Browsing by Author "Sousa, Susana"
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- Detection of Toxoplasma gondii oocysts in fresh vegetables and berry fruitsPublication . Marques, Cláudia S.; Sousa, Susana; Castro, António; da Costa, José Manuel CorreiaBackground: Toxoplasma gondii is the third most important contributor to health burden caused by food-borne illness. Ingestion of tissue cysts from undercooked meat is an important source of horizontal transmission to humans. However, there is an increasing awareness of the consumption of fresh fruit and vegetables, as a possible source for oocyst transmission, since this stage of the parasite can persist and remain infective in soil and water for long time. Herein, we outline findings related with detection of T. gondii oocysts in vegetables and berry fruits, which are usually raw consumed. The procedure includes the estimation of the number of oocysts. Methods: Food samples were collected from local producers and supermarket suppliers. Toxoplasma gondii oocysts were concentrated after washing the samples by applying high resolution water filtration and immunomagnetic separation (method 1623.1: EPA 816-R-12-001-Jan 2012), in order to (i) remove potential Cryptosporidium spp. oocysts and Giardia spp. cysts present in the samples; and (ii) select T. gondii oocysts. Toxoplasma gondii oocyst detection and an estimation of their numbers was performed by conventional PCR and real time qPCR, using specific primers for a 183-bp sequence of the T. gondii repetitive DNA region. All PCR-positive DNA samples were purified and sequenced. Restriction enzyme digestion with EcoRV endonuclease confirmed the presence of the T. gondii DNA fragment. In addition, the presence of the parasite was observed by fluorescent microscopy, taking advantage of the oocysts autofluorescence under UV light. Results: Forty percent of the analysed samples (95% CI: 25.5–56.5%) presented the expected PCR and digested DNA fragments. These fragments were confirmed by sequencing. Microscopic autofluorescence supported the presence of T. gondii-like oocysts. The estimated mean (± SE) oocyst concentration was 23.5 ± 12.1 oocysts/g, with a range of 0.6–179.9 oocysts/g. Conclusions: Our findings provide relevant evidence of contamination of fresh vegetables and berry fruits with T. gondii oocysts.
- Measurement of adsorption constants of laccase on gold nanoparticles to evaluate the enhancement in enzyme activity of adsorbed laccasePublication . Peixoto de Almeida, Miguel; Quaresma, Pedro; Sousa, Susana; Couto, Cláudia; Gomes, Inês; Krippahl, Ludwig; Franco, Ricardo; Pereira, EuláliaAdsorption of enzymes to nanoparticles and the mechanisms responsible for enzyme activity modulation of adsorbed enzymes are not well understood. In this work, gold nanoparticles were used for electrostatic adsorption of a plant-derived laccase. Adsorption constants were determined by four independent techniques: dynamic light scattering, electrophoretic light scattering, agarose gel electrophoresis and fluorescence quenching. Stable bionanoconjugates were formed with log K in the range 6.8-8.9. An increase in enzyme activity was detected, in particular at acidic and close to neutral pH values, a feature that expands the useful pH range of the enzyme. A model for the adsorption was developed, based on geometrical considerations and volume increase data from dynamic light scattering. This indicates that enzymes adsorbed to gold nanoparticles are ca. 9 times more active than the free enzyme.
- rTgOWP1-f, a specific biomarker for Toxoplasma gondii oocystsPublication . Sousa, Susana; Almeida, André; Delgado, Lurdes; Conceição, Antónia; Marques, Cláudia; da Costa, José Manuel Correia; Castro, AntónioToxoplasma gondii oocyst wall protein 1 (TgOWP1) integrates a family of seven proteins, consensually assumed as specific antigens of Toxoplasma gondii oocyst stage, located in the outer layer of the oocyst wall. Herein, we notice the expression of a recombinant antigen, rTgOWP1-f, derived from a fragment selected on basis of its structural homology with Plasmodium MSP1-19. Rabbit polyclonal antibodies anti-rTgOWP1-f evidence ability for specific identification of environmental T. gondii oocysts. We assume, rTgOWP1-f, as a possible biomarker of oocysts. In addition, we present findings supporting this vision, including the development of an immunodetection method for T. gondii oocysts identification.
- Serotyping of naturally Toxoplasma gondii infected meat-producing animalsPublication . Sousa, Susana; Canada, Nuno; Costa, José Manuel Correia da; Dardé, Marie-LaureSerotyping was previously described as a promising method for typing strains of Toxoplasma gondii. The majority of precedent studies utilized serum samples collected from human patients with different T. gondii-associated pathologies. The aim of this work was to study the applicability of the same procedure for serotyping naturally infected meat-producing animals. An ELISA test based on GRA6 and GRA7 C-terminal polymorphic peptides was used. Peptide GRA6II has polymorphisms specific for the archetypal strains type II, GRA6I/III for strains type I and III, GRA7I for strains type I and GRA7III for strains type III. As reference material, and to validate this approach, serum samples from eleven free-range chickens and fifteen pigs used for Toxoplasma genotypes isolation were selected. These strains integrate the Biological Resource Centre (BRC) ToxoBS Bank. Three serum samples from chickens and two from pigs had serotyping results in agreement with genotyping. Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii, from which no isolate was obtained, were also serotyped. Serotype III appeared significantly more frequent among sheep. Our results show that serotyping still need refinement, but may become a valuable tool for typing Toxoplasma strains from animal origin.
- The role of AKT3 copy number changes in brain abnormalities and neurodevelopmental disorders: four new cases and literature reviewPublication . Lopes, Fátima; Torres, Fátima; Soares, Gabriela; van Karnebeek, Clara D.; Martins, Cecília; Antunes, Diana; Silva, João; Muttucomaroe, Lauren; Botelho, Luís Filipe; Sousa, Susana; Rendeiro, Paula; Tavares, Purificação; Van Esch, Hilde; Rajcan-Separovic, Evica; Maciel, PatríciaMicrodeletions at 1q43-q44 have been described as resulting in a clinically recognizable phenotype of intellectual disability (ID), facial dysmorphisms and microcephaly (MIC). In contrast, the reciprocal microduplications of 1q43-q44 region have been less frequently reported and patients showed a variable phenotype, including macrocephaly. Reports of a large number of patients with copy number variations involving this region highlighted the AKT3 gene as a likely key player in head size anomalies. We report four novel patients with copy number variations in the 1q43-q44 region: one with a larger deletion (3.7Mb), two with smaller deletions affecting AKT3 and SDCCAG8 genes (0.16 and 0.18Mb) and one with a quadruplication (1Mb) that affects the entire AKT3 gene. All patients with deletions presented MIC without structural brain abnormalities, whereas the patient with quadruplication had macrocephaly, but his carrier father had normal head circumference. Our report also includes a comparison of phenotypes in cases with 1q43-q44 duplications to assist future genotype-phenotype correlations. Our observations implicate AKT3 as a contributor to ID/development delay (DD) and head size but raise doubts about its straightforward impact on the latter aspect of the phenotype in patients with 1q43-q44 deletion/duplication syndrome.