Recombinant Cryptosporidium parvum GP15 based enzyme linked immunosorbent assay for detection of exposure of bovine populations to Cryptosporidium

Abstract

The serodiagnostic potential of recombinant Cryptosporidium parvum glycoprotein 15 (rCpGP15) was evaluated in Enzyme Linked Immunosorbent Assay (ELISA) in the detection of exposure of bovines to Cryptosporidium species in Himachal Pradesh and Uttarakhand states of North India. The 11.13 kDa rCpGP15 was expressed in pET-32a (+) transformed E. coli BL21 cells and was purified by Ni-NTA affinity chromatography as polyhistidine tagged fusion protein of ∼ 32 kDa. Its immunogenicity was checked in western blot using rabbit antisera raised to the recombinant antigen and bovine sera naturally infected with Cryptosporidium. Two hundred and forty-six bovines were screened for Cryptosporidium spp. oocysts in faecal samples by modified-Ziehl Neelson technique and their sera were used for ELISA standardization. The rCpGP15 based indirect IgG-ELISA was standardized with 83 % sensitivity, 78.3 % specificity where ELISA cut-off and accuracy were decided using ROC curve analysis. The percent accuracy was 79.19 %, with area under the Receiver Operating Characteristic (ROC) curve value 0.827 depicting the moderate accuracy of the assay. Additionally, sera from Eimeria (n = 3), strongyles (n = 3), Babesia (n = 2), Theileria (n = 5), Trypanosoma (n = 2) and Anaplasma (n = 5) positive animals showed no seroreactivity. The diagnostic performance of rCpGP15 protein in differentiating Cryptosporidium species was predicted through in-silico B cell epitope prediction, homology modelling and structural comparison of GP15 protein from C. parvum, C. hominis, C. bovis and C. ryanae. Four linear antigenic epitopes were predicted in CpGP15 protein sequence by SVMTrip. The overall root mean square deviation (RMSD) values during homology modelling and structural comparison of CpGP15 and C. hominis, C. bovis and partial C. ryanae GP15 were 2.093 Å, 3.759 Å and 1.152 Å, respectively. The serodiagnostic assay developed in the present study has moderate accuracy and can be applied in serosurveillance of large bovine populations. It is capable of detecting asymptomatic animals with intermittent oocyst shedding which will further be helpful for better understanding the disease dynamics and for the timely control of cryptosporidiosis.

Keywords: Bovine cryptosporidiosis; Cross-reactivity; ELISA; Recombinant CpGP15; Serodiagnosis; in-silico analysis.