CLONING AND CHARACTERIZATION OF 2 RECOMBINANT NEOSPORA PROTEIN-FRAGMENTS AND THEIR USE IN SERODIAGNOSIS OF BOVINE NEOSPOROSIS

Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle, For the serodiagnosis of this parasitic disease, tw o immunodominant clones from a bovine Neospora lambda gt11 library wer e identified, characterized, and expressed as recombinant proteins for the development of an enzyme linked immunosorbent assay (ELISA), Thes e two clones, designated N54 and N57, were 29 and 20 kDa, respectively , when expressed as histidine fusion proteins from the pRSET expressio n vector, Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecular masses of 97, 8 7, 77, 67, and 64 kDa, Antibodies to recombinant protein N57 recognize d four primary bands with molecular masses of 34, 31, 30, and 28 kDa, When a defined ''gold standard'' panel of bovine sera from confirmed N eospora-positive and Neospora-negative cattle Here characterized by im munoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet, Binding to th e N57 quadruplet was more variable, The same gold standard panel was u sed to evaluate and compare an N54-based ELISA. an N57-based ELISA, an d a whole-tachyzoite lysate-based ELISA. The sensitivities and specifi cities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA), Thus, compared to the whole-tachyzoite lysate-bas ed ELISA, both recombinant-protein-based ELISAs had higher sensitiviti es and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neospor osis.