CPB 697 RESEARCH SEMINAR
Sriveny Dangoudoubiyam, BVSc, MVSc
Graduate Student in Parasitology
Department of Comparative Pathobiology
“Molecular And Serological Detection Of Baylisascaris procyonis Larva Migrans”
Thursday, March 1, 2007
VPTH 112
3:30 p.m.
Abstract:
The raccoon roundworm, Baylisascaris procyonis, is the most commonly recognized cause of clinical larva migrans in animals. It is responsible for neural larva migrans in more than 100 mammal and bird species and is also zoonotic to humans. However, there are several other related and unrelated species of nematodes that cause or have the potential to cause larva migrans. In our laboratory, we are developing molecular and serological techniques to separate B. procyonis larva migrans from infections caused by other nematode species.
Parasite eggs and larvae of these different parasites are sometimes difficult to differentiate morphologically and thus there is a need to develop molecular techniques to be able to specifically identify Baylisascaris species from each other and other related parasites. We are currently studying the species-specific PCR amplification of a 146 bp fragment of cytochrome oxidase II (Cox 2) from this parasite by conventional and real time PCR. Both conventional and realtime PCR were able to amplify this gene fragment from a minimum of 20 B. procyonis eggs. This 146 bp fragment was also successfully amplified from a canine fecal sample spiked with different numbers of B. procyonis eggs. Both PCR techniques could amplify this segment of the gene from a single B. procyonis larva. To date, the test has been performed on a few related and unrelated parasites to rule out amplification of this fragment in other species. Though the test differentiated B. procyonis from several other nematodes, including B. transfuga, it failed to differentiate B. procyonis from B. columnaris, the skunk roundworm. As the sequence of the Cox 2 gene in B. columnaris was not known, a partial length of 529 bp of the gene was cloned and sequenced. Sequence analysis showed that only two bases were different over this region of 529 bp indicating that Cox 2 cannot be used to differentiate between the two parasites.
Currently, the diagnosis of Baylisascaris larva migrans in humans is based on clinical and laboratory findings and results of serological testing (ELISA) developed in our laboratory. This test uses total Excretory-Secretory (ES) proteins from in vitro hatched and cultured B. procyonis larvae as antigen and has shown great utility in clinical cases in children. However, due to the complexity of the antigen, there have been problems with one-way cross-reactivity with Toxocara and perhaps other parasites. Therefore, as an attempt to characterize the immunodominant antigens of this parasite, a B. procyonis L3 cDNA expression library constructed in Uni-ZAPR XR vector was immunoscreened with B. procyonis infection serum raised in baboons. The recombinant phages were identified, the phagemid rescued and clones sequenced. The results of analysis of these partial length sequences, for the presence of functional domains and/or the sequence similarity to known nematode proteins show promise and will be discussed.