Evaluation of recombinant granule antigens GRA1 and GRA7 for serodiagnosis of Toxoplasma gondii infection in dogs.

Xing-Quan Zhu,Jiping Li,Quan Liu,Wei Ge,Si-Yang Huang,Zedong Wang

doi:10.1186/1746-6148-10-158

Abstract

BackgroundToxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii, is an important zoonotic disease worldwide. The precise detection of T. gondii infection in dogs has important public health significance. In this study, recombinant granule antigen proteins GRA1 and GRA7 were evaluated as potential diagnostic markers for T. gondii infection in dogs by an indirect enzyme-linked immunosorbent assay (ELISA).ResultsGRA1 and GRA7 were cloned and expressed in Escherichia coli, and the recombinant GRA1, GRA7- and Toxoplasma lysate antigen (TLA)-based ELISAs were developed and evaluated using the canine positive and negative serum samples for anti-T. gondii antibodies determined by modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT), showing a seroprevalence of 15.1% by TLA- and GRA1-ELISA, and 15.8% by GRA7-ELISA, and no significant difference was observed (P > 0.05). When compared with the two reference assays, MAT and IFAT, the GRA7-ELISA showed the highest co-positivity and co-negativity rates. Receiver operating characteristic (ROC) analysis revealed a largest area under curve (AUC) of 0.973 (95% CI, 0.955 to 0.991), and a highest relative sensitivity (93.2%) and specificity (94.0%) for a cut-off value of 0.809 in GRA7-ELISA.ConclusionsThe results of the present study showed that GRA7-ELISA is highly sensitive and specific, and GRA7 is a potential serodiagnostic marker for the detection of T. gondii infection in dogs.

Highlights

Toxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii, is an important zoonotic disease worldwide
Ethics statement The pet dogs from which blood were collected, were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China
The resulting gene products were cloned into the EcoR1/NcoI site of pET-28a to generate a recombinant plasmids pET28-GRA1 and pET28-GRA7, which were confirmed by restriction enzymes and sequencing, and further processed for the expression of recombinant products in Escherichia coli BL21 (DE3) according to the standard techniques

Summary

Introduction

Toxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii, is an important zoonotic disease worldwide. Dogs are potentially involved in the mechanical transmission of T. gondii oocysts to humans, and Definitive diagnosis of T. gondii infection by mouse inoculation, or immunohistochemical analyses is optimal. These tests are time-consuming, involved in experimental animals, and may have a low sensitivity [8]. Multiple tests, such as indirect haemagglutination (IHA), modified agglutination test (MAT), latex agglutination test (LAT), indirect fluorescent antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA), are useful to demonstrate T. gondii infection in humans and animals. Most conventional tests using tachyzoites grown in mice or in tissue culture are usually difficult to standardize, making the test results difficult to evaluate [9]

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Results

Conclusion