Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

Variation in Venoms of <Emphasis Type="Italic">Polybia Paulista</Emphasis> Von Ihering and <Emphasis Type="Italic">Polybia Occidentalis</Emphasis> Olivier (Hymenoptera: Vespidae), Assessed by the FTIR-PAS Technique

Wasps are able to synthesize toxic compounds known as venoms, which form a part of a mechanism to overcome prey and also to defend their colonies. Study of the compounds that constitute these substances is essential in order to understand how this defense mechanism evolved, since there is evidence that the venoms can vary both intra- and interspecifically. Some studies have used liquid and gas chromatography as a reliable technique to analyze these compounds. However, the use of Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) to analyze the variations in venom’s chemical profile has been proposed recently. This study evaluated whether the FTIR-PAS technique is effective for assessing the role of environmental factors on intra- and interspecific differences in the venom of the wasps Polybia paulista Von Ihering and Polybia occidentalis Olivier by FTIR-PAS. The colonies were collected in three municipalities of Mato Grosso do Sul, Brazil, in different types of environments. The results showed that the venoms of P. paulista and P. occidentalis differed significantly in profile. In addition, the intraspecific differences in the venom’s chemical profile of P. paulista are related to the type of environment where they nested, regardless of the geographical distance between the nests. The FTIR-PAS technique proved to be reliable and effective to evaluate the variations in the venom’s chemical profile in social wasps.     

Polyphasic characterization of rhizobia microsymbionts of common bean [<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> (L.)] isolated in Mato Grosso do Sul,a hotspot of Brazilian biodiversity

Common bean [Phaseolus vulgaris (Linnaeus)] is the key source of protein, carbohydrates and micronutrients for over 300 million people in the tropics. Like many legumes, P. vulgaris can fix atmospheric nitrogen in symbiosis with rhizobia, alleviating the need for the expensive and polluting N-fertilizers. The crop is known to nodulate with a wide range of rhizobia and, although Brazil is not a center of genetic origin/domestication of P. vulgaris, a variety of rhizobial species have been found as symbionts of the legume. Mato Grosso do Sul (MS) is one of the largest common bean producer states in Brazil, with reports of high yields and abundant natural nodulation. The objective of this study was to evaluate the diversity of 73 indigenous rhizobia isolated from common bean grown in 22 municipalities of MS. Great morphophysiological and genetic diversity was found, as indicated by the six and 35 clusters formed, considering the similarity level of 75 and 70%, respectively, for the phenotypic and rep-PCR dendrograms. Eleven representative isolates were selected for detailed genetic characterization using 16S rRNA and three protein-coding housekeeping genes, glnII, gyrB and recA. We identified species originated from the centers of origin/domestication of the legume, R. etli and R. phaseoli, species probably indigenous of Brazil, R. leucaenae and others of the Rhizobium/Agrobacterium clade, in addition to putative new species. The results highlight the great rhizobial diversity of the region.     

Animal Models and Antifungal Agents in Paracoccidioidomycosis: An Overview

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The morbidity and mortality associated with paracoccidioidomycosis necessitate our understanding of fungal pathogenesis and discovering of new agents to treat this infection. Animal models have contributed much to the knowledge of fungal infections and their corresponding therapeutic treatments. This is true for animal models of the primary fungal pathogens such as P. brasiliensis. This review describes the development, details and utility of animal models of paracoccidioidomycosis for studying and developing the current antifungal agents used for therapy of this fungal disease and novel agents with antifungal properties against P. brasiliensis.     

<Emphasis Type="Italic">Parapharyngodon hugoi</Emphasis> n. sp., a new nematode (Oxyuroidea: Pharyngodonidae) of the tree frog <Emphasis Type="Italic">Trachycephalus typhonius</Emphasis> (Linnaeus) from the Brazilian Pantanal,including a key to the congeners from amphibians of the American continent

A new nematode Parapharyngodon hugoi n. sp. (Oxyuroidea: Pharyngodonidae) is described parasitising the large intestine of the tree frog Trachycephalus typhonius (Linnaeus) (Anura: Hylidae) from the wetlands of Pantanal, State of Mato Grosso do Sul, Brazil. The new species exhibits a unique structure of the posterior cloacal lip in males, which is supported by a rigid V-shaped structure. Parapharyngodon hylidae parasitic in hylid frogs, including T. typhonius, from Mexico, is the most similar congener to P. hugoi n. sp. but is distinguished from the new species by the presence of a gubernaculum (vs absence), by the lateral alae in males ending far anterior to cloacal opening (vs near to it) and because in gravid females the ovaries encircle the oesophageal corpus. Additionally, the new species differs from its congeners as well as from species of Thelandros Wedl, 1862, a very closely related genus, by the combination of features such as spicule length, number of caudal papillae, morphology of the anterior cloacal lip, which is echinate, and position of ovaries. The geographical distribution of hosts seems to play an important role in the speciation process of Parapharyngodon spp.; however, due the lack of molecular data this issue along with the validity of both Thelandros and Parapharyngodon are still questions to be solved in the future, after improvement of the genetic database. A key to the species of Parapharyngodon parasitic in amphibians from the American continent is provided.     

The fitness of the Brazilian damsel <Emphasis Type="Italic">Stegastes fuscus</Emphasis> is increased by sharing the territory with the dusky grouper <Emphasis Type="Italic">Epinephelus marginatus</Emphasis>

Stegastes fuscus and Epinephelus marginatus are known for co-habiting shelters. The damselfish S. fuscus uses the territory for nesting and must protect its eggs from grazers; the grouper E. marginatus is an omnivorous sit-and-wait predator. This study aims to evaluate the effect of juvenile groupers on the reproductive success of the Brazilian damsel. Twenty-five hours of underwater observations were done in São Sebastião and Ilhabela, Northern shore of São Paulo, Brazil. Fitness increase was measured by the egg-clutch area and number of contributing females in 130 nests shared by groupers and another 130 where damselfishes stood alone. An egg predator crab was placed into the damselfish territory, and behavioural responses during 2 min were recorded for nests with or without E. marginatus, 80 replicates each. Nests shared by the dusky groupers had more eggs and received eggs from more females too. While fathers who were alone in the territory had to deal with the egg predator crab, in shared nests, the grouper would take care of the intruder, sometimes feeding on it. Therefore, the Brazilian damsel may benefit from the presence of the dusky grouper by increasing the fitness and diminishing the costs of parental care.     

Genetic analysis of forest species <Emphasis Type="Italic">Eugenia uniflora</Emphasis> L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA) _n and (CA) _n microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeirão Preto, Tambaú and São José do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guaçu river basins, in state of São Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H _E  = 0.83) and a low genetic differentiation (mean F _ST = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.     

Antagonistic Activity of <Emphasis Type="Italic">Nocardia brasiliensis</Emphasis> PTCC 1422 Against Isolated Enterobacteriaceae from Urinary Tract Infections

The main drawback of current antibiotic therapies is the emergence and rapid increase in antibiotic resistance. Nocardiae are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Nocardia brasiliensis was reported as antibiotic producer. The purpose of the study was to determine antibacterial activity of N. brasiliensis PTCC 1422 against isolated Enterobacteriaceae from urinary tract infections (UTIs). The common bacteria from UTIs were isolated from hospital samples. Antimicrobial susceptibility test was performed for the isolated pathogens using Kirby–Bauer disk diffusion method according to clinical and Laboratory Standards Institute guideline. Antagonistic activity of N. brasiliensis PTCC 1422 was examined with well diffusion methods. Supernatant of N. brasiliensis PTCC 1422 by submerged culture was analyzed with gas chromatography–mass spectrometry. Isolated strains included Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Proteus mirabilis. The most common pathogen isolated was E. coli (72.5 %). Bacterial isolates revealed the presence of high levels of antimicrobial resistances to ceftriaxone and low levels of resistance to cephalexin. Supernatant of N. brasiliensis PTCC 1422 showed antibacterial activity against all of the isolated microorganisms in well diffusion method. The antibiotic resistance among the uropathogens is an evolving process, so a routine surveillance to monitor the etiologic agents of UTI and the resistance pattern should be carried out timely to choose the most effective empirical treatment by the physicians. Our present investigation indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. Antibacterial resistance among bacterial uropathogen is an evolving process. Therefore, in the field on the need of re-evaluation of empirical treatment of UTIs, our present. The study has demonstrated that N. brasiliensis PTCC 1422 has a high potential for the treatment of UTIs.     

Serological Evidence of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Infection in Chickens from Paraná and Mato Grosso do Sul States,Brazil

The objective of this study was to detect antibodies against Paracoccidioides brasiliensis in free-range and caged chickens Gallus domesticus. Initially, the humoral immune response of two chickens immunized with P. brasiliensis was evaluated. Both animals showed the production of antibodies to gp43, the major P. brasiliensis antigen. The seroepidemiological survey was conducted in chickens from the Pantanal region in Mato Grosso do Sul State (free-range n = 40) and from northern region of Paraná State (free-range n = 100, caged n = 43). The serum samples were analyzed by indirect ELISA using gp43 as antigen. The positivity observed in free-range chickens from Mato Grosso do Sul (55%) was significantly higher (P = 0.0001) than in free-range chickens from Paraná State (16%). In contrast to the free-range chickens, no positivity was observed in the caged chickens (P = 0.003). This is the first report showing serological evidence of P. brasiliensis infection in chickens. The results suggest that free-range chickens are more frequently infected by P. brasiliensis, probably due to the constant contact with soil than caged chickens and could be useful as epidemiological markers of paracoccidioidomycosis.     

Banding and FISH in three species of <Emphasis Type="Italic">Vernonia</Emphasis>, subsection <Emphasis Type="Italic">Macrocephalae</Emphasis> (Asteraceae,Vernonieae)

To complement our knowledge about the karyotypes of the genus Vernonia Schreb., different techniques of chromosome banding, including AgNOR, triple staining with fluorochromes CMA/DA/DAPI (CDD), and fluorescence in situ hybridization (FISH) for the 45S rDNA probe, were applied to three species of subsection Macrocephalae. Vernonia bardanoides was collected from an area of cerrado (savanna) vegetation in Itirapina, São Paulo State, Brazil, and V. linearifolia and V. tomentella were collected from areas of rocky, open altitudinal vegetation in Joaquim Felicio and Diamantina, respectively, in Minas Gerais State. All species showed two terminal CMA⁺ and NOR bands. FISH indicated two terminal 45S rDNA sites in V. linearifolia and V. tomentella, and six in V. bardanoides.     

Diagnoses of and illustrated key to the species of <Emphasis Type="Italic">Ixodes</Emphasis> Latreille, 1795 (Acari: Ixodidae) from Brazil

The current Brazilian Ixodes fauna is composed of the following eight species: I. amarali Fonseca, 1935; I. aragaoi Fonseca, 1935; I. auritulus Neumann, 1904; I. fuscipes Koch, 1844; I. loricatus Neumann, 1899; I. luciae Sénevet, 1940; I. paranaensis Barros-Battesti, Arzua, Pichorim &; Keirans, 2003; and I. schulzei Aragão &; Fonseca, 1951. Further studies are needed to establish the taxonomic status of I. serrafreirei Amorim, Gazeta, Bossi &; Linhares, 2003, a recently proposed species based solely on the nymphal stage. We present an up-to-date key to adults of the currently valid Brazilian species of Ixodes based on scanning electron microscopy. The relationships between Brazilian and other Neotropical Ixodes are also discussed.     

Mouse intestinal microbiota reduction favors local intestinal immunity triggered by antigens displayed in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> biofilm

Background

We previously engineered Bacillus subtilis to express an antigen of interest fused to TasA in a biofilm. B. subtilis has several properties such as sporulation, biofilm formation and probiotic ability that were used for the oral application of recombinant spores harboring Echinococcus granulosus paramyosin and tropomyosin immunogenic peptides that resulted in the elicitation of a specific humoral immune response in a dog model.

Results

In order to advance our understanding of the research in oral immunization practices using recombinant B. subtilis spores, we describe here an affordable animal model. In this study, we show clear evidence indicating that a niche is required for B. subtilis recombinant spores to colonize the densely populated mice intestinal microbiota. The reduction of intestinal microbiota with an antibiotic treatment resulted in a positive elicitation of local humoral immune response in BALB/c mice after oral application of recombinant B. subtilis spores harboring TasA fused to E. granulosus (102-207) EgTrp immunogenic peptide. Our results were supported by a lasting prevalence of spores in mice feces up to 50 days after immunization and by the presence of specific secretory IgA, isolated from feces, against E. granulosus tropomyosin.

Conclusions

The reduction of mouse intestinal microbiota allowed the elicitation of a local humoral immune response in mice after oral application with spores of B. subtilis harboring immunogenic peptides against E. granulosus.

     

A new species of <Emphasis Type="Italic">Senecio</Emphasis> (Asteraceae: Senecionae) from southeastern Brazil

Senecio altimontanus, a new species from the states of Minas Gerais and São Paulo, Brazil, is described and illustrated. Senecio altimontanus appears to be restricted to high-elevation grasslands (campos de altitude). This new species is characterized by its usually simple stems that are densely leafy apically, coriaceous leaves, and 1–5 corymbiform capitulescences. It is closely related to Senecio apensis from which it differs by the size, shape, and margins of the leaves, the number of phyllaries, and pubescence of the cypselae.     

Taxonomic and nomenclatural notes concerning <Emphasis Type="Italic">Ormopteris</Emphasis> and <Emphasis Type="Italic">Pellaea</Emphasis> (Pteridaceae) in Brazil

We typify and clarify the nomenclature and identity of three species originally described in the fern genus Pellaea. We find Pellaea bongardiana to be illegitimate and recognize it as a synonym of Ormopteris riedelii (= P. riedelii). We also consider Pellaea brasiliensis to be a synonym of O. riedelii. The phylogenetic position of Pellaea flavescens, recently combined in Ormopteris (as O. flavescens), is discussed, and a key to all species of Ormopteris in Brazil is provided.     

Role of Host Glycosphingolipids on <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Adhesion

Binding of yeast forms to human lung fibroblast cultures was analyzed, aiming to better understand the initial steps of Paracoccidioides brasiliensis infection in humans. A significant P. brasiliensis adhesion was observed either to fibroblasts or to their Triton X-100 insoluble fraction, which contains extracellular matrix and membrane microdomains enriched in glycosphingolipids. Since human lung fibroblasts express at cell-surface gangliosides, such as GM1, GM2, and GM3, the role of these glycosphingolipids on P. brasiliensis adhesion was analyzed by different procedures. Anti-GM3 monoclonal antibody or cholera toxin subunit B (which binds specifically to GM1) reduced significantly fungal adhesion to fibroblast cells, by 35% and 33%, respectively. Direct binding of GM1 to yeast forms of P. brasiliensis was confirmed using cholera toxin subunit B conjugated to AlexaFluor^®488. It was also demonstrated that P. brasiliensis binds to polystyrene plates coated with galactosylceramide, lactosylceramide, trihexosylceramide, GD3, GM1, GM3, and GD1a, suggesting that glycosphingolipids presenting residues of beta-galactose or neuraminic acid at non-reducing end may act as adhesion molecules for P. brasiliensis. Conversely, no binding was detected when plates were adsorbed with glycosphingolipids that contain terminal residue of beta-N-acetylgalactosamine, such as globoside (Gb4), GM2, and asialo-GM2. In human fibroblast (WI-38 cells), GM3 and GM1 are associated with membrane rafts, which remain insoluble after treatment with Triton X-100 at 4°C. Taken together, these results strongly suggest that lung fibroblast gangliosides, GM3 and GM1, are involved in binding and/or infection by P. brasiliensis.     

Comparative analysis of immune responses in Colorado potato beetle larvae during development of mycoses caused by <Emphasis Type="Italic">Metarhizium robertsii</Emphasis>, <Emphasis Type="Italic">M. brunneum</Emphasis>, and <Emphasis Type="Italic">M. pemphigi</Emphasis>

Development of mycoses and progress of humoral and cellular immune responses were compared in larvae of the Colorado potato beetle Leptinotarsa decemlineata infected with entomopathogenic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found to be highly susceptible to the strains of M. robertsii and M. brunneum but weakly responsive to M. pemphigi. The extent of susceptibility to the pathogens was not related to the stimulating effect of epicuticular extracts on fungal growth. Metarhizium pemphigi, which is non-specific to the Colorado potato beetle, did not cause any significant changes in the immune response and did not colonize the hemocoel. When infected with M. robertsii and M. brunneum, the larvae exhibited an increase in hemocyte count during the early stage of mycosis (day 2) followed by a drastic decrease on day 3. The immunocompetent cells, plasmatocytes and granulocytes, exhibited the greatest decrease. Elevated phenoloxidase activity was recorded in the hemolymph and cuticle on days 2 and 3 post-infection. These changes in the immune responses correlated with strain-specific virulence. Thus, the immune response in Colorado potato beetle larvae is an important factor, which determines differences in the development of mycoses caused by different Metarhizium species.