Abstract
Toxoplasma gondii is a zoonotic parasite that is very common in livestock. Meat products from livestock infected with T. gondii are one of the important transmission routes of toxoplasmosis. Rapid and reliable diagnosis is a prerequisite for the prevention and control of toxoplasmosis. Neospora caninum and T. gondii are similar in morphology and life history, and there are a large number of cross antigens between them, making clinical diagnosis of toxoplasmosis more difficult. In this study, immunoprecipitation-mass spectrometry (IP-MS) was used to screen for T. gondii–specific antigens, and the specific antigen was cloned and expressed in Escherichia coli. The specific antigen was then used to establish an indirect ELISA diagnostic method. A total of 241 specific antigens of T. gondii and 662 cross antigens between T. gondii and N. caninum were screened by IP-MS. Through bioinformatics analysis and homology comparison, seven proteins were selected for gene cloning and prokaryotic expression, and the most suitable antigen, TgGRA54, was selected to establish an indirect ELISA for T. gondii. Compared with the indirect immunofluorescent antibody test (IFAT), the positive coincidence rate of the ELISA based on rTgGRA54 was 100% (72/72) and the negative coincidence rate was 80.95% (17/21). The indirect ELISA method based on TgGRA54 recombinant protein was established to detect T. gondii antibodies in bovine sera, and the recombinant protein reacted well with T. gondii positive sera from sheep, mouse, and swine, indicating that the recombinant protein is a good diagnostic antigen for T. gondii.
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Data supporting the conclusions of this article are included within the article and its additional files.
Abbreviations
- GRAs :
-
dense granule proteins
- MICs :
-
micronemes
- DMEM :
-
Dulbecco’s modified Eagle’s medium
- HFF :
-
human foreskin fibroblast
- BSA :
-
bovine serum albumin
- DMSO :
-
dimethyl sulfoxide
- CRISPR/Cas9 :
-
clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9
- KEGG :
-
Kyoto Encyclopedia of Genes and Genomes
- GO :
-
Gene Ontology
- IP-MS :
-
Immunoprecipitation-Mass Spectrometry
- WB :
-
western blot
References
Baril L, Ancelle T, Goulet V, Thulliez P, Tirard-Fleury V, Carme B (1999) Risk factors for Toxoplasma infection in pregnancy: a case-control study in France. Scand J Infect Dis 31(3):305–309. https://doi.org/10.1080/00365549950163626
Chang PY, Fong MY, Nissapatorn V, Lau YL (2011) Evaluation of Pichia pastoris-expressed recombinant rhoptry protein 2 of Toxoplasma gondii for its application in diagnosis of toxoplasmosis. Am J Trop Med Hyg 85(3):485–489. https://doi.org/10.4269/ajtmh.2011.11-0351
Cook AJ, Gilbert RE, Buffolano W, Zufferey J, Petersen E, Jenum PA, Foulon W, Semprini AE, Dunn DT (2000) Sources of toxoplasma infection in pregnant women: European multicentre case-control study: European multicentre case-control studyCommentary: Congenital toxoplasmosis—further thought for food. Bmj 321(7254):142–147. https://doi.org/10.1136/bmj.321.7254.142
Cygan AM, Theisen TC, Mendoza AG, Marino ND, Panas MW, Boothroyd JC (2020) Coimmunoprecipitation with MYR1 identifies three additional proteins within the Toxoplasma gondii parasitophorous vacuole required for translocation of dense granule effectors into host cells. mSphere 5(1):e00858–e00819. https://doi.org/10.1128/mSphere.00858-19
Dubey JP, Lindsay DS, Speer CA (1998) Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin Microbiol Rev 11(2):267–299. https://doi.org/10.1128/CMR.11.2.267
Dubey JP (2004) Toxoplasmosis - a waterborne zoonosis. Vet Parasitol 126(1-2):57–72. https://doi.org/10.1016/j.vetpar.2004.09.005
Dubey JP (2009) History of the discovery of the life cycle of Toxoplasma gondii. Int J Parasitol 39(8):877–882. https://doi.org/10.1016/j.ijpara.2009.01.005
Holec-Gąsior L, Ferra B, Hiszczyńska-Sawicka E, Kur J (2014) The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis. Vet Parasitol 206(3-4):146–152. https://doi.org/10.1016/j.vetpar.2014.09.01
Kotresha D, Noordin R (2010) Recombinant proteins in the diagnosis of toxoplasmosis. Apmis 118(8):529–542. https://doi.org/10.1111/j.1600-0463.2010.02629
Lau YL, Fong MY, Idris MM, Ching XT (2012) Cloning and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris. Southeast Asian J Trop Med Public Health 43(1):10–16
Liao M, Xuan X, Huang X, Shirafuji H, Fukumoto S, Hirata H, Suzuki H, Fujisaki K (2005) Identification and characterization of cross-reactive antigens from Neospora caninum and Toxoplasma gondii. Parasitology 130(Pt 5):481–488. https://doi.org/10.1017/s0031182004006948
Liao M, Ma L, Bannai H, Lee EG, Xie Z, Tang X, Zhang H, Xuan X, Fujisaki K (2006) Identification of a protein disulfide isomerase of Neospora caninum in excretory-secretory products and its IgA binding and enzymatic activities. Vet Parasitol 139(1-3):47–56. https://doi.org/10.1016/j.vetpar.2006.02.029
Nishikawa Y, Claveria FG, Fujisaki K, Nagasawa H (2002) Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. J Vet Med Sci 64(2):161–164. https://doi.org/10.1292/jvms.64.161
Qi T, Ai J, Sun Y, Ma H, Kang M, You X, Li J (2022) Application of Toxoplasma gondii-specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis. Front Cell Infect Microbiol 12:1029768. https://doi.org/10.3389/fcimb.2022.102976
Reid AJ, Vermont SJ, Cotton JA, Harris D, Hill-Cawthorne GA, Könen-Waisman S, Latham SM, Mourier T, Norton R, Quail MA, Sanders M, Shanmugam D, Sohal A, Wasmuth JD, Brunk B, Grigg ME, Howard JC, Parkinson J, Roos DS et al (2012) Comparative genomics of the apicomplexan parasites Toxoplasma gondii and Neospora caninum: Coccidia differing in host range and transmission strategy. PLoS Pathog 8(3):e1002567. https://doi.org/10.1371/journal.ppat.1002567
Robert-Gangneux F, Dardé ML (2012) Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 25(2):264–296. https://doi.org/10.1128/CMR.05013-11
Thiruvengadam G, Init I, Fong MY, Lau YL (2011) Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris. Trop Biomed 28(3):506–513
van der Puije WN, Bosompem KM, Canacoo EA, Wastling JM, Akanmori BD (2000) The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats. Acta Trop 76(1):21–26. https://doi.org/10.1016/s0001-706x(00)00084-x
Wang Z, Ge W, Huang SY, Li J, Zhu XQ, Liu Q (2014a) Evaluation of recombinant granule antigens GRA1 and GRA7 for serodiagnosis of Toxoplasma gondii infection in dogs. BMC Vet Res 10:158. https://doi.org/10.1186/1746-6148-10-158
Wang Z, Ge W, Li J, Song M, Sun H, Wei F, Liu Q (2014b) Production and evaluation of recombinant granule antigen protein GRA7 for serodiagnosis of Toxoplasma gondii infection in cattle. Foodborne Pathog Dis 11(9):734–739. https://doi.org/10.1089/fpd.2014.1749
Xue J, Jiang W, Chen Y, Liu Y, Zhang H, Xiao Y, Qiao Y, Huang K, Wang Q (2016) Twenty-six circulating antigens and two novel diagnostic candidate molecules identified in the serum of canines with experimental acute toxoplasmosis. Parasit Vectors 9(1):374. https://doi.org/10.1186/s13071-016-1643-x
Yang D, Liu J, Hao P, Wang J, Lei T, Shan D, Liu Q (2015) MIC3, a novel cross-protective antigen expressed in Toxoplasma gondii and Neospora caninum. Parasitol Res 114(10):3791–3799. https://doi.org/10.1007/s00436-015-4609-6
Zhang H, Compaore MK, Lee EG, Liao M, Zhang G, Sugimoto C, Fujisaki K, Nishikawa Y, Xuan X (2007) Apical membrane antigen 1 is a cross-reactive antigen between Neospora caninum and Toxoplasma gondii, and the anti-NcAMA1 antibody inhibits host cell invasion by both parasites. Mol Biochem Parasitol 151(2):205–212. https://doi.org/10.1016/j.molbiopara.2006.11.005
Zhang H, Lee EG, Yu L, Kawano S, Huang P, Liao M, Kawase O, Zhang G, Zhou J, Fujisaki K, Nishikawa Y, Xuan X (2011) Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach. Parasitol Res 109(3):899–911. https://doi.org/10.1007/s00436-011-2332-5
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This work was supported by funds of the National Key Research and Development Program of China (2022YFD1800200) and the Beijing Municipal Natural Science Foundation (6212016).
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J-L conceived the project. Z-XF and H-FL performed the experiments and drafted the manuscript. JL, QL, and L-FW participated in the design of the study and helped to draft the manuscript. Z-PS, X-MW, Y-QP, and Z-FZ participated in the interpretation of the data. All authors read and approved the final manuscript.
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The experiments were performed in strict according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. All experimental animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of China Agricultural University.
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Additional file 1
Table S1. The primer sequence of gene cloning. Additional file 2: Figure S1. Expression of recombinant protein. Additional file 3: Figure S2. The result of immunoprecipitation-mass spectrometry analysis. (DOCX 3583 kb)
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Feng, Z., Ling, H., Zhu, Z. et al. Identification of specific antigens between Toxoplasma gondii and Neospora caninum and application of potential diagnostic antigen TgGRA54. Parasitol Res 122, 2557–2566 (2023). https://doi.org/10.1007/s00436-023-07955-8
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DOI: https://doi.org/10.1007/s00436-023-07955-8