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United States Department of Agriculture

Agricultural Research Service

Title: Identification of Immunodominant Proteins of Neospora Caninum Nc-1 in 2-Dimensional Western Blots Using Serum from a Neospora Endemic Dairy Herd

Authors
item Hill, Dolores
item Scott, Alan - JOHNS HOPKINS, BALTO., MD
item Dyer, Robert - UNIV MARYLAND, COLLEGE PK
item Dubey, Jitender

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 15, 2001
Publication Date: N/A

Interpretive Summary: Neospora caninum is a protozoan parasite which has been shown to cause abortion in dairy and beef cattle, as well as neurological disorders in dogs and horses. Vertical transmission has been documented in cattle, though the fidelity of transmission from dam to offspring is variable (Davison et al., 1999; French et al., 1999; Schares et al., 1998; Dyer et al., 2000). The development of resistance to Neospora infection and transmission of N. caninum from dams to offspring may be affected by immunological recognition of specific N. caninum proteins by infected cattle. Major efforts in the field of parasite proteomics (Banks et al., 2000; Chambers et al., 2000; Gavaghan, 2000) have been undertaken in an attempt to relate genomic information to protein products which may have important roles in disease (Alaiya et al., 1999; Edgar et al., 2000), as well as development, transmission, and resistance to parasite pathogens of humans and livestock (Barrett et al., 2000; Gutierrez, 2000). In this study, we have used 2-dimensional gel electrophoresis, Western blotting, and N-terminal amino acid sequence information to identify specific proteins from N. caninum (NC-1) which are recognized as immunodominant by sera from N. caninum infected cattle.

Technical Abstract: Neospora caninum tachyzoites were isolated from in vitro cell culture and proteins were extracted using a chaotropic detergent solution. Two-dimensional electrophoresis was carried out on the solubilized proteins using immobilized pH gradient (3-10) strips in the first dimension and 4-12% gradient slab gels in the second dimension. Separated proteins were blotted onto nylon membranes and subjected to Western blot analysis using polyclonal antiserum from mature cows (positive at 1:1200 for N. caninum by IFA ) from a N. caninum endemic farm. Proteins identified in Western blots by antisera from infected cows were cut from identical blots run in parallel and were subjected to N-terminal amino acid sequence analysis for identification. Twenty immunodominant proteins were identified using sera from N. caninum infected cattle. Four of the proteins were identified as surface associated proteins (SAG-1, SRS-2, Nc-p35, and Nc-p43); 3 were identified as dense granule-associated proteins (NTPase, Nc-GRA-7, and Nc-GRA- 2), 4 were identified as actin, myosin, or tubulin chains, 2 were identified as microneme associated proteins (Nc-p38 and Nc- MIC-10), and 3 were identified as cytoplasmic proteins (HGPRT, Nc-SUL-1, 14-3-3 antigen). Two proteins were found which showed no homology with existing SwissPro entries, and may represent proteins which are unique to N. caninum

Last Modified: 10/1/2014