Identification and Characterization of Antigenic Proteins of Excretory/Secretory and Somatic Products of Gastrothylax crumenifer from Infected Buffaloes

Abstract

Objectives Gastrothylax crumenifer is also known as amphistome that causes severe damage to ruminants in tropical and sub-tropical countries. Early detection of these parasites cannot be performed through coprology, eggs appeared after 17-20 weeks and have irregular shedding. Many immunodiagnostic tests are successfully developed for detecting trematode infections but such efforts are limited for amphistomes and still in the process of development. The present study was aimed to find prevalence and identified antigenic protein profile of somatic and excretory/secretory products of adult worms of Gastrothylax crumenifer collected from buffaloes. Materials and Methods Prevalence along with different risk factors of age, gender, location, breed, grazing habit, localities and interaction with water bodies was calculated for G. crumenifer infection. Somatic and excretory/secretory products were isolated from worms and protein concentration was estimated through bradford assay. The proteins were characterized through SDS-PAGE. Western Blotting and ELISA was performed to detect immunogenic protein. Data were analysed using Chi-Square test and Image-J Software. Results. The overall infection rate found was 12.1% with a significant (p<0.05) difference in the rates of infections in localities. The association of age, gender, breed, contact with water bodies, and grazing habits did not show significant (p>0.05) association with G. crumenifer infection. The characterized molecular weights of proteins in somatic and excretory secretory products were between 4-123 kDa with somatic extract showing maximum abundance of 4-20 kDa 55-71 kDa, 72-88 kDa, 89- 105 and 106-123 kDa proteins. The excretory secretory product showed maximum occurrence of 72-88 kDa protein. The antigenic analysis of these proteins on Western blot revealed a polypeptide of 55-70 kDa in somatic extract while metabolic extracts had not shown any antigenicity when reacted with sera obtained from infected buffaloes. The results of ELISA showed high immunodetection 90.5% with the 95- 250kDa antigen and 85.7% with 38-72 kDa proteins. The cross reactivity with other trematodes was recorded 10-20%. Conclusions In present study the prevalence of G. crumenifer was 12.1% which had nonsignificant association (p>0.05) with all demographic factors except locality (p<0.05). The protein profile had major bands of 4-20 kDa, 21-37 kDa, 38-54 kDa 55-71 kDa, 72-88 kDa, 89-105 kDa and 106-123 kDa and somatic extracts of sizes 4-20 kDa, 21- 37 kDa, 38-54 kDa, 55-71 kDa, 72-88 kDa, 89-105 kDa and 106-123 kDa by using SDS-PAGE. In western blot 55-70 kDa protein was identified antigenic against sera from naturally infected buffaloes. ELISA detected 90.5% antigenicity for 95-123 kDa protein 85.7% for 38-72 kDa and 9.5 % for 73-94kDa. The study concluded that further elaborative studies are needed to be conducted on prevalence of Gastrothylax crumenifer in all areas of Pakistan. The results of current investigation indicated identified proteins could be used as potential candidates for immunodiagnostic studies and further analysis of these proteins can also provide antigens crucial for parasite survival which can be used for vaccine development