A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

BACKGROUND: Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry. AIM: The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA). MATERIALS AND METHODS: The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA. RESULTS: The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples. CONCLUSION: FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Identification and diagnostic evaluation of cross-reactive antigen between hydatid cyst fluid of Echinococcus granulosus and Trichinella spiralis larval extract.

A cross reactive fraction was isolated from hydatid cyst fluid antigen of E. granulosus using CNBr Sepharose 4B affinity chromatography in which anti-T. spiralis antibodies were coupled with the column. Biochemical characterization of the isolated fraction included the use of SDS-PAGE, isoelectric focusing and amino acid analysis. The fraction showed 5 polypeptides of 165, 95.5, 63.5, 30.6 and 24 KDa. The isoelectric points of these polypeptides were 7.8, 7.2, 6.7, 6.2 and 5.7. The fraction exhibited 17 amino acids and was rich in tyrosine (20.81) and glutamic (15.28) ug/100 mg. The fraction proved higher potency in the diagnosis of experimental trichinellosis in rats than echinococcosis in dogs by ELISA. All experimentally infected animals reacted positively, recording 100% diagnostic sensitivity. Collectively, the present study proved that the hydatid cyst fluid cross-reactive fraction could be used in the diagnosis of trichinellosis at different intervals of infection and as early as 1 week post infection.

Induction of protective antibody response in rabbits against fascioliasis with Toxocara/Fasciola cross- reactive defined antigen.

A method of affinity chromatography purification of Toxocara vitulorum antigen cross- reacts with Fasciola gigantica antiserum is described. Characterization of the isolated cross- reactive fraction by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and amino acid analysis resulted in a fraction consists of five polypeptides of 137.7KDa, 81KDa, 75KDa, 48KDa and 21.6KDa with isoelectric points of 8, 7.5, 7.2, 6.7 and 6.6. Seventeen amino acids were identified in the fraction with high proportions of only two of them (tyrosine and glutamic). Rabbits immunization with this identified T. vitulorum cross- reactive antigen in Freund's adjuvant followed by challenge with F. gigantica metacercariae resulted in 60% reduction in worm burden over control infected rabbits. Higher IgG level was detected in vaccinated rabbits four weeks post first immunization than control infected ones and remained high up to the end of the trial.

Cross-reaction: a common trait among helminthes.

Cross-reaction between three important zoonotic helminths, Fasciola gigantica, Trichinella spiralis and Echinococcus granulosus, was proved by ELISA. Cross-binding activities in the prepared antisera were strongly directed towards protoscolices and hydatid fluid antigens of E. granulosus rather than to F. gigantica and T. spiralis antigens. Two sets of polypeptides were identified in each antigen by immunoblot; species-specific and cross-reactive. Cross-reactive components in F. gigantica antigen were 205 KD, 178 KD, 166 KD, 106 KD, 100 KD, 65 KD, 45 KD and 32 KD. While, cross-reactive molecules in T. spiralis antigen were 205 KD, 191 KD, 166 KD, 148 KD, 132 KD, and 32 KD. In protoscolices antigen six cross-reactive components were identified, 205 KD, 191 KD, 149 KD, 106 KD, 45 and 32 KD. Moreover, 205 KD, 190 KD, 177 KD, 149 KD, 103 KD and 33 KD were detected in hydatid fluid antigen by heterologous antisera. Interestingly, three polypeptides of 205 KD, 149 KD and 32 KD showed broad immunogenicity with the developed antisera raising the prospect of being putative common immunoprophylactic components.

Comparative immunodiagnostic approach of toxocariasis in buffalo calves.

Five Toxocara vitulorum antigens were utilized to diagnose natural toxocariasis in buffalo calves by ELISA. Adult antigen was proved to be the most potent one. The second potent antigen was egg antigen followed by excretory secretory antigen of male worms then female worms. The coproantigen was the least potent one. The electrophoretic make-up of the antigens, examined by SDS-PAGE, revealed different patterns of separation. Common as well as specific component(s) to each antigen were identified. Employing naturally infected buffalo calf sera in immunoblot assay, five immunogenic components were detected in adult antigen of molecular weight 191 KD, 166 KD, 102 KD, 65 KD and 54 KD. The reactive polypeptides in egg antigen were 191 KD, 105 KD, 99 KD and 79 KD. In coproantigen, six bands were identified. These components were of molecular weight 191 KD, 178 KD, 166 KD, 124 KD, 96 KD and 65 KD. Five components of molecular weight 191 KD, 166 KD, 102 KD, 96 KD and 65 KD were immunogenic in excretory/secretory antigen of male worms. Only four polypeptide of 191 KD, 166 KD, 102 KD and 66 KD were identified in excretory/secretory female antigen. Of interest is the immunogenic component of 54 KD expressed only by adult extract in immunoblot assay. This component could be responsible for the immunodiagnostic advantage of adult worm antigen and it deserves further studies to evaluate its diagnostic value.