| Toxoplasmosis is a worldwide parasitic zoonosis caused by Toxoplasma gondii. It is hazardous to the health of human, especially for the pregnant and the immunity deficient patients. Meanwhile, it causes huge economic losses to the livestock industry around the world. Domestic scattered pattern of poultry has an obvious problem that relatively high portion of free ranged chickens are infected,30% on average by T. gondii. Human can be infected if he or she eat the undercooked chicken meat which is contaminated by T. gondii and/or just contact an infected bird. Thus Toxoplasma gondii infection in chickens has an important public health and the prevention remains one of the hot topics.Currently, the major tests of diagnosing toxoplasmosis include etiological diagnosis, immunological and nucleic acid detection. ELISA has many advantages, like high sensitivity, specificity and reproducibility. Moreover, less sophisticated facilities are required. However, available commercial ELISA preparations toxoplasmosis diagnosis in poultry has many setbacks, including high rate of false-positive cases, lack of specificity. Recently, many reports about pigs, cattle, sheep infected by the animal toxoplasma have come out along with the solution of advanced antibody testing skills, however, it rarely has something fresh about the investigation of chicken toxoplasma infection.In our laboratory, we cloned the open reading frame (ORF) of GRA8, after validating with Western blot and ELISA, this protein showed valuable features suitable for diagnosing chicken toxoplasmosis, but when the GRA8 gene was sub cloned into the pET plasmid system, the protein expression was too low.In this study GRA8 gene was analyzed using bioinformatics softwares (TMHMM, SignalP4.1 and DNAstar), to predict the signal peptide and hydrophobic region, to help building the truncated GRA8 prokaryotic expression vector without signal peptide and hydrophobic region. The recombinant protein antigenicity has been proved by western blot. After expression and purification, the recombinant protein was used to coated ELISA plates, through optimizing the antigen concentration. After adjustments in the antigen concentration, titers of first and second antibodies, blocking buffer, substrate buffer and other related conditions, the working concentration of sGRA8 was optimized as 1.7μg/ml, the best positive and negative serum dilution was 1:10, second antibody dilution was 1:6000, the best coating condition of sGRA8 was at 37℃ for 1h and overnight at 4℃, skimmed milk was chosen as a blocking buffer at the concentration of 5%, the best blocking conditions were at 37℃ for 1h, incubation with the first antibody was at 37℃ for 1h, and similarly was the HRP conjugated second antibody, while color development using the chromogen was for 10mins. The comparison result shows that the indirect ELISA has a compliance rate of 98.26% with the RB kits by using serum of 7 to 45 days after infection,but the serum of 60 to 130 days could’t detected by using RB kits.Since the aim of the study was to build a new indirect ELISA antibody test method, testing ELISA based on this antigen revealed that, this protein has a sensitivity rate of 80% to 100% detecting toxoplasmosis in artificially infected chicken, using serum of 7 to 130 days after infection. To evaluate any possibilities of cross-reactivity with other pathogens common in poultry, sera from chicken infected with; 2 viral infections (Newcastle disease and IBDV),7 Eimeria species and E. coli, were collected. sGRA8 showed no reactivity to all these sera, an indication that this protein is specific to T. gondii. This finding confirmed that this protein has high sensitivity, specificity detecting chicken toxoplasmosis. |
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