Search for Borrelia sp. in ticks collected from potential reservoirs in an urban forest reserve in the State of Mato Grosso do Sul, Brazil: a short report

A total of 128 ticks of the genus Amblyomma were recovered from 5 marsupials (Didelphis albiventris) - with 4 recaptures - and 17 rodents (16 Bolomys lasiurus and 1 Rattus norvegicus) captured in an urban forest reserve in Campo Grande, State of Mato Grosso do Sul, Brazil. Of the ticks collected, 95 (78.9%) were in larval form and 22 (21.1%) were nymphs; the only adult (0.8%) was identified as A. cajennense. Viewed under dark-field microscopy in the fourth month after seeding, 9 cultures prepared from spleens and livers of the rodents, blood of the marsupials, and macerates of Amblyomma sp. nymphs revealed spiral-shaped, spirochete-like structures resembling those of Borrelia sp. Some of them showed little motility, while others were non-motile. No such structures could be found either in positive Giemsa-stained culture smears or under electron microscopy. No PCR amplification of DNA from those cultures could be obtained by employing Leptospira sp., B. burgdorferi, and Borrelia sp. primers. These aspects suggest that the spirochete-like structures found in this study do not fit into the genera Borrelia or Leptospira, requiring instead to be isolated for proper identification.

Ecological drivers of a vector borne pathogen: distribution and abundance of Borrelia burgdorferi sensu lato and its vector Ixodes ricinus in Scotland

Vector-borne disease emergence in recent decades has been associated with different environmental drivers including changes in habitat, hosts and climate. Lyme borreliosis is among the most important vector-borne diseases in the Northern hemisphere and is an emerging disease in Scotland. Transmitted by Ixodid tick vectors between large numbers of wild vertebrate host species, Lyme borreliosis is caused by bacteria from the Borrelia burgdorferi sensu lato species group. Ecological studies can inform how environmental factors such as host abundance and community composition, habitat and landscape heterogeneity contribute to spatial and temporal variation in risk from B. burgdorferi s.l. In this thesis a range of approaches were used to investigate the effects of vertebrate host communities and individual host species as drivers of B. burgdorferi s.l. dynamics and its tick vector Ixodes ricinus. Host species differ in reservoir competence for B. burgdorferi s.l. and as hosts for ticks. Deer are incompetent transmission hosts for B. burgdorferi s.l. but are significant hosts of all life-stages of I. ricinus. Rodents and birds are important transmission hosts of B. burgdorferi s.l. and common hosts of immature life-stages of I. ricinus. In this thesis, surveys of woodland sites revealed variable effects of deer density on B. burgdorferi prevalence, from no effect (Chapter 2) to a possible ‘dilution’ effect resulting in lower prevalence at higher deer densities (Chapter 3). An invasive species in Scotland, the grey squirrel (Sciurus carolinensis), was found to host diverse genotypes of B. burgdorferi s.l. and may act as a spill-over host for strains maintained by native host species (Chapter 4). Habitat fragmentation may alter the dynamics of B. burgdorferi s.l. via effects on the host community and host movements. In this thesis, there was lack of persistence of the rodent associated genospecies of B. burgdorferi s.l. within a naturally fragmented landscape (Chapter 3). Rodent host biology, particularly population cycles and dispersal ability are likely to affect pathogen persistence and recolonization in fragmented habitats. Heterogeneity in disease dynamics can occur spatially and temporally due to differences in the host community, habitat and climatic factors. Higher numbers of I. ricinus nymphs, and a higher probability of detecting a nymph infected with B. burgdorferi s.l., were found in areas with warmer climates estimated by growing degree days (Chapter 2). The ground vegetation type associated with the highest number of I. ricinus nymphs varied between studies in this thesis (Chapter 2 & 3) and does not appear to be a reliable predictor across large areas. B. burgdorferi s.l. prevalence and genospecies composition was highly variable for the same sites sampled in subsequent years (Chapter 2). This suggests that dynamic variables such as reservoir host densities and deer should be measured as well as more static habitat and climatic factors to understand the drivers of B. burgdorferi s.l. infection in ticks. Heterogeneity in parasite loads amongst hosts is a common finding which has implications for disease ecology and management. Using a 17-year data set for tick infestations in a wild bird community in Scotland, different effects of age and sex on tick burdens were found among four species of passerine bird (Chapter 5). There were also different rates of decline in tick burdens among bird species in response to a long term decrease in questing tick pressure over the study. Species specific patterns may be driven by differences in behaviour and immunity and highlight the importance of comparative approaches. Combining whole genome sequencing (WGS) and population genetics approaches offers a novel approach to identify ecological drivers of pathogen populations. An initial analysis of WGS from B. burgdorferi s.s. isolates sampled 16 years apart suggests that there is a signal of measurable evolution (Chapter 6). This suggests demographic analyses may be applied to understand ecological and evolutionary processes of these bacteria. This work shows how host communities, habitat and climatic factors can affect the local transmission dynamics of B. burgdorferi s.l. and the potential risk of infection to humans. Spatial and temporal heterogeneity in pathogen dynamics poses challenges for the prediction of risk. New tools such as WGS of the pathogen (Chapter 6) and blood meal analysis techniques will add power to future studies on the ecology and evolution of B. burgdorferi s.l.

Identificação de genoespécies do complexo Borrelia burgdorferi sensu lato (s.l.) circulantes na fauna ixodideológica em portugal

Dissertação para obtenção do grau de Mestre em Microbiologia Médica

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

Swiss Army Survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks

A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.

Should reproductively isolated populations of Lutzomyia longipalpis sensu lato receive taxonomically valid names?

A group of 18 research workers involved in different aspects of the biology of Lutzomyia longipalpis discussed whether or not it is important to give taxonomically valid names to populations that have been defined by biological, biochemical and molecular methods to be reproductively isolated. The type material of this medically important species has been lost and because of this it was recommended that a colony should be established from insects captured in the region of the type area and that their description should serve as the basis for future descriptions. It was pointed out that there is a lack of uniformity in the naming of closely related American sand flies and that some of the differences between populations of Lu. longipalpis are greater than those between accepted species. The majority of the participants agreed that the populations that have been defined in the literature as sibling species should be named.

Floral anatomy of the Lecointea clade (Leguminosae, Papilionoideae, Swartzieae sensu lato)

Flower and inflorescence anatomy and morphology of Exostyles, Harleyodendron, Holocalyx, Lecointea, and Zollernia (Leguminosae, Lecointea clade) were studied. Features common to all genera but otherwise rare within the Leguminosae include: (1) the presence of phenolic compounds in the epidermal cells of the anthers and subepidermal cells of the bracteoles, sepals, petals, and ovaries (absent in Holocalyx balansae); (2) simple trichomes on the adaxial base of the bracteoles and on the surface of the calyx and ovaries; and (3) tapetum persisting until the androspores are formed. Other notable anatomical features are: (1) colleters on the adaxial bases of the bracts and bracteoles of Holocalyx balansae and Zollernia ilicifolia; (2) trichomes on the anthers of Harleyodendron unifoliolatum, Holocalyx balansae, Lecointea hatschbachii, Zollernia ilicifolia and Z. magnifica; (3) osmophores on the petals of Exostyles godoyensis; (4) asynchronous pollen development in the anthers of Holocalyx balansae and Zollernia magnifica; and (5) vascular bundles surrounded by lignified fibers in Harleyodendron unifoliolatum. These anatomical characters are discussed according to their possible phylogenetic implications.

KuhlmanniodendronFiaschi & Groppo, a new eastern Brazilian genus of Achariaceae sensu lato segregated from Carpotroche Endl. (formerly included in Flacourtiaceae)

Kuhlmanniodendron Fiaschi & Groppo, a new genus of the tribe Lindackerieae (Achariaceae sensu lato) is created to accommodate Carpotroche apterocarpa Kuhlm, a species previously described based on incomplete floriferous and fruiting material from the rainforests of Espirito Santo state, eastern Brazil. The genus is defined by a unique set of character states, including leaves with Clusia-like venation and scaly trichomes, flowers with glabrous stamen filaments and three free styles, and indehiscent fruits with a smooth surface (sometimes with vertical ribs when dried). A description and illustration are provided, with comments on geographical distribution, phenology and generic relationships, and an emended key to neotropical genera of Lindackerieae. (c) 2008 The Linnean Society of London.

Mitochondrial genome data alone are not enough to unambiguously resolve the relationships of Entognatha, Insecta and Crustacea sensu lato (Arthropoda)

An analysis of the relationships of the major arthropod groups Was undertaken using mitochondrial genome data to examine the hypotheses that Hexapoda is polyphyletic and that Collembola is more closely related to branchiopod crustaceans than insects. We sought to examine the sensitivity of this relationship to outgroup choice, data treatment. gene choice and optimality criteria used in the phylogenetic analysis of mitochondrial genome data. Additionally we sequenced the mitochondrial genome of ail archaeognathan, Nesomachilis australica. to improve taxon selection in the apterygote insects, a group poorly represented in previous mitochondrial phylogenies. The sister group of the Collembola was rarely resolved in our analyses with a significant level of support. The use of different outgroups (myriapods, nematodes, or annelids + mollusks) resulted in many different placements of Collembola. The way in which the dataset was coded for analysis (DNA, DNA with the exclusion of third codon position and as amino acids) also had marked affects on tree topology. We found that nodal Support was spread evenly throughout the 13 mitochondrial genes and the exclusion of genes resulted in significantly less resolution in the inferred trees. Optimality criteria had a much lesser effect on topology than the preceding factors; parsimony and Bayesian trees for a given data set and treatment were quite similar. We therefore conclude that the relationships of the extant arthropod groups as inferred by mitochondrial genomes are highly vulnerable to outgroup choice, data treatment and gene choice, and no consistent alternative hypothesis of Collembola's relationships is supported. Pending the resolution of these identified problems with the application of mitogenomic data to basal arthropod relationships, it is difficult to justify the rejection of hexapod monophyly, which is well supported on morphological grounds. (c) The Willi Hennig Society 2004.

Estudos taxonômicos e filogenéticos de Tenuipalpus Donnadieu (sensu stricto e sensu lato) e Colopalpus Pritchard & Baker (Acari, Tenuipalpidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

Amazonian Foliicolous Fungi III. A preliminary list of Ascomycotina, mostly Dothideales, sensu lato

This paper records 65 leaf-inhabiting sac fungi collected in Amazonas and Roraima during 1977-78 as part of Projeto Flora Amazônica. Lembosia miconiae (Ryan) Farr is a new combination.

Cryptic species in Pagamea coriacea sensu lato (Rubiaceae): evidence from morphology, ecology and reproductive behavior in a sympatric context

In this study we explore morphological and ecological variation in sympatric populations of Pagamea coriacea s.l. - a species complex from white-sand vegetation in the Amazon. A total of 147 trees were sampled and monitored at three nearby sites in Central Amazon, Brazil. Multivariate analyses of morphology indicated two distinct groups (A and B), which also differed in bark type, each containing subgroups associated with sexual dimorphism. However, a single hermaphroditic individual was observed within group B. As expected, all pistillate plants produced fruits, but 23% of the staminate plants of group B, and 5% of group A also produced fruits. This variation suggests that the sexual systems of both groups are between dioecy and gynodioecy. There was an overlap in flowering phases between the two groups, but the pattern of floral maturation differed. Ecologically, plants of group B were found in more shaded habitats and over sandstone bedrocks, while group A was prevalent in deeper sandy soils as canopy plants. The significances of morphological and environmental differences were tested by a multivariate analysis of variance, and a canonical discriminant analysis assessed the importance of variables. The coexistence in sympatry of two discrete morphological groups in the P. coriacea s.l., with different habitat preferences and reproductive behaviors, indicates they represent distinct species.

Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera:Psychodidae:Phlebotominae) in Brazil

Phylogenetic analysis of morphometric and biological characters indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the River Amazonas in the State of Pará while the other occurs in northeast Brazil, in the State of Ceará, and further south, including the type locality in State of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le.(V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

Should reproductively isolated populations of Lutzomyia longipalpis sensu lato receive taxonomically valid names?

A group of 18 research workers involved in different aspects of the biology of Lutzomyia longipalpis discussed whether or not it is important to give taxonomically valid names to populations that have been defined by biological, biochemical and molecular methods to be reproductively isolated. The type material of this medically important species has been lost and because of this it was recommended that a colony should be established from insects captured in the region of the type area and that their description should serve as the basis for future descriptions. It was pointed out that there is a lack of uniformity in the naming of closely related American sand flies and that some of the differences between populations of Lu. longipalpis are greater than those between accepted species. The majority of the participants agreed that the populations that have been defined in the literature as sibling species should be named.