A Novel Designed Sandwich ELISA for the Detection of Echinococcus granulosus Antigen in Camels for Diagnosis of Cystic Echinococcosis.

Echinococcus spp. are important cosmopolitan zoonotic parasitic tapeworms that cause a disease called hydatidosis or cystic echinococcosis (CE), which has remarkable economic losses. The objective of our study was to develop a specific IgG polyclonal antigen-based ELISA (Sandwich ELISA; capture ELISA) method for the detection of circulating Echinococcus granulosus (E. granulosus) antigens in camels infected with hydatid cysts before slaughtering and its application in serodiagnosis of CE in animals to assess the positive rate of hydatidosis in camels slaughtered in Giza governorate abattoirs in Egypt. In this study, molecular identification of Echinococcus sp. isolate was performed based on the NADH dehydrogenase subunit 1 (NAD1) gene, revealing the isolate (GenBank: OQ443068.1), which is identical to the G6 E. granulosus sensu lato genotype. The positive rate of hydatid cysts was determined in slaughtered camels' organs (n = 587). The results revealed that hydatid cysts were found in 46.5% (273/587) of the examined camels. Pulmonary echinococcosis was significantly more prevalent in the slaughtered camels (60%, 164/273) than hepatic echinococcosis (39.9%, 109/273), (p = 0.001, Chi Square = 11.081). Cyst fertility rates were higher in hepatic (90.8%, 99/109) than in pulmonary cysts (83.5%, 137/164) and the most viable protoscoleces were recorded from fertile the hepatic cysts (67.85 ± 12.78). In this study, hydatid cyst germinal layer antigen (GlAg) was isolated and used for the immunization of rabbits to raise IgG polyclonal antibodies (anti-Echinococcus GlAb IgG). These IgG polyclonal antibodies were purified by affinity chromatography using a protein A column, then labeled with horseradish peroxidase. Electrophoretic analysis of IgG polyclonal antibodies and crude GlAg was performed in 10% polyacrylamide gels. The SDS-PAGE revealed four bands at molecular weights of 77 kDa, 65 kDa, 55 kDa, and 25 kDa. The Sandwich ELISA was performed to evaluate the sensitivity and specificity and cross-reactivity of the prepared IgG polyclonal antibodies. The circulating hydatid antigen was found in 270 out of the 273 samples with hydatidosis, with a sensitivity of 98.9% (270/273), a specificity of 94.9% (296/312) and a diagnostic efficacy of 96.8%. Regarding the cross reactivity, anti-Echinococcus GlAb IgG showed a low cross-reactivity with Fasciola gigantica infected camel sera (3/8), and Myiasis (Cephalopina titillator larvae; 3/20). No cross-reactivity was recorded with uninfected camel sera (negative sera for E. granulosus), and no cross-reactivity was found with antigens of Eimeria spp., Toxoplasma gondii, Cryptosporidium sp., and Hyalomma dromedarii (ticks' infestation). Then, Sandwich ELISA was conducted again to detect E. granulosus antigen in all the collected camel sera, which resulted in a 48.7% (286/587) positive rate of CE compared to 46.5% (273/587) using a postmortem inspection (PM diagnosis) (p = 0.5, Chi Square = 0.302). In conclusion, the Sandwich ELISA technique introduced in this study appears to be a sufficiently sensitive diagnostic assay for the detection of camels' echinococcosis using anti-Echinococcus GlAb IgG. In addition, it might offer a significant medical and veterinary importance in helping the early detection of hydatidosis, as well as its early treatment.

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.

Cross-protection induced by cross-reactive antigen against Fasciola gigantica and Trichinella spiralis infections.

Trichinella spiralis /Fasciola gigantica cross-reactive fraction was purified from T. spiralis larval extract by affinity column chromatography in which CNBr-Sepharose 4B was coupled with F. gigantica antibodies. The fraction consisted of six polypeptides of 191KDa, 178KDa, 149KDa, 106KDa, 101KDa and 32 KDa as revealed by SDS-PAGE. Analysis of the free amino acids of the fraction revealed 17 amino acids with high proportions of tyrosine and glutamic. Immunization of rabbits subcutaneously with the cross-reactive fraction in Freund's adjuvant followed by challenge with F. gigantica metacercariae resulted in reduction in worm burdens reached to 47.8%. While immunization of rats with the same fraction in Freund's adjuvant followed by infection with T. spiralis larvae resulted in reduction in worm count reached to 74%. IgG antibody response in rabbits increased due to immunization to reach its maximum value at the time of infection and then decreased gradually up to the end of the experiments. But. remained higher than the level in non vaccinated control animals. In rat sera, IgG level increased due to vaccination but the level recorded its optimum value one week post infection and then decreased. Thus the cross-reactive antigen proved cross-protection with the protection inducing capability against both diseases.

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.