An aspartyl protease inhibitor orthologue expressed by Parelaphostrongylus tenuis is immunogenic in an atypical host

Abstract

Parelaphostrongylus tenuis is a neurotropic nematode common in white-tailed deer (Odocoileus virginianus) of eastern North America. This parasite is the causative agent of a debilitating neurologic disease in atypical hosts, including domestic livestock. In order to identify proteins of potential significance in the host-parasite relationship, a cDNA library was produced from adult P. tenuis mRNA. Screening the library with antisera from infected red deer (Cervus elaphus elaphus) and immunized AO strain rats, we identified clones with sequence similarities to aspartyl protease inhibitors from several parasitic nematodes. Antibody that was generated against this recombinant protein of P. tenuis (Pt-API-1) detected the native protein in E/S products, in muscle and gonad, and on the surface of the cuticle of adult male and female P. tenuis. The native protein was detected in internal structures of first-stage (L1) and third-stage (L3) larvae. Reverse transcription-PCR confirmed expression of Pt-api-1 in L1, L3, and adult male and female worms. Expression of Pt-API-1 throughout the life cycle of P. tenuis suggests an essential function. Antibodies specific for recombinant Pt-API-1 were detected by enzyme-linked immunosorbent assay in sera from 12 red deer experimentally infected with P. tenuis. Antibodies were detected within 28 to 56 days postinfection. Responses were sustained or biphasic in animals with patent infections, consistent with expression of Pt-API-1 by L1. Our results are compatible with findings in other parasitic nematodes showing that aspartyl protease inhibitors are highly immunogenic.

FIG. 1.

ClustalW alignment (MacVector 6.5.3) of the deduced amino acid sequence of rPt-API-1 with orthologous sequences from other nematodes identified by BLASTp and tBLASTn searches. Amino acid positions are numbered along the margins. Conserved residues are indicated by the shaded boxed areas. The percent identities of orthologues to Pt-API-1 are indicated in the box at the lower right. Asterisks indicate four invariant conserved cysteine residues. The overline at the N terminus indicates the predicted signal peptide of P. tenuis.

FIG. 2.

Native Pt-API-1 is present in the E/S products of adult P. tenuis worms. Serum antibodies from deer infected with P. tenuis (A) or from rats immunized with rPt-API-1 (B) specifically recognize a 27-kDa molecule in the E/S products of adult P. tenuis. E/S products were blotted and incubated with pooled sera from 11 red deer at 112 to 140 days postexperimental infection (lane 1), antibody (from white-tailed deer) affinity-purified against rPt-API-1 (lane 2), pooled sera collected from rats immunized with rDHFR (lane 3), pooled sera collected from rats immunized with rPt-API-1 (lane 4), and pooled sera collected from rats immunized with E/S products from adult P. tenuis (lane 5).

FIG. 3.

Immunohistochemical detection of Pt-API-1 in adult and larval stages of P. tenuis. (A and C) Sections were treated with pooled sera collected from rats immunized with rDHFR. (B and D) Sections were treated with pooled sera collected from rats immunized with rPt-API-1. (A and B) Adult female; (C and D) infective L3 in situ within foot of terrestrial gastropod. M, muscle; G, gonad; C, cuticle; I, intestine.

FIG. 4.

rPt-API-1 is specifically recognized by serum antibody from red deer (C. elaphus elaphus) infected with P. tenuis in an ELISA. Deer were uninoculated controls (A) or were inoculated orally with 10 (B), 25 (C), or 100 (D) infective larvae (L3) of P. tenuis. Arrows indicate time of equivalent secondary inoculation of L3. Thicker lines indicate periods during which infections were patent by fecal examination. Legend lists individual animals and presence (+) or absence (−) of adult P. tenuis at necropsy. NC, animals did not receive a secondary inoculation of P. tenuis L3.