Molecular detection and development of improved serological assays for the diagnosis of Baylisascaris larva migrans
Abstract
Larva migrans caused by the raccoon roundworm, Baylisascaris procyonis, occurs in more than 100 mammal and bird species and is also an emerging public health concern in America, Europe and parts of Asia. The objective of this research was to develop molecular and improved serological tests to assist the diagnosis of Baylisascaris larva migrans. Identification of Baylisascaris eggs in fecal and soil samples, and larvae in tissue samples were previously based entirely on microscopic examination. In the present studies, we have optimized conventional and real-time PCR assays using B. procyonis mitochondrial cytochrome oxidase 2 (cox2) gene as the target of amplification for detection of the parasite's eggs and larvae in fecal, soil and tissue samples. Currently, serological diagnosis of this disease is performed using ELISA, utilizing B. procyonis larval excretory-secretory antigen (BPES), a complex mixture of proteins that results in cross-reactivity with Toxocara spp. and other parasites. In addition, the preparation of BPES antigen has other shortcomings such as batch to batch variation, handling of infective eggs and larvae, etc. A Western blot assay using BPES antigen and an ELISA using B. procyonis recombinant antigen, Bp1 (identified by immunoscreening a B. procyonis larva cDNA expression library), were developed to overcome the limitations of BPES antigen-based ELISA. Results of the Western blot assay showed that BPES antigens between 30-45 kDa were specifically identified only by serum from individuals with Baylisascaris larva migrans and not by serum from patients with other parasitic diseases tested so far, including larva migrans caused by Toxocara spp. The recBp1-based ELISA showed no cross-reactivity with serum samples from rabbits experimentally infected with different ascarids. The recBp1-based ELISA showed high sensitivity of 84.21% and specificity of 86.86% when serum samples from patients with Baylisascaris or Toxocara spp. larva migrans and other parasitic diseases were tested. The PCR assays, BPES antigen-based Western blot and recBp1-based ELISA together can be used for specific diagnosis as well as for molecular and serological surveillance of this important disease.
Degree
Ph.D.
Advisors
Kazacos, Purdue University.
Subject Area
Molecular biology|Parasitology|Veterinary services
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