Márcia M. Silva

Determination of lead in blood by tungsten coil electrothermal atomic absorption spectrometry

Abstract A method for the determination of lead in blood using a tungsten coil atomizer is described. A 100 μl volume of the whole blood sample is transferred to a sampler cup containing 100 μl of water plus 300 μl of 0.25% v/v Triton X-100. After lysis of blood cells, 500 μl of 10% w/v trichloroacetic acid is added for protein precipitation and 10 μl of the supernatant solution is automatically delivered into the tungsten coil. The furnace heating program is implemented in 41 s. It is shown by the paired t -test that there is no significant difference at the 5% probability level between results obtained by the proposed method and by using a transversely heated graphite atomizer with a longitudinal Zeeman background correction. Accuracy is also assessed by employing reference materials. The proposed tungsten coil procedure presents a characteristic mass of 15 pg Pb and a detection limit of 1.9 μg Pb dl −1 .

Separation and preconcentration by flow injection coupled to tungsten coil electrothermal atomic absorption spectrometry

Abstract A flow injection system coupled to a tungsten coil electrothermal atomizer has been developed for on-line separation and preconcentration, using lead as a model element. The system utilizes three-way solenoid valves for sampling, buffering, washing and reconditioning solution management, and the resin column is inserted in the tip of the autosampler arm of a Varian GTA-96. The solenoid valves and tungsten coil power supply were controlled by a computer program written in Visual Basic, interfaced with the built-in Varian software. The system performance was tested by loading the resin column with the sample flowing at 3 ml min −1 for 60 s. Elution was performed automatically by sampling 20 μl of the eluent from a sample cup of the autosampler, and this aliquot was delivered into a 150 W tungsten coil. With Chelex-100 resin, the separation of concomitants was tested with lead in the presence of as much as 1000 mg l −1 of Ca, Mg, Na or K. The model system presented an enrichment factor of 64 at a sampling rate of 30 samples per hour.

Henneguya cuniculator sp nov., a parasite of spotted sorubim Pseudoplatystoma corruscans in the Sao Francisco Basin, Brazil

Henneguya cuniculator sp. nov. was found infecting spotted sorubim catfish Pseudoplatystoma corruscans from the São Francisco River, Minas Gerais, Brazil. The parasites form elongated plasmodia of up to 1 cm in length in the gill filaments. Mature spores were ellipsoidal from the frontal view, with total length of 29.4 ± 2.4 (mean ± SD, range 23.3-32.4) µm, body length of 12.1 ± 1.0 (10.0-14.7) µm, width of 4.8 ± 0.4 (4.0-5.9) µm, and tail length of 16.7 ± 2.0 (12.3-19.4) µm. From the lateral view, spores were biconvex, with thickness of 4.2 ± 0.7 (3.9-4.9) µm. The polar capsules were elongated and equal in size, 6.2 ± 0.3 (5.2-6.2) µm in length, and 1.8 ± 0.1 (1.4-1.9) µm in width. Ultrastructural analysis showed that the plasmodial wall had delicate projections towards the host tissue and a thin layer that prevented contact between the host cells and the parasite. In the ectoplasm, few mitochondria were observed, while generative cells, early stages of sporogenesis, and advanced spore development occurred in the plasmodial periphery, and more mature spores in internal regions. Histopathological analysis showed that plasmodia developed in the sub-epithelial connective tissue of gill filaments, causing compression of the adjacent tissues, deformation of gill filaments, and lamellar fusion. Phylogenetic analysis, based on 18S rDNA genes and using only Henneguya/Myxobolus species parasites of siluriform fish, showed grouping according to the fish family.

Host-parasite and phylogenetic relationships of Myxobolus filamentum sp. n. (Myxozoa: Myxosporea), a parasite of Brycon orthotaenia (Characiformes: Bryconidae) in Brazil.

Myxobolus filamentum sp. n. was found infecting gill filaments of three of 39 Brycon orthotaenia Günther specimens examined (8%), which were taken from the river São Francisco in Minas Gerais state, Brazil. Plasmodia of the parasite were white and long, measuring 5 mm in lenght. Mature spores of M. filamentum sp. n. were oval from the frontal view and biconvex from the lateral view, measuring 7.5-9.7 µm (9.0 ± 0.3 µm) in length and 5.2-7.3 µm (6.2 ± 0.4 µm) in width. The polar capsules were elongated and equal in size, measuring 3.8-5.5 µm (4.7 ± 0.3 µm) in length and 1.3-2.2 µm (1.7 ± 0.1 µm) in width. The development of the parasite led to compression of the adjacent tissues and inflammatory infiltrate with granulocytic cells. Ultrastructural observation revealed that the plasmodia were delimited by two membranes, which had numerous and extensive pinocytotic channels extending into the wide ectoplasm zone. The plasmodial wall exhibited abundant villi-like projections and a thin layer of granular material prevented direct contact between the plasmodial wall and the host tissue. Phylogenetic analysis, based on 18S rDNA, showed M. filamentum sp. n. as a sister species of Myxobolus oliveirai Milanin, Eiras, Arana, Maia, Alves, Silva, Carriero, Ceccarelli et Adriano, 2010, a parasite of other fish species of the genus Brycon Müller et Troschel from South America.

Prevalence, intensity, and phylogenetic analysis of Henneguya piaractus and Myxobolus cf. colossomatis from farmed Piaractus mesopotamicus in Brazil.

Henneguya piaractus and Myxobolus colossomatis (Myxosporea: Myxobolidae) are commonly found in the characid Piaractus mesopotamicus, an important fish farm species in Brazil. This paper describes the prevalence, mean intensity, molecular phylogeny, ultrastructure, and histology of H. piaractus and M. cf. colossomatis found infecting specimens of P. mesopotamicus collected from fish farms in the state of São Paulo, Brazil. A total of 278 fish were collected from 3 fish farms between February 2008 and July 2010. Parasite prevalence and mean intensity varied throughout the study period, and according to location and year. A phylogenetic tree, placing South American species in a global context, showed a clear tendency among myxosporean species to cluster according to host families. Ultrastructural analysis for M. cf. colossomatis showed the plasmodial wall with numerous projections toward host cells and phagocytic activity. Histopathological data showed hyperplasia caused by H. piaractus in highly infected fish. Histological and ultrastructural analysis of H. piaractus showed results similar to those that have previously been reported.

Supplementary data of Henneguya leporinicola (Myxozoa, Myxosporea) a parasite of Leporinus macrocephalus from fish farms in the state of São Paulo, Brazil

Abstract Henneguya leporinicola is a parasite of the gill filament of Leporinus macrocephalus, a characiform fish belonging to the Anostomidae family, which is of major economic importance. Despite the damage it causes in fish, little is known about this parasite. Therefore, a study was undertaken with fourteen specimens of L. macrocephalus taken from fish farms in the state of Sao Paulo. The fish were collected and examined searching for lesions and/or myxosporean plasmodia. One of the specimens (7.14%) contained white elongated plasmodia in the gill filament. The mature spores had elongated bodies with polar capsules of equal size and a caudal length greater than body length. Morphological characteristics identified the parasite as H. leporinicola. Molecular analysis of the 18S rDNA sequence resulted in a 1954 bp, demonstrating significant genetic differences with previously described species of Henneguya/Myxobolus. Phylogenetic analysis comparing the 18S rDNA sequence of H. leporinicola with other species, previously described in South America, and the 20 closest species as indicated by BLASTn Max Score showed H. leporinicola as a basal branch of a subclade composed by Henneguya spp. parasite of characiform hosts.

New myxosporeans parasitizing Phractocephalus hemioliopterus from Brazil: morphology, ultrastructure and SSU-rDNA sequencing

Myxozoans are a diverse group of parasitic cnidarians, with some species recognized as serious pathogens to their hosts. The present study describes 2 new myxobolid species (Myxobolus figueirae sp. nov. and Henneguya santarenensis sp. nov.) infecting skin and gill filaments of the Amazonian pimelodid fish Phractocephalus hemioliopterus, based on ultrastructural, histology and phylogenetic analysis. The fish were caught in the Amazon River, Pará, Brazil. The plasmodial development of M. figueirae sp. nov. was in the dermis and those of H. santarenensis sp. nov. were of the intralamellar type. For both species, the plasmodia were surrounded by a connective tissue layer, but there was no inflammatory infiltrate. For M. figueirae sp. nov., mature spores were ovoid measuring 9.1 to 10 (9.5 ± 0.3) µm in length, 5.8 to 6.9 (6.4 ± 0.3) µm in width and 4.4 to 4.5 (4.5 ± 0.1) µm in thickness. Two polar capsules were elongated and of unequal size. For H. santarenensis sp. nov., mature spores were ellipsoidal in the frontal view, measuring 26.3 to 36.1 (31.9 ± 3) µm in total length, 9.6 to 11.9 (10.8 ± 0.5) µm in body length, 3.7 to 4.9 (4.3 ± 0.3) µm in width and 16.6 to 25.6 (21 ± 3.1) µm in caudal process. The polar capsules were elongated and of equal size. Phylogenetic analysis, based on partial small subunit ribosomal DNA (SSU rDNA) sequences and using the closest myxozoan sequences to each one of the species studied here based on previous GenBank data, showed M. figueirae sp. nov. and H. santarenensis sp. nov. clustering in distinct lineages. While H. santarenensis sp. nov. clustered in a well-supported subclade composed of Henneguya species that infect gills of South American pimelodid hosts, M. figueirae sp. nov. clustered in a weakly supported subclade containing parasite species of bryconid hosts.

Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon

The involvement of oligochaetes in the life cycles of fresh water myxozoan parasites in Brazil was investigated. Of 333 oligochaetes collected in a fish farm in the State of São Paulo, three (0.9%) released Aurantiactinomyxon type spores. From 86 worms collected in a fish farm in Mato Grosso do Sul State, 1 (0.9%) released actinospores with a novel morphology for which we propose the name Seisactinomyxon. Infected oligochaetes were identified by morphology: all belonged to family Naididae, with Pristina americana the host for Aurantiactinomyxon and Slavina evelinae the host of Seisactinomyxon. This is the first report of the involvement these two species of oligochaetes in the life cycle of myxozoans. Small subunit rDNA sequences of the Aurantiactinomyxon (1204 nt) and Seisactinomyxon (1877 nt) did not match any previously sequenced myxozoan. Phylogenetic analyses showed that both actinospore types fell in a clade formed by six Myxobolus spp. that parasitize Characiformes fishes.

Molecular phylogeny and ultrastructure of Myxobolus cf. cuneus, a parasite of patinga hybrid and Henneguya pseudoplatystoma, a parasite of pintado hybrid.

Abstract Through morphological, histopathological and ultrastructural analysis of Myxobolus cuneus Adriano, Arana et Cordeiro, 2006 and Henneguya pseudoplatystoma Naldoni, Arana, Maia, Ceccarelli, Tavares, Borges, Pozo et Adriano, 2009 were identified infecting pacu respectively (Piaractus mesopotamicus) and hybrid pintado (Pseudoplatystoma corruscans x Pseudoplatystoma reticulatum) taken from Brazilian fish farms. The present study describes 18S rDNA sequencing of Myxobolus cf. cuneus infecting the spleen of farmed patinga, a hybrid fish resulting from the crossing of P. mesopotamicus x Piaractus brachypomus, and H. pseudoplatystoma found in farmed hybrid pintado from the state of Sao Paulo, Brazil. The study also provides new details of the host-parasite interface of M. cf. cuneus, which reveal that the plasmodial wall is composed of a single membrane connected to the plasmodium ectoplasm by numerous pinocytic canals. The plasmodia also displayed asynchronous development but had disporic pansporoblasts at different developmental stages; immature and mature spores were found at different depth levels of the plasmodium. Maximum likelihood phylogenetic analysis showed that M. cf. cuneus appeared as a sister species of Henneguya pellucida Adriano, Arana et Cordeiro, 2005 in a sub-clade composed mainly of myxosporean parasites of characiforms, and that H. pseudoplatystoma clustered in a sub-clade composed of Henneguya/Myxobolus spp. parasites of siluriform fish.

Prevalence, geographic and seasonal distribution of protozoan and myxozoan parasites of jau (Zungaro jahu)

Adriano E.A., Ceccarelli P.S., Silva M.R.M. & Maia A.A.M. 2012. [Prevalence, geographic and seasonal distribution of protozoan and myxozoan parasites of jaú (Zungaro jahu) in the Pantanal of Mato Grosso, Brazil.] Prevalência, distribuição geográ ica e sazonal de protozoários e mixozoários parasitos de jaú (Zungaro jahu) no Pantanal Matogrossense. Pesquisa Veterinária Brasileira 32(12):1341-1344. Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Av. Duque de Caxias Norte 225, Pirassununga, SP 13635-900, Brazil. E-mail: antomaia@usp.br In a study carried out in the Pantanal of Mato Grosso, Brazil, the prevalence, geographic and seasonal distribution of protozoan and myxozoan parasites of Zungaro jahu was evaluated. The ish were captured in the southern region of Pantanal Mato-grossense (Aquidauana, Miranda and Paraguay rivers) in 2001, 2002 and 2003, in the central region (Pantanal National Park PARNA Pantanal) in 2003, 2004, 2005 and 2008, and in the northern region (Cuiabá and Manso rivers, in the municipality of Nobres) in 2003, 2004 and 2005. Trichodina sp. was identi ied parasitized skin and gills of jaú in the three regions studied. Epistylis sp. parasitized skin and Cryptobia sp. the gills and were restricted to the Central region, whilst Ichthyophthirius multi iliis parasitized skin in the three regions studied. The occurrence of myxozoans was also observed: Myxobolus cordeiroi parasitized several organs and Henneguya sp. parasitized the gills of jaú in the three regions studied.In a study carried out in the Pantanal of Mato Grosso, Brazil, the prevalence, geographic and seasonal distribution of protozoan and myxozoan parasites of Zungaro jahu was evaluated. The fish were captured in the southern region of Pantanal Mato-grossense (Aquidauana, Miranda and Paraguay rivers) in 2001, 2002 and 2003, in the central region (Pantanal National Park - PARNA Pantanal) in 2003, 2004, 2005 and 2008, and in the northern region (Cuiaba and Manso rivers, in the municipality of Nobres) in 2003, 2004 and 2005. Trichodina sp. was identified parasitized skin and gills of jau in the three regions studied. Epistylis sp. parasitized skin and Cryptobia sp. the gills and were restricted to the Central region, whilst Ichthyophthirius multiiiliis parasitized skin in the three regions studied. The occurrence of myxozoans was also observed: Myxobolus cordeiroi parasitized several organs and Henneguya sp. parasitized the gills of jau in the three regions studied.