Construction of a complementary DNA library for Parelaphostrongylus tenuis and identification of a potentially sero-diagnostic recombinant antigen.
Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.
Ogunremi
Oladele
O
Ontario Animal Health Laboratory, Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, 3851 Fallowfield road, Ottawa, Ontario KsH 8P9, Canada. dogunremi@inspection.gc.ca
Benjamin
Jane
J
MacDonald
Lily
L
Schimpf
Robert
R
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood / Q000276:immunology; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D003429:Cross Reactions; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D003670:Deer / Q000469:parasitology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005786:Gene Expression Regulation; D015723:Gene Library; D008681:Metastrongyloidea / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity; D051379:Mice; D018510:RNA, Helminth / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid; D017206:Strongylida Infections / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D018510:RNA, Helminth; D011994:Recombinant Proteins