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Abstract
Fascioliasis caused by F. hepatica and F. gigantica is one the major public health problems in the world and in Egypt. Studies in some villages in the Delta have revealed prevalence rates varying between 2% and 17%. The number of infected cases amounts to 830,000 individuals (WHO, 1995). The prevalence is believed to be higher than reported cases.
The parasitological diagnosis is based on identification of eggs in stool, duodenal contents or bile, also by the recovery of adult worm during surgical exploration or at autopsy. However, the eggs may be present in very small number at irregular intervals, hence difficult to be found.
The samples tested in this study were taken from Fasciola infected animals collected from local abattoir, where the parasitological examination reached 73.74% true positive. As regards infected human living in endemic area only 64.29% had Fasciola eggs in stool examined by Kato-Katz technique.
Many immunological techniques have been developed over the past years for diagnosis of parasitic infections and to replace the parasitological techniques which are time-consuming and usually lack sensitivity and reproducibility with a possibility of missed diagnosis. Among these immunological techniques, the ELISA and its different modification systems held great promise for application in a wide variety of conditions and receiving most attention as immunodiagnostic methods for various parasitic infections.
Antigen detection assays are considered of prime importance for immunodiagnosis, as the detection of circulating antigens can indicate an active infection. In addition coproantigen detection should considered being non-invasive in field surveys for antigen detection assays; purified antibody and antigen were needed.
The present study described the immuno-affinity purification of fatty acid binding protein (FABP) from the crude extract of adult liver flukes by ion exchange chromatography on sephadex A-50 (DEAE column chromatography method) followed by gel filtration chromatography on sephacryl-S-200 HR column. The eluted proteins, from the gel filtration column chromatography analyzed by 12.5% SDS PAGE under reducing condition, showed only on band at 14.5 kDa which was FABP.
Also we had generated rabbit polyclonal anti-fatty acid binding protein antibodies by immunized New Zealand white rabbits with the prepared FABP antigen. The produced polyclonal antibodies (PAb) gave strong reactivity to Fasciola antigen (by indirect ELISA) compared to antigens from other parasites. The IgG fraction of rabbit anti-Fasciola antibody was purified using ammonium sulfate precipitation method followed by 7% caprylic acid precipitation method and finally ion exchange chromatography method. The protein content of highly purified anti-Fasciola IgG antibody, detected by Bio-Rad protein assay, was 1.1 mg/ml.
Conjugation of purified rabbit anti-Fasciola IgG with horse reddish peroxidase (HRP) was carried out, 10 g/ml concentration of the conjugate gave the highest OD reading against different concentrations of Fasciola antigen.
Standardization of sandwich ELISA used for detection of Fasciola antigen was carried out to determine the preliminary standardization and optimization work before the application of the technique on individuals and animals (stools and sera).
This work was conducted on 116 animal blood samples and 77 human blood samples. The animal blood samples were classified according to clinical and parasitological investigations in to three main group; fascioliasis (99 cases), healthy control (10 cases) and other parasitic (17 cases). Fascioliasis group were classified according to the detection of parasitic eggs in stool samples into high (75.4±16.06), moderate (34.6±5.63), and low (11.81±6.59) infected groups. While, the human blood samples were classified according to clinical and parasitological investigations in to three main group; fascioliasis (28 cases), healthy control (10 cases) and other parasitic (39 cases).
Each case was subjected to the following investigations:
1) Fasciola egg count by Kato method in sheep stool samples
2) Detection of the circulating Fasciola antigens in sheep sera by sandwich ELISA.
3) Detection of coproantigens in sheep stool by sandwich ELISA.
For detection of Fasciola antigen in stool and sera of both patients and animals sandwich ELISA was undertaken (using PAb to F. gigantica FABP antigen). Data revealed high rates of sensitivity (96.97% and 96.43% in stools of infected animals and patients respectively and 94.95% and 94.74% in sera of infected animals and patients, respectively) and specificity (94.12% and 94.87 in stools of infected animals and patients, respectively and 82.35% and 84.62% in sera of infected animals and patients, respectively), in addition there was a strong correlation between Fasciola egg count and both stool and serum antigen level detected.