ITS2 sequences; mitochondrial COII, ND2 and ND6 sequences} revealed different topologies when each data set was analyzed separately but no heterogeneity between the data sets using the arn test. Consequently, the most accurate estimate of the phylogeny was obtained when all the data were combined. This new phylogeny supports a monophyletic Nyssorhynchus subgenus but both previously recognized sections in the subgenus (Albimanus and Argyritarsis) were demonstrated to be paraphyletic relative to each other and four of the seven clades included species previously placed in both sections. One of these clades includes both Anopheles darlingi and An. albimanus, suggesting that the ability to vector malaria effectively may have originated once in this subgenus. Both a conserved (315 bp) and a variable (425 bp) region of the mitochondrial COI gene from 15 populations of An. darlingi from Belize, Bolivia, Brazil, French Guiana, Peru and Venezuela were used to examine the evolutionary history of this species and to test several analytical assumptions. Results demonstrated (1) parsimony analysis is equally informative compared to distance analysis using NJ; (2) clades or clusters are more strongly supported when these two regions are combined compared to either region separately; (3) evidence (in the form of remnants of older haplotype lineages) for two colonization events; and (4) significant genetic divergence within the population from Peixoto de Azevedo (State of Mato Grosso, Brazil). The oldest lineage includes populations from Peixoto, Boa Vista (State of Roraima) and Dourado (State of São Paulo).

Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA) gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied). Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc.) of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.