Skip to main content

Advertisement

SpringerLink
Log in

Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Aguado-Martinez A, Alvarez-Garcia G, Fernandez-Garcia A, Risco-Castillo V, Arnaiz-Seco I, Rebordosa-Trigueros X, Navarro-Lozano V, Ortega-Mora LM (2008) Usefulness of rNcGRA7- and rNcSAG4-based ELISA tests for distinguishing primo-infection, recrudescence, and chronic bovine neosporosis. Vet Parasitol 157:182–195

    Article  PubMed  CAS  Google Scholar 

  • Aguado-Martinez A, Alvarez-Garcia G, Fernandez-Garcia A, Risco-Castillo V, Marugan-Hernandez V, Ortega-Mora LM (2009a) Failure of a vaccine using immunogenic recombinant proteins rNcSAG4 and rNcGRA7 against neosporosis in mice. Vaccine 27:7331–7338

    Article  PubMed  CAS  Google Scholar 

  • Aguado-Martinez A, Ortega-Mora LM, Alvarez-Garcia G, Rodriguez-Marco S, Risco-Castillo V, Marugan-Hernandez V, Fernandez-Garcia A (2009b) Stage-specific expression of Nc SAG4 as a marker of chronic Neospora caninum infection in a mouse model. Parasitology 136:757–764

    Article  PubMed  CAS  Google Scholar 

  • Anderson ML, Andrianarivo AG, Conrad PA (2000) Neosporosis in cattle. Anim Reprod Sci 60–61:417–431

    Article  PubMed  Google Scholar 

  • Andrianarivo AG, Choromanski L, McDonough SP, Packham AE, Conrad PA (1999) Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants. Int J Parasitol 29:1613–1625

    Article  PubMed  CAS  Google Scholar 

  • Andrianarivo AG, Barr BC, Anderson ML, Rowe JD, Packham AE, Sverlow KW, Conrad PA (2001) Immune responses in pregnant cattle and bovine fetuses following experimental infection with Neospora caninum. Parasitol Res 87:817–825

    Article  PubMed  CAS  Google Scholar 

  • Baszler TV, Long MT, McElwain TF, Mathison BA (1999) Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice. Int J Parasitol 29:1635–1646

    Article  PubMed  CAS  Google Scholar 

  • Baszler TV, McElwain TF, Mathison BA (2000) Immunization of BALB/c mice with killed Neospora caninum tachyzoite antigen induces a type 2 immune response and exacerbates encephalitis and neurological disease. Clin Diagn Lab Immunol 7:893–898

    PubMed  CAS  Google Scholar 

  • Bohne W, Gross U, Ferguson DJ, Heesemann J (1995) Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants. Mol Microbiol 16:1221–1230

    Article  PubMed  CAS  Google Scholar 

  • Debache K, Guionaud C, Alaeddine F, Hemphill A (2010) Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites. Parasitology 137:229–240

    Article  PubMed  CAS  Google Scholar 

  • Dubey JP (2003) Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 41:1–16

    Article  PubMed  CAS  Google Scholar 

  • Dubey JP, Barr BC, Barta JR, Bjerkas I, Bjorkman C, Blagburn BL, Bowman DD, Buxton D, Ellis JT, Gottstein B, Hemphill A, Hill DE, Howe DK, Jenkins MC, Kobayashi Y, Koudela B, Marsh AE, Mattsson JG, McAllister MM, Modry D, Omata Y, Sibley LD, Speer CA, Trees AJ, Uggla A, Upton SJ, Williams DJ, Lindsay DS (2002) Redescription of Neospora caninum and its differentiation from related coccidia. Int J Parasitol 32:929–946

    Article  PubMed  CAS  Google Scholar 

  • Dubey JP, Buxton D, Wouda W (2006) Pathogenesis of bovine neosporosis. J Comp Pathol 134:267–289

    Article  PubMed  CAS  Google Scholar 

  • Eastick FA, Elsheikha HM (2010) Stress-driven stage transformation of Neospora caninum. Parasitol Res 106:1009–1014

    Article  PubMed  Google Scholar 

  • Ellis J, Miller C, Quinn H, Ryce C, Reichel MP (2008) Evaluation of recombinant proteins of Neospora caninum as vaccine candidates (in a mouse model). Vaccine 26:5989–5996

    Article  PubMed  CAS  Google Scholar 

  • Fernandez-Garcia A, Risco-Castillo V, Zaballos A, Alvarez-Garcia G, Ortega-Mora LM (2006) Identification and molecular cloning of the Neospora caninum SAG4 gene specifically expressed at bradyzoite stage. Mol Biochem Parasitol 146:89–97

    Article  PubMed  CAS  Google Scholar 

  • Gondim LF, McAllister MM, Pitt WC, Zemlicka DE (2004) Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int J Parasitol 34:159–161

    Article  PubMed  Google Scholar 

  • Guionaud C, Hemphill A, Mevissen M, Alaeddine F (2010) Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix. Parasitology 137:1605–1619

    Article  PubMed  CAS  Google Scholar 

  • Haldorson GJ, Mathison BA, Wenberg K, Conrad PA, Dubey JP, Trees AJ, Yamane I, Baszler TV (2005) Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice. Int J Parasitol 35:1407–1415

    Article  PubMed  CAS  Google Scholar 

  • Hemphill A, Gottstein B (2000) A European perspective on Neospora caninum. Int J Parasitol 30:877–924

    Article  PubMed  CAS  Google Scholar 

  • Hemphill A, Vonlaufen N, Naguleswaran A (2006) Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum. Parasitology 133:261–278

    Article  PubMed  CAS  Google Scholar 

  • Hohdatsu T, Eiguchi Y, Ide S, Baba K, Yamagishi H, Kume T, Matumoto M (1987) Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies. Vet Microbiol 13:93–97

    Article  PubMed  CAS  Google Scholar 

  • Hu J, Ferroglio E, Trisciuoglio A (2011) Immunoblot diagnosis of infection with Neospora caninum in cattle based on recombinant NcSAG4 Antigen. Parasitol Res 108:1055–1058

    Article  PubMed  Google Scholar 

  • Ike K, Uchida Y, Nakamura T, Imai S (2005) Induction of interferon-gamma (IFN-gamma) and T helper 1 (Th1) immune response by bitter gourd extract. J Vet Med Sci 67:521–524

    Article  PubMed  Google Scholar 

  • Innes EA, Vermeulen AN (2006) Vaccination as a control strategy against the coccidial parasites Eimeria, Toxoplasma and Neospora. Parasitology 133(Suppl):S145–168

    Article  PubMed  Google Scholar 

  • Innes EA, Andrianarivo AG, Bjorkman C, Williams DJ, Conrad PA (2002) Immune responses to Neospora caninum and prospects for vaccination. Trends Parasitol 18:497–504

    Article  PubMed  CAS  Google Scholar 

  • Jimenez-Ruiz E, Alvarez-Garcia G, Aguado-Martinez A, Salman H, Irache JM, Marugan-Hernandez V, Ortega-Mora LM (2012) Low efficacy of NcGRA7, NcSAG4, NcBSR4 and NcSRS9 formulated in poly-varepsilon-caprolactone against Neospora caninum infection in mice. Vaccine 30:4983–4992

    Article  PubMed  CAS  Google Scholar 

  • Kang SW, Kweon CH, Lee EH, Choe SE, Jung SC, Quyen DV (2008a) The differentiation of transcription between tachyzoites and bradyzoites of in vitro cultured Neospora caninum. Parasitol Res 103:1011–1018

    Article  PubMed  Google Scholar 

  • Kang SW, Lee EH, Jean YH, Choe SE, Van Quyen D, Lee MS (2008b) The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured Neospora caninum. Parasitol Res 103:905–913

    Article  PubMed  Google Scholar 

  • Khan IA, Schwartzman JD, Fonseka S, Kasper LH (1997) Neospora caninum: role for immune cytokines in host immunity. Exp Parasitol 85:24–34

    Article  PubMed  CAS  Google Scholar 

  • Klevar S, Kulberg S, Boysen P, Storset AK, Moldal T, Bjorkman C, Olsen I (2007) Natural killer cells act as early responders in an experimental infection with Neospora caninum in calves. Int J Parasitol 37:329–339

    Article  PubMed  CAS  Google Scholar 

  • Kobayashi T, Narabu S, Yanai Y, Hatano Y, Ito A, Imai S, Ike K (2013) Gene cloning and characterization of the protein encoded by the Neospora caninum bradyzoite-specific antigen gene bag1. J Parasitol 99: in press

  • Lindsay DS, Dubey JP (1989) In vitro development of Neospora caninum (Protozoa: Apicomplexa) from dogs. J Parasitol 75:163–165

    Article  PubMed  CAS  Google Scholar 

  • Long MT, Baszler TV, Mathison BA (1998) Comparison of intracerebral parasite load, lesion development, and systemic cytokines in mouse strains infected with Neospora caninum. J Parasitol 84:316–320

    Article  PubMed  CAS  Google Scholar 

  • Lunden A, Marks J, Maley SW, Innes EA (1998) Cellular immune responses in cattle experimentally infected with Neospora caninum. Parasite Immunol 20:519–526

    Article  PubMed  CAS  Google Scholar 

  • Marugan-Hernandez V, Ortega-Mora LM, Aguado-Martinez A, Jimenez-Ruiz E, Alvarez-Garcia G (2011) Transgenic Neospora caninum strains constitutively expressing the bradyzoite NcSAG4 protein proved to be safe and conferred significant levels of protection against vertical transmission when used as live vaccines in mice. Vaccine 29:7867–7874

    Article  PubMed  CAS  Google Scholar 

  • McAllister MM, Parmley SF, Weiss LM, Welch VJ, McGuire AM (1996) An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen. J Parasitol 82:354–355

    Article  PubMed  CAS  Google Scholar 

  • McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM (1998) Dogs are definitive hosts of Neospora caninum. Int J Parasitol 28:1473–1478

    Article  PubMed  CAS  Google Scholar 

  • Nielsen HV, Di Cristina M, Beghetto E, Spadoni A, Petersen E, Gargano N (2006) Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts. Exp Parasitol 112:274–279

    Article  PubMed  CAS  Google Scholar 

  • Nishikawa Y, Inoue N, Makala L, Nagasawa H (2003) A role for balance of interferon-gamma and interleukin-4 production in protective immunity against Neospora caninum infection. Vet Parasitol 116:175–184

    Article  PubMed  CAS  Google Scholar 

  • Okeoma CM, Stowell KM, Williamson NB, Pomroy WE (2005) Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR. Exp Parasitol 110:48–55

    Article  PubMed  CAS  Google Scholar 

  • Parmley S, Slifer T, Araujo F (2002) Protective effects of immunization with a recombinant cyst antigen in mouse models of infection with Toxoplasma gondii tissue cysts. J Infect Dis 185(Suppl 1):S90–95

    Article  PubMed  CAS  Google Scholar 

  • Ramamoorthy S, Duncan R, Lindsay DS, Sriranganathan N (2007) Optimization of the use of C57BL/6 mice as a laboratory animal model for Neospora caninum vaccine studies. Vet Parasitol 145:253–259

    Article  PubMed  CAS  Google Scholar 

  • Reichel MP, Ellis JT (2009) Neospora caninum—how close are we to development of an efficacious vaccine that prevents abortion in cattle? Int J Parasitol 39:1173–1187

    Article  PubMed  Google Scholar 

  • Risco-Castillo V, Fernandez-Garcia A, Zaballos A, Aguado-Martinez A, Hemphill A, Rodriguez-Bertos A, Alvarez-Garcia G, Ortega-Mora LM (2007) Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum. Int J Parasitol 37:887–896

    Article  PubMed  CAS  Google Scholar 

  • Risco-Castillo V, Marugan-Hernandez V, Fernandez-Garcia A, Aguado-Martinez A, Jimenez-Ruiz E, Rodriguez-Marco S, Alvarez-Garcia G, Ortega-Mora LM (2011) Identification of a gene cluster for cell-surface genes of the SRS superfamily in Neospora caninum and characterization of the novel SRS9 gene. Parasitology 138:1832–1842

    Article  CAS  Google Scholar 

  • Ritter DM, Kerlin R, Sibert G, Brake D (2002) Immune factors influencing the course of infection with Neospora caninum in the murine host. J Parasitol 88:271–280

    PubMed  Google Scholar 

  • Schares G, Heydorn AO, Cuppers A, Conraths FJ, Mehlhorn H (2001) Cyclic transmission of Neospora caninum: serological findings in dogs shedding oocysts. Parasitol Res 87:873–877

    Article  PubMed  CAS  Google Scholar 

  • Tunev SS, McAllister MM, Anderson-Sprecher RC, Weiss LM (2002) Neospora caninum in vitro: evidence that the destiny of a parasitophorous vacuole depends on the phenotype of the progenitor zoite. J Parasitol 88:1095–1099

    PubMed  CAS  Google Scholar 

  • Vonlaufen N, Muller N, Keller N, Naguleswaran A, Bohne W, McAllister MM, Bjorkman C, Muller E, Caldelari R, Hemphill A (2002) Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures. Int J Parasitol 32:1253–1265

    Article  PubMed  CAS  Google Scholar 

  • Vonlaufen N, Guetg N, Naguleswaran A, Muller N, Bjorkman C, Schares G, von Blumroeder D, Ellis J, Hemphill A (2004) In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites. Infect Immun 72:576–583

    Article  PubMed  CAS  Google Scholar 

  • Wapenaar W, Jenkins MC, O'Handley RM, Barkema HW (2006) Neospora caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans). J Parasitol 92:1270–1274

    Article  PubMed  CAS  Google Scholar 

  • Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412

    PubMed  CAS  Google Scholar 

  • Weiss LM, Ma YF, Halonen S, McAllister MM, Zhang YW (1999) The in vitro development of Neospora caninum bradyzoites. Int J Parasitol 29:1713–1723

    Article  PubMed  CAS  Google Scholar 

  • Williams DJ, Guy CS, McGarry JW, Guy F, Tasker L, Smith RF, MacEachern K, Cripps PJ, Kelly DF, Trees AJ (2000) Neospora caninum-associated abortion in cattle: the time of experimentally-induced parasitaemia during gestation determines foetal survival. Parasitology 121:347–358

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research to K. Ike (no. 22580342) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest

The authors declare no conflicts of interest.

Author information

Authors and Affiliations

  1. Laboratory of Veterinary Parasitology, Department of Veterinary Medicine, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan, Musashino, Tokyo, 180-8602, Japan

    Masaki Uchida, Kotomi Nagashima, Yui Akatsuka, Soichi Imai & Kazunori Ike

  2. Nemuro Agricultural Mutual Aid Association, Hokkaido, 088-2725, Japan

    Takashi Murakami

  3. Advanced Industrial Science and Technology (AIST), Plant Molecular Technology Research Group, Bioproduction Research Institute, Hokkaido, 062-8517, Japan

    Akira Ito

Authors
  1. Masaki Uchida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kotomi Nagashima
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yui Akatsuka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takashi Murakami
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Akira Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Soichi Imai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kazunori Ike
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kazunori Ike.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uchida, M., Nagashima, K., Akatsuka, Y. et al. Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection. Parasitol Res 112, 655–663 (2013). https://doi.org/10.1007/s00436-012-3182-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-012-3182-5

Keywords

Search

Navigation