The currently available ES antigen-based ELISA for serological detection of B. procyonis
larva migrans exhibits low specificity, primarily because of antigenic cross-reactivity with related ascarids (such as T. canis
and A. suum
) resulting from potentially conserved proteins, a phenomenon well documented in other parasites (9
). Despite this, ES antigens are widely used in serological assays in either crude or purified (native or recombinant) form to diagnose parasite infections (23
), and recombinant antigen-based serological assays show increased diagnostic specificity (23
). However, production of recombinant proteins requires knowledge about the genes encoding the specific antigens. The present study was undertaken to identify genes that code for BPES antigens. To the best of our knowledge, this is the first report of molecular cloning and expression of an antigenic protein of Baylisascaris procyonis
In the absence of posttranslational modifications, native RAG1 can be expected to have a molecular mass of about 25 kDa, on the basis of full-length rag1
cDNA (assuming that the signal peptide is cleaved and all 12 repeats are present). However, the presence of multiple cysteines favoring protein-protein interaction and potential sites of glycosylation can result in a protein with a higher molecular mass. In addition, rRAG1 migrated slowly in polyacrylamide gels and showed an unexpected apparent mass, possibly due to the rigidity conferred by proline in polypeptides. This has been documented in proline-rich proteins from different organisms (19
). The identification of the native RAG1 antigen was not possible because both monospecific and anti-rRAG1 antibodies reacted with multiple proteins (52 kDa and higher) in the BPES antigen. However, the anti-rRAG1 antibodies alone reacted to an ~37-kDa protein in the BPES antigens, suggesting that this protein could be the native RAG1; further studies are needed to confirm the actual molecular size of the native RAG1. Although blastn analysis did not show any significant identity of the protein sequence to any known parasite nucleotide sequences, the blastp analysis showed that it has limited similarity to different proteins with repeats rich in proline, glycine, glutamine, and asparagine such as the S antigen and phosphatidylcholine-sterol acyltransferase from Plasmodium falciparum
and a hypothetical protein from Branchyostoma floridae
. Therefore, an identity could not be assigned to RAG1. However, on the basis of the reactivity of monospecific and anti-rRAG1 mouse antibodies to several components of the BPES antigen complex, reactivity of the antibodies to proteins transcribed from a multigene family cannot be ruled out and requires further investigation. In addition to the repeats, rRAG1 contains two tandemly located cystiene-rich motifs at the C terminus, which has been described in several nematodes, including Toxocara canis
and Caenorhabditis elegans
). This domain has been cited under different names (ShkT domain/NC6 motif/SXC motif) but is commonly recognized as the SXC motif in nematodes. The SXC motif homology arises only by virtue of the position of the six cysteine residues and not other amino acids. Scientific literature shows that the SXC motif has largely been associated with the functional domain of diverse proteins, viz.
, phosphatidylethanolamine-binding protein, zinc metalloprotease, tyrosinases, lectins, mucins, etc. The domain is also known to associate with signal peptides or in an extracellular milieu facilitates protein-protein interaction (22
). Initially identified in T. canis
, the SXC motif has been identified in several other nematode parasites, including A. suum
spp., and Necator americanus.
In addition, no homology to the 12-mer amino acid repeat could be identified, and therefore, a putative function of this protein is not known. At the least, the presence of a secretory signal and the reactivity of RAG1-monospecific and anti-rRAG1 mouse antibodies to components of BPES antigen suggest that RAG1 is a component of the ES antigen. Excretory-secretory antigens in parasites serve several functions, such as in feeding, penetration of the host, tissue migration, reproduction, and immune evasion (5
). Functional assays are required to determine the function of this protein in B. procyonis
. Also, the rag1
cDNA described here has been identified from B. procyonis
L3, a tissue migration stage of the parasite in the paratenic host and also the parasite stage seen in larva migrans. Whether the expression of this gene is parasite stage specific is not known.
Anti-rRAG1 mouse antibodies did not recognize any proteins in A. suum or T. canis ES antigens, suggesting the absence of RAG1 homologues or cross-reactive epitopes in the ES antigens of A. suum and T. canis. The antibodies raised against rRAG1 strongly reacted to a similar group of BPES antigens (37 to 52 kDa), further supporting the idea of these proteins being specific to B. procyonis. The absence of cross-reactivity of this protein in ELISA to serum raised against related ascarids like Toxocara and Ascaris also suggests that this protein might be unique to Baylisascaris spp. Among the Baylisascaris spp., strong reactivity was observed with serum against B. procyonis, B. columnaris, and B. melis. These parasites are closely related and are considered to be highly pathogenic causes of larva migrans compared to the somewhat distantly related and less pathogenic species B. transfuga. The anti-B. transfuga serum did not react as strongly as serum against other Baylisascaris spp. The presence of the RAG1 gene in related parasites remains to be investigated.
The B. procyonis RAG1 reported here is a potential diagnostic antigen that appears to be highly specific for Baylisascaris spp. and that does not cross-react with antibodies to either Toxocara canis or Ascaris suum, two common ascarid parasites to which humans may also be exposed. Ongoing studies in our laboratory using this rRAG1 protein for the diagnosis of B. procyonis larva migrans in humans by ELISA also suggest that there is no cross-reactivity with antibodies to Toxocara canis or other parasites. This recombinant antigen should be useful in various assay formats for the development of improved serological tests for the diagnosis, seroepidemiology, and serosurveillance of B. procyonis larva migrans.