OCCURRENCE OF CRYPTOSPORIDIUM SP. IN DOGS AND CATS FROM CURITIBA AND ITS METROPOLITAN AREA

The present study was carried out with the aim of assess the occurrence of Cryptosporidium sp. infection in dogs and cats in Curitiba and its metropolitan area, state of Paraná, Brazil. Techniques employed to detect the protozoan in fecal samples were: staining by Ziehl-Neelsen for oocysts search and nested polymerase chain reaction (nPCR) targeting the 18SSU rDNA gene. To attempt the proposed aim, 91 feces samples of dogs and 25 of cats were collected and analyzed. Ziehl-Neelsen technique was unable to detect any oocyst in both groups analyzed, showing a very low sensitivity. Results of nPCR showed an infection rate of 13.2% (12/91) and 4% (1/25) in dogs and cats respectively.  The implications of these epidemiological data are discussed in this work

Métodos de desinfecção em água contendo Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) e sua detecção por técnica de biologia molecular /

Orientadora : Profª Drª Vanete Thomas SoccolCo-orientadora : Profª Drª Adriana Oliveira CostaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 2007Inclui bibliografiaÁrea de concentração: Parasitologi

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred