Characterization of the external female genitalia of six species of Triatominae (Hemiptera: Reduviidade) by scanning electron microscopy

By macroscopic and microscopic dorsal side observation, it was noted that the IX and X segments of two species each of Panstrongylus and Triatoma terminate in an elongated way, whereas they terminate abruptly in the two species of Rhodnius. Scanning observation of the dorsal, ventral, lateral and posterior sides of the female genitalia of Panstrongylus herreri, Panstrongylus megistus, Rhodnius colombiensis, Rhodnius prolixus, Triatoma infestans and Triatoma vitticeps revealed that these six species are generally and specifically distinguished based on these elements. We describe several components that distinguish P. herreri from P. megistus: four on the dorsal side: the VII, VIII, IX and X segments, on the ventral view, three: VII sternite, VIII gonocoxite and VIII gonapophyse, on the lateral view one character, VIII gonocoxite and on the posterior view three characters: VIII and IX gonocoxite and XI gonopophyse. Comparing R. colombiensis and R. prolixus, there were three distinct characters on the dorsal side: the VII, VIII and X segments, on the ventral view three characters: the IX and X segments and VIII gonocoxite and on the posterior view four characters: the VIII, IX, X segments and VIII gonapophyse that distinguish the two species. T. infestans and T. vitticeps have four different characters on the dorsal side: the VII, VIII, IX and X segments, on the ventral view four characters: the VII and X segments, VIII gonocoxite and VIII gonapophyse, on the lateral view two characters, IX and X segments and on the posterior view four characters: the IX and X segments, VIII gonocoxite and VIII gonapophyse that distinguish the two species. Examination of the external female genitalia of six triatomine species by scanning suggests that these components are useful for taxonomical studies.

The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil

Over the last 20 years, there has been an increase in the number of leishmaniasis cases in Brazil. Belo Horizonte (BH) is one of the most highly populated Brazilian cities that is affected by visceral leishmaniasis (VL). The health services in BH are coordinated by a central nucleus that is subdivided into nine sanitary districts. Historically, the highest level of human VL cases was found in the northeast sanitary district (NSD). The objective of our study was to detect Leishmania infection in the phlebotomine sand flies collected in the NSD by dissection and molecular approaches. Following the occurrence of human VL cases in 2005, entomological captures were performed from July 2006-June 2007. Out of the 245 sand flies dissected, only three Lutzomyia longipalpis spp contained flagellates. The female sand flies were grouped into 120 pools according to date, collection site and species, with approximately 10 individual sand flies in each pool. Subsquently, the DNA was extracted and Leishmania spp and other parasites were detected and identified by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorfism. Leishmania infantum was present in at least 19% of the Lu. longipalpis collected, in 3.8% of the Nyssomiya whitmani collected, in 33.3% of the Evandromiya termitophila collected and in 14.3% of the Nyssomiya intermedia collected. When the females of the cortelezzii complex were compared with each other, 3.2% of the females were infected with Leishmania braziliensis, whereas 3.2% of the females were infected with trypanosomatids.

Full-length genomic sequence of hepatitis B virus genotype C2 isolated from a native Brazilian patient

The hepatitis B virus (HBV) is among the leading causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma. In Brazil, genotype A is the most frequent, followed by genotypes D and F. Genotypes B and C are found in Brazil exclusively among Asian patients and their descendants. The aim of this study was to sequence the entire HBV genome of a Caucasian patient infected with HBV/C2 and to infer the origin of the virus based on sequencing analysis. The sequence of this Brazilian isolate was grouped with four other sequences described in China. The sequence of this patient is the first complete genome of HBV/C2 reported in Brazil.

The phylogeny of Simulium (Chirostilbia) (Diptera: Simuliidae) and perspectives on the systematics of the genus in the Neotropical Region

Recently, knowledge of Neotropical Simuliidae has been accumulating quickly. However, information about supra-specific relationships is scarce and diagnoses of Simulium subgenera are unsatisfactory. To investigate the relationships among Simulium (Chirostilbia) species and test the subgenus monophyly, we performed a cladistic analysis. The ingroup included all species of this subgenus and the outgroup included representatives of the 17 species groups of Neotropical Simulium and three Holarctic species. The study was based on a data matrix with 31 terminal taxa and 45 morphological characteristics of adult, pupa and larva. The phylogenetic analysis under equal weights resulted in eight most-parsimonious trees (length = 178, consistency index = 34, retention index = 67). The monophyly of the S. (Chirostilbia) was not supported in our analysis. The Simulium subpallidum species group was closer to Simulium (Psilopelmia) and Simulium (Ectemnaspis) than to the Simulium pertinax species group. Additionally, we describe the three-dimensional shape of the terminalia of male and female of Simulium (Chirostilbia) for the first time and provide comments about the taxonomic problems involving some species of the subgenus: Simulium acarayense, Simulium papaveroi, S. pertinax, Simulium serranum, Simulium striginotum and S. subpallidum.

Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil

Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB) polymerase chain reaction (PCR) was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs). We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human), respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia) braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.

Description of Lutzomyia (Helcocyrtomyia) tolimensis, a new species of phlebotomine sandfly (Diptera: Psychodidae) from Colombia

A description is presented of Lutzomyia tolimensis sp. nov., a new species of the subgenus Helcocyrtomyia, series sanguinaria. It was collected in dwellings, peridomestic environment and in nearby forest patches located in the foothills of the Andean Central Cordillera, where in 2004-2006 occurred the largest epidemic ever recorded of leishmaniasis in Colombia. The male of this species is differentiated from other members of the series sanguinaria based on the following combination of characters: (i) base of coxite with 0-3 subequal setae, (ii) spines of gonostyle organized in positions 2.1.2, (iii) spines inserted on distal half of gonostyle and (iv) relationship of alar indices. The female is recognized principally by the following characters: (i) palpomere V longer than III, (ii) length of labro-epipharynx and (iii) relationship of the alar indices.

Morphological and molecular characterisation of Heliconema hainanensis sp. nov. (Spirurina: Physalopteridae) from congers in the South China Sea, with a key to the species of Heliconema

Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Anthropophilic Anopheles species composition and malaria in Tierradentro, Córdoba, Colombia

Malaria is still a primary health problem in Colombia. The locality of Tierradentro is situated in the municipality of Montelíbano, Córdoba, in the northwest of Colombia, and has one of the highest annual parasite index of malaria nationwide. However, the vectors involved in malaria transmission in this locality have not yet been identified. In this study, the local anthropophilic Anopheles composition and natural infectivity with Plasmodium were investigated. In August 2009, 927 female Anopheles mosquitoes were collected in eight localities using the human landing catch method and identified based on their morphology. Cryptic species were determined by restriction fragment length polymorphism-internal transcribed spacer (ITS)2 molecular analysis. Eight species [Anopheles nuneztovari s.l. (92.8%), Anopheles darlingi (5.1%), Anopheles triannulatus s.l. (1.8%), Anopheles pseudopunctipennis s.l. (0.2%), Anopheles punctimacula s.l. (0.2%), Anopheles apicimacula (0.1%), Anopheles albimanus (0.1%) and Anopheles rangeli (0.1%)] were identified and species identity was confirmed by ITS2 sequencing. This is the first report of An. albimanus, An. rangeli and An. apicimacula in Tierradentro. Natural infectivity with Plasmodium was determined by ELISA. None of the mosquitoes was infectious for Plasmodium. An. nuneztovari s.l. was the predominant species and is considered the primary malaria vector; An. darlingi and An. triannulatus s.l. could serve as secondary vectors.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Brazilian Angiostrongylus cantonensis haplotypes, ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.