CPB 697 RESEARCH SEMINAR
Sriveny Dangoudoubiyam, BVSc, MVSc
Graduate Student in Parasitology
Department of Comparative Pathobiology
“Immunoscreening
Of Baylisascaris procyonis L3 cDNA Expression Library
For Identification Of Potential Diagnostic Antigens”
Thursday, April 17, 2008
VPTH
112
3:30
pm
Abstract:
Larva migrans caused by the raccoon roundworm, Baylisascaris procyonis, occurs in more than 100 animal and bird species and is also zoonotic to humans. Currently, the diagnosis is based on clinical and laboratory findings and results of serological testing (ELISA) developed in our laboratory, using the larval excretory–secretory (ES) antigens of this parasite. The ES antigens are prepared by the standard procedure of parasite egg-collection, in vitro-embryonation, hatching and culture of B.procyonis larvae. Preparation of such ES antigens depends largely on the availability of parasite eggs, and has certain drawbacks, viz. lengthy procedures, risk of handling the infective larvae, batch variation in the antigen composition, and cross reactivity. Use of recombinant B.procyonis antigens in the ELISA would be an appropriate choice to circumvent these drawbacks. However, none of the excretory-secretory antigens of this parasite have been characterized and the lack of genetic information about B.procyonis hinders generation of recombinant proteins. In order to identify the genes encoding ES antigens, a B.procyonis third-stage larvae (L3) cDNA expression library was constructed in Lambda ZAP® II vector. Immunoscreening of the library was performed using serum from a baboon experimentally infected with embryonated B.procyonis eggs. Immunoreactive plaques were identified, purified and monospecific antibodies corresponding to each of the different purified recombinant phages were prepared. The reactivity of these monospecific antibodies to B.procyonis ES antigens was assessed on western blots. The phagemids carrying the inserts were then excised from the phage and sequenced to ascertain the identity of B.procyonis cDNAs coding for ES antigens. Selected cDNAs were expressed in a bacterial expression system and the recombinant proteins purified using affinity chromatography. The utility of these purified recombinant proteins in the specific diagnosis of B.procyonis larva migrans is being evaluated.