ABSTRACT
An enzyme-linked immunosorbent assay (ELISA) based on a recombinant Theileria uilenbergi immunodominant protein (rTuIP) was validated for detection of antibodies in 188 positive and 198 negative reference serum samples, respectively. The cut-off value was determined at 32.7% with 95% and 90% accuracy levels by two-graphic receiver-operating characteristic (TG-ROC). The equal diagnostic sensitivity (Se) and specificity (Sp) were calculated to be 98.4%. Further validation of the repeatability with positive and negative reference samples indicated the reliable performance of the assay. Monitoring the antibody dynamics of sheep experimentally infected with Theileria luwenshuni showed the efficient detection of antibody response against the pathogen at the early infection stage and up until two months post infection. Application of this assay for detection of antibody in field sera from previous unknown Theileria endemic regions in Suizhou and Guiyang showed 17.8% and 11.6% seroprevalence, respectively, and presence of the pathogen was confirmed by identification of the 18S rRNA gene in the corresponding blood of the seropositive animals. These data support that the rTuIP ELISA could be a useful tool to study the epidemiology of theileriosis caused by T. uilenbergi and/or T. luwenshuni.
Subject(s)
Antibodies, Protozoan/blood , Protozoan Proteins/immunology , Sheep Diseases/epidemiology , Theileria/immunology , Theileriasis/epidemiology , Animals , China/epidemiology , Enzyme-Linked Immunosorbent Assay/veterinary , Recombinant Proteins , Reproducibility of Results , Ruminants , Sensitivity and Specificity , Seroepidemiologic Studies , Sheep , Sheep Diseases/immunology , Sheep Diseases/parasitology , Theileria/genetics , Theileriasis/immunology , Theileriasis/parasitologyABSTRACT
Isolation and characterization of the Theileria annulata macroshizont stage protein TaSP showed that this parasite surface membrane protein is a highly antigenic protein suitable for the development of diagnostic tools for tropical theileriosis. An enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antibodies against Theileria annulata was established and validated using the recombinantly expressed TaSP protein. The ELISA has subsequently been applied for cross-sectional surveys to determine the distribution and prevalence of tropical theileriosis in Sudan.
Subject(s)
Protozoan Proteins/genetics , Theileria annulata , Theileriasis/diagnosis , Amino Acid Sequence , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/genetics , Antigens, Protozoan/metabolism , Antigens, Surface/genetics , Antigens, Surface/metabolism , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Genes, Protozoan , Intracellular Space/metabolism , Molecular Sequence Data , Protozoan Proteins/metabolism , Recombinant Proteins/biosynthesis , Sequence Alignment , Seroepidemiologic Studies , Sudan/epidemiology , Theileria annulata/genetics , Theileria annulata/immunology , Theileria annulata/metabolism , Theileriasis/blood , Theileriasis/epidemiologyABSTRACT
An ELISA based on a recombinant Theileria annulata surface protein (TaSP) was evaluated for detection of antibodies in sera from cattle exposed to tropical theileriosis in Sudan. The reference positive samples, used in this study, were from Theileria-infected populations and consisted of 80 cattle from an endemic area in Khartoum State, with high antibody titers in the indirect fluorescent antibody test (IFAT). The reference negative samples were taken from non-exposed populations and consisted of 120 cattle maintained under strict tick control at a commercial farm in Sudan. The cut-off value determined by Two-Graph Receiver-Operating Characteristic (TG-ROC) curves was set at 31.6%, based on the positive reference samples. Further diagnostic validation was performed, which consisted of the measurement of the area under the ROC (AUC) and by valid range proportion (VRP), which was 0.97 and 0.98 for the cut-off, respectively. There were no cross-reactions with antibodies raised against Babesia spp. It is concluded that the TaSP ELISA is a useful test for the diagnosis of T. annulata infection in cattle under field conditions.
