Development of a Serological Diagnostic assay for Elaeophora schneideri infection in moose (Alces alces)
Date of Award
Master of Science
Comparative and Experimental Medicine
Stephen Kania, Lisa Muller
Elaeophora schneideri is a nematode which inhabits the arteries of its domestic and free-ranging ungulate hosts. It is the etiological agent of elaeophorosis, a proven cause of morbidity and mortality in ungulates, including moose. Moose numbers are declining in several U.S. states and Canadian provinces, and recent research suggests that Elaeophora schneideri is a factor in these declines. At this time, the only conclusive method for diagnosing elaeophorosis requires finding nematodes or arterial lesions in recently deceased moose. The lack of a serological diagnostic test has made the prevalence, significance, and geographical distribution of this parasite difficult to assess. This research is an investigation of antigenic potential of E. schneideri proteins. Ultimately, these proteins will be utilized in developing an enzyme-linked immunosorbent assay (ELISA) to detect anti-Elaeophora antibodies in moose sera. Genome sequencing and transcriptome analysis were effective in identifying two genes encoding peptides that are predicted to be immunogenic.We prepared the synthetic genes encoding these peptides and used them to transform TOPO-brand E. coli cells (Invitrogen, Carlsbad, California). Despite successful transformation and culturing in LB Broth with 0.05 % [percent] ampicillin, attempts to isolate the polyhistidine-tagged proteins were unsuccessful. However, one western blot did result in distinct bands when probed with 6x anti-histidine monoclonal antibody. One of these bands was the approximate weight of the smaller of the two recombinant proteins (8.94 kDa); this band also appeared when the blot was probed with E. schneideri-positive moose serum, but did not appear when probed with E. schneideri-negative deer serum. This result was unable to be replicated, but may indicate this protein is a useful antigen. Future research will utilize sub-cloning procedures to insert PCR-amplified synthetic, optimized genes into pETBlue-2 cells. This should produce effective recombinant proteins which can be isolated using the his-tags on the proteins. These proteins will be able to be used in an ELISA to detect current or previous E. schneideri infection in moose.
Miller, Megan D., "Development of a Serological Diagnostic assay for Elaeophora schneideri infection in moose (Alces alces). " Master's Thesis, University of Tennessee, 2019.