RESUMO
The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrheal diseases worldwide. The small size of oocysts under the microscope and the possibility of changes in characteristics of oocysts, mainly in environmental samples, make the taxonomy of the genus difficult if morphologic characteristics are considered. This limitation encouraged the application of molecular methods to identify this microorganism. The aim of this study was to detect and identify by nested-polymerase chain reaction oocysts of Cryptosporidium present in water samples in the state of São Paulo, Brazil. Water samples were concentrated through a membrane filter, DNA was extracted by using a standard technique, and both amplification reactions used forward and reverse oligonucleotides that were complementary to Cryptosporidium 18S ribosomal RNA gene sequences. Thirty water samples from different sites of collection in the state of São Paulo were evaluated. Cryptosporidium oocysts were detected in 30% of the samples. By genoptyping, C. hominis and Cryptosporidium sp. were identified in recreational water and C. meleagridis was identified in surface water samples. This is the first report of C. hominis in environmental samples in Brazil. Although identification of Cryptosporidium is still a difficult task, molecular methods are essential for specific identification and are a helpful tool to aid to understand the epidemiology of this parasite in Brazil.
Assuntos
Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Água/parasitologia , Brasil , Cryptosporidium/classificação , Genótipo , Humanos , FilogeniaRESUMO
Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 degrees C for up to six years and the low number of oocysts may be overcome by repetitions of extraction.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium/genética , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Oocistos , Animais , Protocolos Clínicos , Cryptosporidium/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.
A caracterização molecular de oocistos de Cryptosporidium spp. em amostras clínicas é útil à saúde pública, pois permite estudo das fontes de contaminação e a transmissão em determinadas regiões geográficas. Apesar de largamente utilizada em países desenvolvidos, no Brasil está restrita aos estudos acadêmicos, na maioria utilizando kits comerciais para extração do DNA genômico, ou em colaborações com centros de referência externos, o que torna o método caro e limitado. Este estudo propõe a introdução de modificações nos métodos existentes para melhorar a viabilidade e baixar custos. O método proposto foi eficiente em amostras clínicas preservadas a -20 °C por até seis anos e o baixo número de oocistos pode ser contornado por replicadas extrações de DNA.
Assuntos
Animais , Humanos , Criptosporidiose/diagnóstico , Cryptosporidium/genética , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Oocistos , Protocolos Clínicos , Cryptosporidium/isolamento & purificação , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Immunodeficient animals are important research models for studies in parasitology, oncology and immunology. Immunosuppressive drugs have been experimentally used to obtain a state of immunodeficiency in mice. This investigation aimed to quantify the circulating T and B cells of mice treated with the immunosuppressive agents dexamethasone (Dx), cyclosporine (CsA) and cyclophosphamide (CY), as well as to observe the behaviour of lymphocytic populations in the spleen of these animals. Blood samples were collected for counting the total peripheral blood leukocytes and T and B lymphocytes using flow cytometry. Total leukocytes of mice treated with the three drugs during all study showed a significant decrease when compared to the results of the control group. The proportion of B and T lymphocytes from the treated animals also decreased significantly. Spleen sections revealed a moderate decrease in the cellularity of the white pulp and the development of lymphocyte apoptosis in mice from groups treated with CY and Dx. Results showed that the proposed experimental models demonstrated to be suitable for studies of murine immunodeficiency.