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Evaluation of Toxoplasma gondii-recombinant dense granular protein (GRA2) for serodiagnosis by western blot

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Abstract

Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the food industry. Commonly used serological tests involve preparation of whole Toxoplasma lysate antigens from tachyzoites which are costly and hazardous. An alternative method for better antigen production involving the prokaryotic expression system was therefore used in this study. Recombinant dense granular protein, GRA2, was successfully cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of this purified antigen for diagnosis of human infections was evaluated through western blot analysis against 100 human serum samples. Results showed that the rGRA2 protein has 100 and 61.5 % sensitivity towards acute and chronic infection, respectively, in T. gondii-infected humans, indicating that this protein is useful in differentiating present and past infections. Therefore, it is suitable to be used as a sensitive and specific molecular marker for the serodiagnosis of Toxoplasma infection in both humans and animals.

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References

  • Ahn HJ, Kim S, Nam HW (2005) Host cell binding of GRA10, a novel, constitutively secreted dense granular protein from Toxoplasma gondii. Biochem Biophys Res Commun 331(2):614–620. doi:10.1016/j.bbrc.2005.03.218

    Article  PubMed  CAS  Google Scholar 

  • Anderson T, DeJardin A, Howe DK, Dubey JP, Michalski ML (2007) Neospora caninum antibodies detected in Midwestern white-tailed deer (Odocoileus virginianus) by Western blot and ELISA. Vet Parasitol 145(1–2):152–155. doi:10.1016/j.vetpar.2006.11.012

    Article  PubMed  Google Scholar 

  • Aubert D, Maine GT, Villena I, Hunt JC, Howard L, Sheu M, Brojanac S, Chovan LE, Nowlan SF, Pinon JM (2000) Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J Clin Microbiol 38(3):1144–1150

    PubMed  CAS  Google Scholar 

  • Beghetto E, Spadoni A, Bruno L, Buffolano W, Gargano N (2006) Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. J Clin Microbiol 44(6):2133–2140. doi:10.1128/JCM.00237-06

    Article  PubMed  CAS  Google Scholar 

  • Cesbron-Delauw MF (1994) Dense-granule organelles of Toxoplasma gondii: their role in the host–parasite relationship. Parasitol Today 10(8):293–296, 0169-4758(94)90078-7 [pii]

    Article  PubMed  CAS  Google Scholar 

  • Gatkowska J, Hiszczynska-Sawicka E, Kur J, Holec L, Dlugonska H (2006) Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model. Exp Parasitol 114(3):220–227. doi:10.1016/j.exppara.2006.03.011

    Article  PubMed  CAS  Google Scholar 

  • Golkar M, Rafati S, Abdel-Latif MS, Brenier-Pinchart MP, Fricker-Hidalgo H, Sima BK, Babaie J, Pelloux H, Cesbron-Delauw MF, Mercier C (2007) The dense granule protein GRA2, a new marker for the serodiagnosis of acute Toxoplasma infection: comparison of sera collected in both France and Iran from pregnant women. Diagn Microbiol Infect Dis 58(4):419–426. doi:10.1016/j.diagmicrobio.2007.03.003

    Article  PubMed  CAS  Google Scholar 

  • Golkar M, Azadmanesh K, Khalili G, Khoshkholgh-Sima B, Babaie J, Mercier C, Brenier-Pinchart MP, Fricker-Hidalgo H, Pelloux H, Cesbron-Delauw MF (2008) Serodiagnosis of recently acquired Toxoplasma gondii infection in pregnant women using enzyme-linked immunosorbent assays with a recombinant dense granule GRA6 protein. Diagn Microbiol Infect Dis 61(1):31–39. doi:10.1016/j.diagmicrobio.2007.09.003

    Article  PubMed  CAS  Google Scholar 

  • Holec-Gasior L, Kur J (2010) Toxoplasma gondii: recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. Exp Parasitol 124(3):272–278. doi:10.1016/j.exppara.2009.10.010

    Article  PubMed  CAS  Google Scholar 

  • Holec-Gasior L, Kur J, Hiszczynska-Sawicka E (2009) GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in humans with acute toxoplasmosis. Clin Vaccine Immunol 16(4):510–514. doi:10.1128/CVI.00341-08

    Article  PubMed  CAS  Google Scholar 

  • Huang X, Xuan X, Hirata H, Yokoyama N, Xu L, Suzuki N, Igarashi I (2004) Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats. J Clin Microbiol 42(1):351–353

    Article  PubMed  CAS  Google Scholar 

  • Hughes HP, van Knapen F (1982) Characterisation of a secretory antigen from Toxoplasma gondii and its role in circulating antigen production. Int J Parasitol 12(5):433–437

    Article  PubMed  CAS  Google Scholar 

  • Igarashi M, Kano F, Tamekuni K, Machado RZ, Navarro IT, Vidotto O, Vidotto MC, Garcia JL (2008) Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice. Exp Parasitol 118(3):386–392. doi:10.1016/j.exppara.2007.10.002

    Article  PubMed  CAS  Google Scholar 

  • Ismael AB, Sekkai D, Collin C, Bout D, Mevelec MN (2003) The MIC3 gene of Toxoplasma gondii is a novel potent vaccine candidate against toxoplasmosis. Infect Immun 71(11):6222–6228

    Article  PubMed  CAS  Google Scholar 

  • Jacobs D, Vercammen M, Saman E (1999) Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain. Clin Diagn Lab Immunol 6(1):24–29

    PubMed  CAS  Google Scholar 

  • Jones JL, Kruszon-Moran D, Won K, Wilson M, Schantz PM (2008) Toxoplasma gondii and Toxocara spp. co-infection. AmJTrop Med Hyg 78(1):35–39, 78/1/35 [pii]

    Google Scholar 

  • Labruyere E, Lingnau M, Mercier C, Sibley LD (1999) Differential membrane targeting of the secretory proteins GRA4 and GRA6 within the parasitophorous vacuole formed by Toxoplasma gondii. Mol Biochem Parasitol 102(2):311–324, S0166-6851(99)00092-4 [pii]

    Article  PubMed  CAS  Google Scholar 

  • Li S, Maine G, Suzuki Y, Araujo FG, Galvan G, Remington JS, Parmley S (2000) Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen. J Clin Microbiol 38(1):179–184

    PubMed  CAS  Google Scholar 

  • Mercier C, Howe DK, Mordue D, Lingnau M, Sibley LD (1998) Targeted disruption of the GRA2 locus in Toxoplasma gondii decreases acute virulence in mice. Infect Immun 66(9):4176–4182

    PubMed  CAS  Google Scholar 

  • Mercier C, Adjogble KD, Daubener W, Delauw MF (2005) Dense granules: are they key organelles to help understand the parasitophorous vacuole of all Apicomplexa parasites? Int J Parasitol 35(8):829–849. doi:10.1016/j.ijpara.2005.03.011

    Article  PubMed  Google Scholar 

  • Mercier C, Cesbron-Delauw MF, Ferguson DJP (2007) Dense granules of the infectious stages of Toxoplasma gondii: their central role in the host/parasite relationship. In: Soldati D, Ajioka J (eds) Toxoplasma: molecular and cellular biology. Horizon Scientific Press, Norwich

    Google Scholar 

  • Michelin A, Bittame A, Bordat Y, Travier L, Mercier C, Dubremetz JF, Lebrun M (2009) GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network. Int J Parasitol 39(3):299–306. doi:10.1016/j.ijpara.2008.07.011

    Article  PubMed  CAS  Google Scholar 

  • Murray A, Mercier C, Decoster A, Lecordier L, Capron A, Cesbron-Delauw MF (1993) Multiple B-cell epitopes in a recombinant GRA2 secreted antigen of Toxoplasma gondii. Appl Parasitol 34(4):235–244

    PubMed  CAS  Google Scholar 

  • Nam HW (2009) GRA proteins of Toxoplasma gondii: maintenance of host–parasite interactions across the parasitophorous vacuolar membrane. Korean J Parasitol 47(Suppl):S29–37. doi:10.3347/kjp.2009.47.S.S29

    Article  PubMed  CAS  Google Scholar 

  • Nockler K, Reckinger S, Broglia A, Mayer-Scholl A, Bahn P (2009) Evaluation of a Western Blot and ELISA for the detection of anti-Trichinella-IgG in pig sera. Vet Parasitol 163(4):341–347. doi:10.1016/j.vetpar.2009.04.034

    Article  PubMed  CAS  Google Scholar 

  • Prigione I, Facchetti P, Lecordier L, Deslee D, Chiesa S, Cesbron-Delauw MF, Pistoia V (2000) T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: characterization of the fine antigenic specificity of the clones and implications for vaccine development. J Immunol 164(7):3741–3748, ji_v164n7p3741 [pii]

    PubMed  CAS  Google Scholar 

  • Redlich A, Muller WA (1998) Serodiagnosis of acute toxoplasmosis using a recombinant form of the dense granule antigen GRA6 in an enzyme-linked immunosorbent assay. Parasitol Res 84(9):700–706

    Article  PubMed  CAS  Google Scholar 

  • Rome ME, Beck JR, Turetzky JM, Webster P, Bradley PJ (2008) Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect Immun 76(11):4865–4875. doi:10.1128/IAI.00782-08

    Article  PubMed  CAS  Google Scholar 

  • Sadeghiani G, Zare M, Babaie J, Shokrgozar MA, Azadmanesh K, Fard-Esfahani P, Golkar M (2009) Heterologous production of dense granule GRA7 antigen of Toxoplasma gondii in Escherichia coli. Southeast Asian J Trop Med Public Health 40(4):692–700

    PubMed  CAS  Google Scholar 

  • Zhou H, Gu Q, Zhao Q, Zhang J, Cong H, Li Y, He S (2007) Toxoplasma gondii: expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris. Parasitol Res 100(4):829–835. doi:10.1007/s00436-006-0341-6

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

Serum samples were kindly provided by the Diagnostic Laboratory (Para: SEAD), Department of Parasitology, University of Malaya and University Malaya Medical Centre. Special thanks to the UMCPR for carrying out the MALDI-TOF MS analysis. We would also like to thank Claudia Nisha Anthony for proofreading this article. This project was supported by the University of Malaya under the Postgraduate Research Grant (PS197-2009C) and High Impact Research Fund (E000051-20001).

Ethical standards

The author(s) declared that the experiments comply with the current laws of the country in which they were performed (Malaysia).

Conflict of interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Authors and Affiliations

  1. Department of Parasitology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia

    Xiao Teng Ching, Yee Ling Lau, Mun Yik Fong & Veeranoot Nissapatorn

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  1. Xiao Teng Ching
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  2. Yee Ling Lau
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  3. Mun Yik Fong
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  4. Veeranoot Nissapatorn
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Correspondence to Yee Ling Lau.

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Ching, X.T., Lau, Y.L., Fong, M.Y. et al. Evaluation of Toxoplasma gondii-recombinant dense granular protein (GRA2) for serodiagnosis by western blot. Parasitol Res 112, 1229–1236 (2013). https://doi.org/10.1007/s00436-012-3255-5

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