A RECOMBINANT PROTEIN BASED COMPETITIVE-INHIBITION ELISA FOR DETECTION OF NEOSPOROSIS IN CATTLE

NOVOA, MARÍA B.; VALENTINI, BEATRIZ S.; SARLI, MACARENA; PRIMO, MARÍA E.; ECHAIDE, IGNACIO E.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias

Infection with Neospora caninum, an apicomplexan protozoan parasite,causes significant economic losses in cattle worldwide due toabortions. The reference method for serodiagnosis of neosporosis isthe indirect fluorescent antibody test (IFAT), in spite of its complexityand subjectivity. The aim of this study was to develop and validatea competitive-inhibition ELISA (cELISA) based on major surface antigenprotein (SAG1) of N. caninum and the monoclonal antibody(mAb) P1C2D8F8, for detection of antibodies to N. caninum in cattle.A truncated variant of NcSAG1 was expressed as a recombinantprotein (rSAG1) in Escherichia coli. The mAb was generated byimmunizing mice with sonicated N. caninum tachyzoites. cELISAr-SAG1 was standardized under different physicochemical conditions.Sera (n=1085) from 264 dairy cows, with known status of neosporosisinfection were used as gold standard. Infected and uninfectedpopulation were defined as those with at least three consecutivepositive (n=385) or negative (n=700) results by IFAT and cELISAtach(in-house test based on lysed tachyzoites), over four years monitoring.The prevalence of neosporosis in 262 beef cows from 12 herdsof Santa Fe province was evaluated using the cELISArSAG1, and theresults compared with IFAT, cELISAtach and commercial cELISAVMRD.The efficiency of the cELISArSAG1 and agreement (kappa) with othertests was evaluated by Receiver Operating Characteristic analysis.The rSAG1protein had high yield, solubility and stability. cELISArSAG1 showed 97.1% sensitivity and 97.6% specificity with a cutoff point of28% of inhibition. The prevalence of neosporosis in beef cattle was36%. The agreement between cELISArSAG1 and IFAT or cELISAtachor cELISAVMRD was 92% (k= 0,820); 95% (k= 0.880); and 96% (k=0.905) respectively. The cELISArSAG1 is simple, fast, and could beused for large-scale epidemiological studies or disease-monitoringprograms of multiple animal species.