Advertisement

Parasitology Research

, Volume 115, Issue 2, pp 615–622 | Cite as

Screening and characterization of early diagnostic antigens in excretory–secretory proteins from Trichinella spiralis intestinal infective larvae by immunoproteomics

  • Ruo Dan Liu
  • Peng Jiang
  • Hui Wen
  • Jiang Yang Duan
  • Li Ang Wang
  • Jie Feng Li
  • Chun Ying Liu
  • Ge Ge Sun
  • Zhong Quan Wang
  • Jing Cui
Original Paper

Abstract

The excretory–secretory (ES) antigens from Trichinella spiralis muscle larvae are the most commonly used diagnostic antigens for trichinellosis, but specific IgG antibodies were not detected in early stage of infection. The aim of this study was to identify early diagnostic antigens from ES proteins of intestinal infective larvae (IIL), the first invasive stage of T. spiralis. Six bands (92, 52, 45, 35, 32, and 29 kDa) of IIL ES proteins were recognized by infection sera in Western blotting as early as 10 days post infection. Total of 54 T. spiralis proteins in six bands were identified by shotgun LC-MS/MS, 30 proteins were annotated, and 27 had hydrolase activity. Several proteins (serine protease, putative trypsin, deoxyribonuclease II family protein, etc.) could be considered as the potential early diagnostic antigens for trichinellosis. Our study provides new insights for screening early diagnostic antigens from intestinal worms of T. spiralis.

Keywords

Trichinella spiralis Intestinal infective L1 larvae (IIL1) Immunoproteomics Diagnostic antigens 

Notes

Acknowledgments

The authors wish to thank Shanghai Applied Protein Technology Co. Ltd. for the technology support. This work was supported by National Natural Science Foundation of China (nos. 81471981, 81271860).

Conflict of interest

All authors declare that they have no competing interests.

Supplementary material

436_2015_4779_MOESM1_ESM.doc (192 kb)
ESM 1 (DOC 191 kb)

References

  1. Bolas-Fernandez F, Dea-Ayuela MA, Connolly B, Robinson MW (2009) Micro-environmental conditions modulate protein secretion and infectivity of the Trichinella spiralis L1 larva. Vet Parasitol 159:236–239. doi: 10.1016/j.vetpar. 2008.10.049 CrossRefPubMedGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  3. Bruschi F, Tassi C, Pozio E (1990) Parasite-specific antibody response in Trichinella sp. 3 human infection: a one year follow-up. Am J Trop Med Hyg 43:186–193PubMedGoogle Scholar
  4. Campbell WC (1983) Trichinella and trichinosis. Plenum Press, New York, p 75CrossRefGoogle Scholar
  5. Conlan JV, Vongxay K, Khamlome B, Gomez-Morales MA, Pozio E, Blacksell SD et al (2014) Patterns and risks of Trichinella infection in humans and pigs in northern Laos. PLoS Negl Trop Dis 8:e3034. doi: 10.1371/journal.pntd.0003034 PubMedCentralCrossRefPubMedGoogle Scholar
  6. Cui J, Wang ZQ, Xu BL (2011) The epidemiology of human trichinellosis in China during 2004–2009. Acta Trop 118:1–5. doi: 10.1016/j.actatropica.2011.02.005 CrossRefPubMedGoogle Scholar
  7. Cui J, Jiang P, Liu LN, Wang ZQ (2013a) Survey of Trichinella infections in domestic pigs from northern and eastern Henan, China. Vet Parasitol 194:133–135. doi: 10.1016/j.vetpar.2013.01.038 CrossRefPubMedGoogle Scholar
  8. Cui J, Liu RD, Wang L, Zhang X, Jiang P, Liu MY, Wang ZQ (2013b) Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry. Parasite Vector 6:355. doi: 10.1186/1756-3305- 6–355 CrossRefGoogle Scholar
  9. Cui J, Li LG, Jiang P, Liu RD, Yang X, Liu LN, Liu P, Zhang SB, Wang ZQ (2015a) Biochemical and functional characterization of the glutathione S-transferase from Trichinella spiralis. Parasitol Res 114:2007–2013. doi: 10.1007/s00436-015-4410-6 CrossRefPubMedGoogle Scholar
  10. Cui J, Wang L, Sun GG, Liu LN, Zhang SB, Liu RD, Xi Z, Jiang P, Wang ZQ (2015b) Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis. Acta Trop 142:57–63. doi: 10.1016/j.actatropica.2014.10.017 CrossRefPubMedGoogle Scholar
  11. Dupouy-Camet J, Kociecka W, Bruschi F, Bolas-Fernandez F, Pozio E (2002) Opinion on the diagnosis and treatment of human trichinellosis. Expert Opin Pharmacother 3:1117–1130. doi: 10.1517/14656566.3.8.1117 CrossRefPubMedGoogle Scholar
  12. Gamble HR, Bessonov AS, Cuperlovic K, Gajadhar AA, van Knapen F, Noeckler K, Schenone H, Zhu X (2000) International commission on trichinellosis: recommendations on methods for the control of Trichinella in domestic and wild animals intended for human consumption. Vet Parasitol 93:393–408CrossRefPubMedGoogle Scholar
  13. Gamble HR, Pozio E, Bruschi F, Nockler K, Kapel CM, Gajadhar AA (2004) International commission on trichinellosis: recommendations on the use of serological tests for the detection of Trichinella infection in animals and man. Parasite 11:3–13CrossRefPubMedGoogle Scholar
  14. Gottstein B, Pozio E, Nockler K (2009) Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev 22:127–145. doi: 10.1128/CMR.00026-08 PubMedCentralCrossRefPubMedGoogle Scholar
  15. Kennedy ED, Hall RL, Montgomery SP, Pyburn DG, Jones JL (2009) Trichinellosis surveillance—United States, 2002–2007. MMWR Surveill Summ 58:1–7PubMedGoogle Scholar
  16. Lee HJ, Chung OS, Kim JL, Lee SH, Yoo YB, Seo M (2015) Seroprevalence of Trichinella sp. in wild boars (Sus scrofa) from Yanggu-gun, Gangwon-do, Korea. Korean J Parasitol 53:233–236. doi: 10.3347/kjp.2015.53.2.233 PubMedCentralCrossRefPubMedGoogle Scholar
  17. Li F, Cui J, Wang ZQ, Jiang P (2010) Sensitivity and optimization of artificial digestion in the inspection of meat for Trichinella spiralis. Foodborne Pathog Dis 7:879–885. doi: 10.1089/fpd.2009.0445 CrossRefPubMedGoogle Scholar
  18. Liu LN, Jing FJ, Cui J, Fu GY, Wang ZQ (2013a) Detection of circulating antigen in serum of mice infected with Trichinella spiralis by an IgY-IgM mAb sandwich ELISA. Exp Parasitol 133:150–155. doi: 10.1016/j.exppara.2012.11.001 CrossRefPubMedGoogle Scholar
  19. Liu RD, Wang ZQ, Wang L, Long SR, Ren HJ, Cui J (2013b) Analysis of differentially expressed genes of Trichinella spiralis larvae activated by bile and cultured with intestinal epithelial cells using real-time PCR. Parasitol Res 112:4113–4120. doi: 10.1007/s00436-013-3602-1 CrossRefPubMedGoogle Scholar
  20. Liu RD, Cui J, Wang L, Long SR, Zhang X, Liu MY, Wang ZQ (2014a) Identification of surface proteins of Trichinella spiralis muscle larvae using immunoproteomics. Trop Biomed 31:579–591PubMedGoogle Scholar
  21. Liu XD, Wang XL, Bai X, Liu XL, Wu XP, Zhao Y et al (2014b) Oral administration with attenuated salmonella encoding a Trichinella cystatin-like protein elicited host immunity. Exp Parasitol 141:1–11. doi: 10.1016/j.exppara.2014.03.015 CrossRefPubMedGoogle Scholar
  22. Liu LN, Zhang X, Jiang P, Liu RD, Zhou J, He RZ, Cui J, Wang ZQ (2015a) Serodiagnosis of sparganosis by ELISA using recombinant cysteine protease of Spirometra erinaceieuropaei spargana. Parasitol Res 114:753–757. doi: 10.1007/s00436-014- 4270–5 CrossRefPubMedGoogle Scholar
  23. Liu RD, Cui J, Liu XL, Jiang P, Sun GG, Zhang X, Long SR, Wang L, Wang ZQ (2015b) Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae. Acta Trop 150:79–86. doi: 10.1016/j.actatropica.2015.07.002 CrossRefPubMedGoogle Scholar
  24. Lv Z, Wu Z, Zhang L, Ji P, Cai Y, Luo S, Wang H, Li H (2015) Genome mining offers a new starting point for parasitology research. Parasitol Res 114:399–409. doi: 10.1007/ s00436-014-4299-5 CrossRefPubMedGoogle Scholar
  25. ManWarren T, Gagliardo L, Geyer J, McVay C, Pearce-Kelling S, Appleton J (1997) Invasion of intestinal epithelia in vitro by the parasitic nematode Trichinella spiralis. Infect Immun 65:4806–4812PubMedCentralPubMedGoogle Scholar
  26. Mitreva M, Jasmer DP, Zarlenga DS, Wang Z, Abubucker S, Martin J et al (2011) The draft genome of the parasitic nematode Trichinella spiralis. Nat Genet 43:228–235. doi: 10.1038/ng.769 PubMedCentralCrossRefPubMedGoogle Scholar
  27. Murrell KD, Pozio E (2011) Worldwide occurrence and impact of human trichinellosis, 1986–2009. Emerg Infect Dis 17:2194–2202. doi: 10.3201/eid1712.110896 PubMedCentralCrossRefPubMedGoogle Scholar
  28. Nagano I, Wu Z, Takahashi Y (2008) Species-specific antibody responses to the recombinant 53-kilodalton excretory and secretory proteins in mice infected with Trichinella spp. Clin Vaccine Immunol 15:468–473. doi: 10.1128/CVI.00467-07 PubMedCentralCrossRefPubMedGoogle Scholar
  29. Nagano I, Wu Z, Takahashi Y (2009) Functional genes and proteins of Trichinella spp. Parasitol Res 104:197–207. doi: 10.1007/s00436-008-1248-1 CrossRefPubMedGoogle Scholar
  30. 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 163:341–347. doi: 10.1016/j.vetpar.2009.04.034 CrossRefPubMedGoogle Scholar
  31. Ren HJ, Cui J, Wang ZQ, Liu RD (2011) Normal mouse intestinal epithelial cells as a model for the in vitro invasion of Trichinella spiralis infective larvae. Plos One 6:e27010. doi: 10.1371/journal.pone.0027010 PubMedCentralCrossRefPubMedGoogle Scholar
  32. Ren HJ, Cui J, Yang W, Liu RD, Wang ZQ (2013) Identification of differentially expressed genes of Trichinella spiralis larvae after exposure to host intestine milieu. Plos One 8:e67570. doi: 10.1371/journal.pone.0067570 PubMedCentralCrossRefPubMedGoogle Scholar
  33. Robinson MW, Connolly B (2005) Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle. Proteomics 5:4525–4532. doi: 10.1002/pmic.200402057 CrossRefPubMedGoogle Scholar
  34. Robinson MW, Massie DH, Connolly B (2007) Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis. Mol Biochem Parasitol 151:9–17. doi: 10.1016/j.molbiopara.2006.09.008 CrossRefPubMedGoogle Scholar
  35. Sun GG, Liu RD, Wang ZQ, Jiang P, Wang L, Liu XL, Liu CY, Zhang X, Cui J (2015) New diagnostic antigens for early trichinellosis: the excretory–secretory antigens of Trichinella spiralis intestinal infective larvae. Parasitol Res. doi: 10.1007/s00436- 015-4709-3 Google Scholar
  36. Tang B, Liu M, Wang L, Yu S, Shi H, Boireau P, Cozma V, Wu X, Liu X (2015) Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage. Parasite Vector 8:78. doi: 10.1186/s13071-015-0689-5 CrossRefGoogle Scholar
  37. Van De N, Thi Nga V, Dorny P, Vu Trung N, Ngoc Minh P, Trung Dung D, Pozio E (2015) Trichinellosis in Vietnam. Am J Trop Med Hyg 92:1265–1270. doi: 10.4269/ajtmh.14–0570 CrossRefPubMedGoogle Scholar
  38. Wang ZQ, Cui J, Wu F, Mao FR, Jin XX (1998) Epidemiological, clinical and serological studies on trichinellosis in Henan province, China. Acta Trop 71:255–268CrossRefPubMedGoogle Scholar
  39. Wang SW, Wang ZQ, Cui J (2011) Protein change of intestinal epithelial cells induced in vitro by Trichinella spiralis infective larvae. Parasitol Res 108:593–599. doi: 10.1007/s00436- 010-2102-9 CrossRefPubMedGoogle Scholar
  40. Wang ZQ, Wang L, Cui J (2012) Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach. J Proteome 75:2375–2383. doi: 10.1016/j.jprot.2012.02.005 CrossRefGoogle Scholar
  41. Wang B, Wang ZQ, Jin J, Ren HJ, Liu LN, Cui J (2013a) Cloning, expression and characterization of a Trichinella spiralis serine protease gene encoding a 35.5 kDa protein. Exp Parasitol 134:148–154. doi: 10.1016/j.exppara.2013.03.004 CrossRefPubMedGoogle Scholar
  42. Wang L, Wang ZQ, Cui J (2013b) Proteomic analysis of the changed proteins of Trichinella spiralis infective larvae after co-culture in vitro with intestinal epithelial cells. Vet Parasitol 194:160–163. doi: 10.1016/j.vetpar.2013.01.045 CrossRefPubMedGoogle Scholar
  43. Wang L, Wang ZQ, Hu DD, Cui J (2013c) Proteomic analysis of Trichinella spiralis muscle larval excretory-secretory proteins recognized by early infection sera. BioMed Res Int 2013:139745. doi: 10.1155/2013/139745 PubMedCentralPubMedGoogle Scholar
  44. Wang L, Cui J, Hu DD, Liu RD, Wang ZQ (2014) Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics. Parasite Vector 7:40. doi: 10.1186/1756-3305-7-40 CrossRefGoogle Scholar
  45. Wang ZQ, Zhang SB, Jiang P, Liu RD, Long SR, Zhang X, Ren HJ, Cui J (2015) The siRNA-mediated silencing of Trichinella spiralis nudix hydrolase results in reduction of larval infectivity. Parasitol Res 114:3551–3557. doi: 10.1007/s00436-015-4650-5 CrossRefPubMedGoogle Scholar
  46. Washburn MP, Wolters D, Yates JR 3rd (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247. doi: 10.1038/85686 CrossRefPubMedGoogle Scholar
  47. Wilson NO, Hall RL, Montgomery SP, Jones JL (2015) Trichinellosis surveillance—United States, 2008–2012. MMWR Surveill Summ 64(Suppl 1):1–8PubMedGoogle Scholar
  48. Wu XP, Fu BQ, Wang XL, Yu L, Yu SY, Deng HK et al (2009) Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries. Vet Parasitol 159:272–275. doi: 10.1016/j.vetpar.2008.10.035 CrossRefPubMedGoogle Scholar
  49. Yang J, Pan W, Sun X, Zhao X, Yuan G, Sun Q, Huang J, Zhu X (2015) Immunoproteomic profile of Trichinella spiralis adult worm proteins recognized by early infection sera. Parasite Vector 8:20. doi: 10.1186/s13071-015-0641-8 CrossRefGoogle Scholar
  50. Zarlenga DS, Gamble HR (1995) Molecular cloning and expression of an immunodominant 53-kDa excretory-secretory antigen from Trichinella spiralis muscle larvae. Mol Biochem Parasitol 72:253CrossRefPubMedGoogle Scholar
  51. Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848. doi: 10.1093/bioinformatics/17.9.847 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ruo Dan Liu
    • 1
  • Peng Jiang
    • 1
  • Hui Wen
    • 1
  • Jiang Yang Duan
    • 1
  • Li Ang Wang
    • 1
  • Jie Feng Li
    • 1
  • Chun Ying Liu
    • 1
  • Ge Ge Sun
    • 1
  • Zhong Quan Wang
    • 1
  • Jing Cui
    • 1
  1. 1.Department of Parasitology, Medical CollegeZhengzhou UniversityZhengzhouPeople’s Republic of China

Personalised recommendations