Screening Of Diagnostic Antigens For Toxoplasma Gondii Infection In Chicken

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii. Its worldwide distribution is hazardous to the health of human, especially for the pregnant and the immunity deficient patient. Relatively high portion of free ranged chickens in China are infected by T. gondii,26%on average. Infected chickens contain Toxoplasma gondii cysts in their tissues. If the cooked foods are contaminated by uncooked chicken or infected chickens are under-cooked, an infection of T. gondii is possible. In China, we consume a lot of free ranged chickens, so the detection of T. gondii infection of chickens is important to food security.The detection methods for T. gondii in animals include etiology methods, DNA sequence based methods and antibody based methods. As an antibody based detection method, ELISA has the advantage of easy sampling, low requirement of equipments, specific and sensitive. ELISA is a good way to detect T. gondii infection. However, right now, the chicken T. gondii antibody detection ELISA kit on the market has the disadvantage of relatively short effective detection period after chicken infection of T. gondii:after39days the IgM kit and after53days the IgG kit cannot distinguish the infected chicken with the healthy chicken. This is very possibly because of the inefficiency of antigen chosen by those kits. For some ELISA kits coated by crude natural Toxoplasma gondii protein, the specificity is low and the false positive rate is high. At the mean time, research of T. gondii antibody ELISA detection methods is mainly focused on human and stocks, avian research is relatively not as much especially for chickens.To prolong the detection period and enhance the specificity, this research used Western blot methods,2D-protein electrophoresis methods and MALDI-TOF-TOF-MS-MS method to screen for the efficient antigen suitable for chicken T. gondii antibody detection. GRA8is selected, expressed and proved efficient in ELISA. In the experiment,25chicken of1day’s age was infected with107tachyzoites and chicken serum was sampled from0days to139days after infection. Western blot was conducted with Toxoplasma gondii crude protein and serum samples collected.35KD and38KD antigen bands are detected, which showed strong positive result from7days to95days after infection. In the next step, proteins of T. gondii was separated by SDS-PAGE, proteins between molecular weight35KD and38KD are cut from the gel, electro-eluted and condensed in a dialysis bag. Then the purified protein sample was added into IPG Strip to perform IEF and2D protein electrophoreses. One set of gel is stained by Coomassie brilliant blue G-250and the other is examined by Western blot. Protein spots matched to the positive spots in western blot are cut from the stained gel and analyzed by MALDI-TOF-TOF-MS-MS and detected as Porin and GRA8.Design specific primers based on the mRNA sequence of Porin and GRA8published on NCBI. Use the cDNA of T. gondii as template, the ORF of Porin and GRA8was amplified by RT-PCR and cloned into pET-28a vector and transfected into E.coli strain BL21for protein expression. After His purification, relatively pure recombinant rPorin protein and rGRA8are collected. In the following Western blot test between recombinant protein and infected serum sample, rPorin didn’t show positive result and rGRA8showed high positive results. In the following ELISA test, recombinant proteins are used as antigens and serum sample are used as first antibody. rPorin didn’t show significant difference between positive and negative results and rGRA8showed significant difference between them.After adjustment in the antigen amount, concentration of first and second antibody, ELISA diagnostic method proved to have a100%sensitivity in detecting experimentally infected chicken in chicken-anti-Toroploswa gondii antibody in7-28days after infection,95%sensitivity in45days,90%in63days and85%in139days. In specificity, recombinant rGRA8protein didn’t show cross reaction with other major chicken diseases:coccidiosis (Eimeria tenella, E.necatrix, E.acervulina, E.maxima, E.branetti, E.mitis, E.praecox), chicken colibacillosis, bird flu(type H5and type H9) or new castle disease. This rGRA8-based ELISA method has a high sensitivity and specificity in chicken-anti-Toxoplasma gondii antibody detection. GRA8is a highly efficient diagnostic antigen. Its application is good news for the investigation and control of the potential danger chicken Toxoplasmosis brought to human health.