الفهرس 
ACKNOLEDGMENTOnly 14 pages are availabe for public view
 |  | from | 141 |  |  |
| | | from | 141 | | |
Abstract
Facioliasis is one of the major public health problems in the world caused by F. hepatica and F. gigantica or ‘liver flukes’ particularly, in Egypt. Epidemics of fascioliasis have caused extensive suffering and economic losses. Diagnosis of fascioliasis is performed by conventional method through the demonstration of the fluke’s eggs in the faeces. However, this is possible only after 13–14 weeks post infection. While, major damage to the host hepatic system has already occurred. So, early diagnosis of fascioliasis is necessary for institution of prompt treatment before irreparable damage of the liver occurs.
In the present study, immunodiagnosis techniques of Fasciola gigantica were applied by using excretory/ secretory antigens in naturally infected cattle sera by Sandwich ELISA and in human infected sera by Dot blot technique. The detection of Fasciola antigens in serum could be more valuable in diagnosis, that because it has an advantage over antibody detection through indicating recent active infection.
In this study, viable adult Fasciola gigantica worms were collected from the bile ducts of infected cattle, and then incubated in RPMI culture medium (15worms/20ml medium). Medium was collected after 6hours and used to extract crude excretory /secretory (E/S) antigen. Protein content was measured by Bio-Rad protein assay (6.8mg/ml). E/S antigen was resolved by SDS-PAGE (12.5%) under reducing conditions (2-mercaptoethanol) and stained with Coomassie blue. Protein bands were appeared at 15, 17, 28, 32, 40, 60 and 84 kDa. E/S antigens were used to immunize rabbit and mouse to raise polyclonal antibodies against Fasciola gigantica E/S antigens. The IgG fraction of rabbit and mouse anti-Fasciola antibodies were purified using different purification steps including ammonium sulfate precipitation method followed by 7% caprylic acid precipitation method. The protein content of highly purified anti-Fasciola IgG antibodies, detected by Bio-Rad protein assay, were (8.8mg/ml, 5.2mg/ml), respectively for rabbit and for mouse was (4.1mg/ml, 2.7mg/ml), respectively. The purity of IgG after each step of purification was assayed by 12.5% SDS-PAGE under reducing conditions. Then, purified antibodies were further used as primary capture to coat ELISA plates. The secondary capture of antibodies was by conjugation with horse reddish peroxidase. Reactivity of anti-Fasciola ES antigens polyclonal antibodies that produced in both rabbit and mouse with Fasciola antigens and with different parasite antigens such as S. mansoni, hydatid and hook worm were strongly reactive with sera of cattle naturally infected with F. gigantica worms while no cross reactions were recorded with sera of other parasites Schistosoma mansoni, hydatid and hook worm by indirect ELISA.
Western blotting analyses revealed that immunization with E/S antigens induced in rabbit and mouse a very uniform antibody recognition of all E/S antigens bands for mouse, namely of 62–66, 28 & 15-17 KDa and for rabbit 62, 28, 43, 17 & 97 KDa. Then Sandwich ELISA was performed to detect Fasciola gigantica E/S antigens in serum samples collected from a total of 198 cattle. After slaughtering, gross inspection of liver. Collected samples were divided into Fasciola positive group (168 cattle) and healthy control group (30 cattle). Fasciola gigantica E/S antigens detected in serum of cattle by ELISA showed when rabbit and mouse anti- E/S antigens IgGs were each separately or alternatively with the other used as both coating and as conjugated antibody. Four cases were recorded with different cut-off values and with different results for sensitivity, specificity, positive and negative predictive values for each case. The highest sensitivity was found in the first three cases 100%, 100% and 94%, respectively, but the lowest one was in the last case 73.23%. While the highest specificity was in the last case (100%) and the other three cases also give high specificity 96.67%, 93.34% and 96.67%, respectively. Positive predictive values were high in the four cases 99.4%, 98.83%, 99.38% and 100%, respectively. While the highest negative predictive values in the first two cases were 100%, the lowest one was in the last case 66.67% and the third case was 74.36%.
In order to verify if antigens from other parasites could be detected by the assay (sandwich ELISA), using rabbit anti- F. gigantica IgG for coating and conjugation (where it gave the best result in this study) using 50 co-infected samples with parasites other than Fasciola such as S. mansoni (n=16), hydatid (n=20), hook worms (n=12) and trichostrongyloid (n= 15). All of these parasites give 100% negativity except S. mansoni that only 2 samples from 16 samples were positive and this due to the cross reactivity between Fasciola and Schistosoma.
Finally Dot blot technique was performed to detect Fasciola gigantica E/S antigens in serum collected from a total of 38 human samples after parasitological stool examination. Human samples were divided into Fasciola positive group (26 samples) and healthy control group (12 samples). This technique was applied for naturally infected cattle, which giving good results that encourage applying this technique on human samples, while all Fasciola infected patients gave positive results, which detected through appearance of reddish brown dots on nitrocellulose. Compared to healthy control individuals that showed disappearance of reddish brown color of dots. So, proved the ability of anti-rabbit IgG a raised against ESP antigens of F. gigantica to bind with ESP of a patient infected with Fascioliasis, using for diagnosis of human Fascioliasis.
The importance of this technique is referred to: this assay is very sensitive, simple to perform, needs short time, applied with high laboratory facilities with very sheep components and has long time stability. Nitrocellulose membrane is very sensitive and efficient for this assay; it allows direct detection of the antigen in a test sample, full analysis can be completed in just few hours thus saves time. Nitrocellulose papers spotted with antigen are stable for at least three months at -20°C, all incubation steps are performed at room temperature, and the results can be read with the naked eye, thus an expensive spectrophotometer is not required, only five microlitters of serum is required for the dot-blot, a distinct advantage when serum is difficult to obtain.