Toxoplasmosis is an important zoonotic disease caused by Toxoplasma gondii that can infect almost all warm-blooded animals worldwide, including humans. The high prevalence of T. gondii infection and its ability to cause serious harm to humans and animals, especially immunodeficient individuals, make it a key public health issue. Accurate diagnostic tools with high sensitivity are needed for controlling T. gondii infection. In the current study, we compared the performance of recombinant SAG2, GRA6, and GRA7 in ELISA for the serological diagnosis of T. gondii infection in cats. We further investigated the antigenicity of recombinant dense granule protein 3 (rGRA3), rGRA5, rGRA8, and rSRS29A expressed in a plant-based, cell-free expression system for detecting antibodies in T. gondii-infected cats. In summary, our data suggest that GRA7 is more sensitive than the other two antigens for the serodiagnosis of T. gondii infection in cats, and GRA3 expressed in the cell-free system is also a priming antigen in serological tests for detecting T. gondii infection in cats.

Toxoplasma gondii (T. gondii) is an obligatory intracellular parasite that can infect varieties of warm-blooded animals, including humans and birds. Heparan sulfate (HS) is widely distributed on the eukaryotic cell surface of vertebrates and can inhibit T. gondii invasion. In this study, we investigated the transcription and expression of the level of TgROP9, TgMIC3, and TgSAG2 in T. gondii RH strain, and found that the expression levels of these three proteins in invading parasites were higher compared to those free ranging parasites. The recombinant proteins showed specific binding activity to both heparin and host cell surface. Incubation of these proteins with the host cells could block T. gondiiinvasion. Furthermore, protein-specific antibodies also blocked parasite invasion. Antibodies in the sera of T. gondii infected individuals recognized the recombinant TgROP9, TgMIC3, and TgSAG2, which suggested the exposure of these proteins to human immune system. Mice immunized with the three proteins exhibited protective immunity against lethal challenge. The data collectively suggested that these parasitic proteins may be used as candidate antigens for development of anti-toxoplasmosis vaccine.

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5\' and 3\' ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.