title: Detection of antibodies to Toxoplasma gondii in domesticated ruminants by recombinant truncated SAG2 enzyme-linked immunosorbent assay.
abstract: An antibody detection recombinant enzyme-linked immunosorbent assay (ELISA) specific for Toxoplasma gondii was laboratory standardized using recombinant truncated surface antigen 2 (SAG2) protein of T. gondii. A 483-bp sequence coding for truncated tachyzoite stage-specific SAG2 protein was amplified and ligated in pPROExHT-b expression vector to transform Escherichia coli DH5α cells. A high-level expression of the histidine-tagged fusion protein was obtained after 8 h of incubation. The recombinant protein was affinity purified using Ni-NTA agarose column and characterized by SDS-PAGE and Western blot analysis. Subsequently, the diagnostic potential of the recombinant protein was assessed with 168 field sera samples from sheep, goats and cattle. Among the small ruminants, 50% (n = 60) sheep sera samples and 41.26% (n = 63) goat samples were detected positive for T. gondii-specific antibodies. As far as seroprevalence of toxoplasmosis in cattle is concerned, 64.44% (n = 45) of sera samples assayed were found to be positive. When compared to indirect fluorescent antibody test (IFAT), the sensitivity of the recombinant truncated SAG2 antigen-based ELISA (rec-SAG2-ELISA) ranged from 81.25 to 87.10% while the specificity was 85.71 to 91.43% with substantial agreement between the tests.
authors.affiliation: Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243122, India, drharkiratsingh@hotmail.com.
authors.forename: Harkirat
authors.initials: H
authors.lastname: Singh
authors.forename: Anup Kumar
authors.initials: AK
authors.lastname: Tewari
authors.forename: Ashok Kumar
authors.initials: AK
authors.lastname: Mishra
authors.forename: Biswaranjan
authors.initials: B
authors.lastname: Maharana
authors.forename: Vikrant
authors.initials: V
authors.lastname: Sudan
authors.forename: Opinder Krishan
authors.initials: OK
authors.lastname: Raina
authors.forename: Jammi Raghavendra
authors.initials: JR
authors.lastname: Rao
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000276:immunology*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D006041:Goats / Q000276:immunology; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D012418:Ruminants / Q000276:immunology*; D012680:Sensitivity and Specificity; D016036:Seroepidemiologic Studies; D012756:Sheep / Q000276:immunology; D014122:Toxoplasma / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C067547:surface antigen P22, Toxoplasma