Immune based detection of Angiostrongylus vasorum infection in dogs and molecular characterization of the antigen

Angiostrongylus vasorum (French heartworm) is a metastrongylid nematode which infects the pulmonary artery and right ventricle of wild and domestic canids and can lead to fatal cardiopulmonary disease in infected dogs. In North America, there is an endemic focus of infection in Newfoundland, Canada. Diagnosis is made by detection of first-stage larvae by Baermann fecal examination, however, fecal diagnosis can lack sensitivity due to intermittent shedding of larvae. In this study we developed serological tests to detect French Heartworm similar to those used for the diagnosis of North American Heartworm (Dirofilaria immitis) infection. An initial attempt to use an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to the parasite was confounded by significant cross-reactivity to antigens of Crenosoma vulpis, which is a metastrongylid nematode that is also common in Atlantic Canada. We therefore developed a sandwich ELISA to detect circulating antigens of A. vasorum using rabbit polyclonal antiserum prepared against whole-worm adult antigen. A test positive cut-off value and test sensitivity and specificity were determined by two-graph receiver operating characteristic curve (ROC) analysis using sera from 24 Baermann positive A. vasorum dogs from Newfoundland and sera from 52 Baermann negative A. vasorum dogs from non endemic areas. An optical density of 0.19 was used as the positive cutoff and the test specificity was 100% with a sensitivity of 92%. Furthermore, sera from 30 Crenosoma vulpis positive dogs were tested and found to be negative on the sandwich ELISA. A survey of dogs from Newfoundland with signs of cardiopulmonary disease compared Baermann fecal results with the sandwich ELISA and indicated that fecal testing may have missed almost half of the infections. To standardize the test, increase sensitivity, and develop a dependable source of diagnostic antigen(s), this study characterized some immunoreactive antigen(s) detected in the sandwich ELISA test. We developed a cDNA library from adult A. vasorum and after screening with rabbit anti-A. vasorum serum we identified 8 immunoreactive clones. Sequence analysis showed these clones to contain sequences homologous to 3 proteins, which shared homology to similar proteins in other parasites: vitellogenin, tropomyosin and heat shock protein 70 (Hsp70).

For production of specific monoclonal or polyclonal antibodies to be used in the sandwich ELISA test, it is necessary to produce a pure protein from the A. vasorum cDNA library. In the current study the most commonly occurring sequence encoded for a vitellogenin protein was chosen for expression and was inserted into a pGEX plasmid that was used to transform E. coli strain BL21. This vector incorporated glutathione S-transferase (GST) at the N-terminus and vitellogenin was synthesized as a fusion protein with GST. Western blotting with rabbit anti-A. vasorum polyclonal antiserum was able to identify the recombinant protein. This fusion protein had a molecular mass of 77 kDa which represented the combined mass of the GST (26 kDa) and the vitellogenin protein (51 kDa). The fusion protein was purified using GST affinity resin. Several modifications to the protocol for eluting the protein of interest from an affinity column had to be made including adjustments to pH, reduced glutathione buffer, salt concentration and Triton X-100 concentration.

In conclusion, a sandwich ELISA was developed and found to be more sensitive than the current Baermann test. Specific gene fragments from A. vasorum antigens were identified and sequenced. The most common sequence encoding for vitellogenin protein was purified. The generation of specific proteins should result in the development of antiserum that will enable higher test sensitivity than that obtained using crude A. vasorum somatic antigen. Furthermore, this will potentially increase sensitivity of the sandwich ELISA test and allow dependable production of reagents.