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Genetic manipulation of Neospora caninum to express the bradyzoite-specific protein NcSAG4 in tachyzoites

Published online by Cambridge University Press:  14 January 2011

V. MARUGÁN-HERNÁNDEZ ,
L. M. ORTEGA-MORA ,
A. AGUADO-MARTÍNEZ  and
G. ÁLVAREZ-GARCÍA
V. MARUGÁN-HERNÁNDEZ
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
L. M. ORTEGA-MORA
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
A. AGUADO-MARTÍNEZ
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
G. ÁLVAREZ-GARCÍA*
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
*
*Corresponding author: Tel: +34 91 3944095. Fax: +34 91 3944098. E-mail: gemaga@vet.ucm.es
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Summary

Neospora caninum is an apicomplexan parasite and the aetiological agent of bovine neosporosis, one of the main causes of reproductive failure worldwide. We have generated 2 independent transgenic knock-in clones, Nc-1SAG4c1.1 and Nc-1SAG4c2.1, that express the bradyzoite stage-specific protein NcSAG4 in the tachyzoite stage. These clones have similar growth rates in vitro as the wild-type (WT) strain Nc-1. Studies in a cerebral mouse model of infection revealed a slightly lower rate of detection of the transgenic strains in brains during the chronic phase of infection. However, a pregnant mouse model of infection revealed a reduction in the virulence of the Nc-1SAG4c1.1 strain despite the same tachyzoite expression of NcSAG4 and a similar anti-NcSAG4 response displayed by mice inoculated with Nc-1 SAG4c1.1 or Nc-1 SAG4c2.1 parasites. This behaviour may be related to the reduced ability of the Nc-1SAG4c1.1 parasites to invade host cells, which was observed in in vitro assays. The apparent reduction in persistence and the high growth rate of the transgenic strains, together with their constitutive expression of the protein NcSAG4, may be useful features for future immunoprophylaxis trials based on a safe live attenuated vaccine.

Keywords

Neospora caninumNcSAG4knock-in strainvirulencevaccine
Type
Research Article
Information
Parasitology , Volume 138 , Issue 4 , April 2011 , pp. 472 - 480
Copyright
Copyright © Cambridge University Press 2011

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References

REFERENCES

Aguado-Martinez, A., Alvarez-Garcia, G., Fernandez-Garcia, A., Risco-Castillo, V., Arnaiz-Seco, I., Rebordosa-Trigueros, X., Navarro-Lozano, V. and Ortega-Mora, L. M. (2008). Usefulness of rNcGRA7- and rNcSAG4-based ELISA tests for distinguishing primo-infection, recrudescence, and chronic bovine neosporosis. Veterinary Parasitology 157, 182–195. doi: 10.1016/j.vetpar.2008.08.002.CrossRefGoogle ScholarPubMed
Aguado-Martinez, A., Ortega-Mora, L. M., Alvarez-Garcia, G., Rodriguez-Marco, S., Risco-Castillo, V., Marugan-Hernandez, V. and Fernandez-Garcia, A. (2009). Stage-specific expression of Nc SAG4 as a marker of chronic Neospora caninum infection in a mouse model. Parasitology 136, 757–764. doi: 10.1017/S0031182009006076.CrossRefGoogle ScholarPubMed
Aguado-Martínez, A., Álvarez-García, G., Schares, G., Risco-Castillo, V., Fernández-García, A., Marugán-Hernández, V. and Ortega-Mora, L. M. (2010). Characterisation of NcGRA7 and NcSAG4 proteins: immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites. Acta Parasitologica 55, 304–312. doi 10.2478/s11686-010-0056-9.CrossRefGoogle Scholar
Alvarez-Garcia, G., Pitarch, A., Zaballos, A., Fernandez-Garcia, A., Gil, C., Gomez-Bautista, M., Aguado-Martinez, A. and Ortega-Mora, L. M. (2007). The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum. Parasitology 134, 41–50. doi: 10.1017/S0031182006001284.CrossRefGoogle ScholarPubMed
Appleby, P. and Catty, D. (1983). Transmission of immunoglobulin to foetal and neonatal mice. Journal of Reproductive Immunology 5, 203–213.CrossRefGoogle ScholarPubMed
Bartley, P. M., Wright, S., Sales, J., Chianini, F., Buxton, D. and Innes, E. A. (2006). Long-term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo. Parasitology 133, 421–432. doi: 10.1017/S0031182006000539.CrossRefGoogle ScholarPubMed
Beckers, C. J., Wakefield, T. and Joiner, K. A. (1997). The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein. Molecular and Biochemical Parasitology 89, 209–223.CrossRefGoogle ScholarPubMed
Bland, J. M. and Altman, D. G. (2004). The logrank test. British Medical Journal (Clinical research ed.) 328, 1073. doi: 10.1136/bmj.328.7447.1073.CrossRefGoogle ScholarPubMed
Bland, J. M. and Altman, D. G. (1998). Survival probabilities (the Kaplan-Meier method). British Medical Journal (Clinical research ed.) 317, 1572.CrossRefGoogle ScholarPubMed
Bohne, W., Hunter, C. A., White, M. W., Ferguson, D. J., Gross, U. and Roos, D. S. (1998). Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii. Molecular and Biochemical Parasitology 92, 291–301.CrossRefGoogle Scholar
Buxton, D., Maley, S. W., Wright, S., Thomson, K. M., Rae, A. G. and Innes, E. A. (1998). The pathogenesis of experimental neosporosis in pregnant sheep. Journal of Comparative Pathology 118, 267–279.CrossRefGoogle ScholarPubMed
Cerede, O., Dubremetz, J. F., Soete, M., Deslee, D., Vial, H., Bout, D. and Lebrun, M. (2005). Synergistic role of micronemal proteins in Toxoplasma gondii virulence. The Journal of Experimental Medicine 201, 453–463. doi: 10.1084/jem.20041672.CrossRefGoogle ScholarPubMed
Collantes-Fernandez, E., Lopez-Perez, I., Alvarez-Garcia, G. and Ortega-Mora, L. M. (2006). Temporal distribution and parasite load kinetics in blood and tissues during Neospora caninum infection in mice. Infection and Immunity 74, 2491–2494. doi: 10.1128/IAI.74.4.2491-2494.2006.CrossRefGoogle ScholarPubMed
Collantes-Fernandez, E., Zaballos, A., Alvarez-Garcia, G. and Ortega-Mora, L. M. (2002). Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR. Journal of Clinical Microbiology 40, 1194–1198.CrossRefGoogle ScholarPubMed
Donald, R. G. and Roos, D. S. (1993). Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria. Proceedings of the National Academy of Sciences, USA 90, 11703–11707.CrossRefGoogle ScholarPubMed
Dubey, J. P., Schares, G. and Ortega-Mora, L. M. (2007). Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews 20, 323–367. doi: 10.1128/CMR.00031-06.CrossRefGoogle ScholarPubMed
Dzierszinski, F., Mortuaire, M., Cesbron-Delauw, M. F. and Tomavo, S. (2000). Targeted disruption of the glycosylphosphatidylinositol-anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice. Molecular Microbiology 37, 574–582.CrossRefGoogle ScholarPubMed
Fernandez-Garcia, A., Risco-Castillo, V., Zaballos, A., Alvarez-Garcia, G. and Ortega-Mora, L. M. (2006). Identification and molecular cloning of the Neospora caninum SAG4 gene specifically expressed at bradyzoite stage. Molecular and Biochemical Parasitology 146, 89–97. doi: 10.1016/j.molbiopara.2005.08.019.CrossRefGoogle ScholarPubMed
Howe, D. K., Mercier, C., Messina, M. and Sibley, L. D. (1997). Expression of Toxoplasma gondii genes in the closely-related apicomplexan parasite Neospora caninum. Molecular and Biochemical Parasitology 86, 29–36.CrossRefGoogle ScholarPubMed
Howe, D. K. and Sibley, L. D. (1997). Development of molecular genetics for Neospora caninum: A complementary system to Toxoplasma gondii. Methods 13, 123–133. doi: 10.1006/meth.1997.0505.CrossRefGoogle ScholarPubMed
Huynh, M. H., Opitz, C., Kwok, L. Y., Tomley, F. M., Carruthers, V. B. and Soldati, D. (2004). Trans-genera reconstitution and complementation of an adhesion complex in Toxoplasma gondii. Cellular Microbiology 6, 771–782. doi: 10.1111/j.1462-5822.2004.00403.x.CrossRefGoogle ScholarPubMed
Innes, E. A. (2007). The host-parasite relationship in pregnant cattle infected with Neospora caninum. Parasitology 134, 1903–1910. doi: 10.1017/S0031182007000194.CrossRefGoogle ScholarPubMed
Innes, E. A. and Mattsson, J. G. (2007). Neospora caninum emerges from the shadow of Toxoplasma gondii. Trends in Parasitology 23, 43–4; discussion 44–5. doi: 10.1016/j.pt.2006.12.004.CrossRefGoogle Scholar
Kim, S. K. and Boothroyd, J. C. (2005). Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence. Journal of Immunology 174, 8038–8048.CrossRefGoogle ScholarPubMed
Kim, S. K., Karasov, A. and Boothroyd, J. C. (2007). Bradyzoite-specific surface antigen SRS9 plays a role in maintaining Toxoplasma gondii persistence in the brain and in host control of parasite replication in the intestine. Infection and Immunity 75, 1626–1634. doi: 10.1128/IAI.01862-06.CrossRefGoogle Scholar
Lindsay, D. S., Lenz, S. D., Blagburn, B. L. and Brake, D. A. (1999). Characterization of temperature-sensitive strains of Neospora caninum in mice. The Journal of Parasitology 85, 64–67.CrossRefGoogle ScholarPubMed
Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. doi: 10.1006/meth.2001.1262.CrossRefGoogle ScholarPubMed
Lopez-Perez, I. C., Collantes-Fernandez, E., Aguado-Martinez, A., Rodriguez-Bertos, A. and Ortega-Mora, L. M. (2008). Influence of Neospora caninum infection in BALB/c mice during pregnancy in post-natal development. Veterinary Parasitology 155, 175–183. doi: 10.1016/j.vetpar.2008.05.018.CrossRefGoogle ScholarPubMed
Lopez-Perez, I. C., Risco-Castillo, V., Collantes-Fernandez, E. and Ortega-Mora, L. M. (2006). Comparative effect of Neospora caninum infection in BALB/c mice at three different gestation periods. The Journal of Parasitology 92, 1286–1291.CrossRefGoogle ScholarPubMed
Matrajt, M., Donald, R. G., Singh, U. and Roos, D. S. (2002). Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii. Molecular Microbiology 44, 735–747.CrossRefGoogle ScholarPubMed
Miller, C. M., Quinn, H. E., Windsor, P. A. and Ellis, J. T. (2002). Characterisation of the first Australian isolate of Neospora caninum from cattle. Australian Veterinary Journal 80, 620–625.CrossRefGoogle ScholarPubMed
Pereira, Garcia-Melo, D., Regidor-Cerrillo, J., Collantes-Fernandez, E., Aguado-Martinez, A., Del Pozo, I., Minguijon, E., Gomez-Bautista, M., Aduriz, G. and Ortega-Mora, L. M. (2010). Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves. Parasitology 137, 1057–1068. doi: 10.1017/S0031182009991855.CrossRefGoogle ScholarPubMed
Perez-Zaballos, F. J., Ortega-Mora, L. M., Alvarez-Garcia, G., Collantes-Fernandez, E., Navarro-Lozano, V., Garcia-Villada, L. and Costas, E. (2005). Adaptation of Neospora caninum isolates to cell-culture changes: an argument in favor of its clonal population structure. The Journal of Parasitology 91, 507–510.CrossRefGoogle ScholarPubMed
Ramamoorthy, S., Lindsay, D. S., Schurig, G. G., Boyle, S. M., Duncan, R. B., Vemulapalli, R. and Sriranganathan, N. (2006). Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum. The Journal of Eukaryotic Microbiology 53, 151–156. doi: 10.1111/j.1550-7408.2005.00083.x.CrossRefGoogle ScholarPubMed
Regidor-Cerrillo, J., Gomez-Bautista, M., Del Pozo, I., Jimenez-Ruiz, E., Aduriz, G. and Ortega-Mora, L. M. (2010). Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model. Veterinary Research 41, 52. doi: 10.1051/vetres/2010024.CrossRefGoogle Scholar
Regidor-Cerrillo, J., Gomez-Bautista, M., Pereira-Bueno, J., Aduriz, G., Navarro-Lozano, V., Risco-Castillo, V., Fernandez-Garcia, A., Pedraza-Diaz, S. and Ortega-Mora, L. M. (2008). Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain. Parasitology 135, 1651–1659. doi: 10.1017/S003118200800509X.CrossRefGoogle ScholarPubMed
Reichel, M. P. and Ellis, J. T. (2006). If control of Neospora caninum infection is technically feasible does it make economic sense? Veterinary Parasitology 142, 23–34. doi: 10.1016/j.vetpar.2006.06.027.CrossRefGoogle ScholarPubMed
Risco-Castillo, V., Fernandez-Garcia, A. and Ortega-Mora, L. M. (2004). Comparative analysis of stress agents in a simplified in vitro system of Neospora caninum bradyzoite production. The Journal of Parasitology 90, 466–470.CrossRefGoogle Scholar
Risco-Castillo, V., Fernandez-Garcia, A., Zaballos, A., Aguado-Martinez, A., Hemphill, A., Rodriguez-Bertos, A., Alvarez-Garcia, G. and Ortega-Mora, L. M. (2007). Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum. International Journal for Parasitology 37, 887–896. doi: 10.1016/j.ijpara.2007.02.003.CrossRefGoogle ScholarPubMed
Rojo-Montejo, S., Collantes-Fernandez, E., Blanco-Murcia, J., Rodriguez-Bertos, A., Risco-Castillo, V. and Ortega-Mora, L. M. (2009 a). Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy. Veterinary Research 40, 40–49. doi: 10.1051/vetres/2009032.CrossRefGoogle Scholar
Rojo-Montejo, S., Collantes-Fernandez, E., Regidor-Cerrillo, J., Alvarez-Garcia, G., Marugan-Hernandez, V., Pedraza-Diaz, S., Blanco-Murcia, J., Prenafeta, A. and Ortega-Mora, L. M. (2009 b). Isolation and characterization of a bovine isolate of Neospora caninum with low virulence. Veterinary Parasitology 159, 7–16. doi: 10.1016/j.vetpar.2008.10.009.CrossRefGoogle ScholarPubMed
Saeij, J. P., Arrizabalaga, G. and Boothroyd, J. C. (2008). A cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence. Infection and Immunity 76, 2402–2410. doi: 10.1128/IAI.01494-07.CrossRefGoogle ScholarPubMed
Saeij, J. P., Boyle, J. P. and Boothroyd, J. C. (2005 a). Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends in Parasitology 21, 476–481. doi: 10.1016/j.pt.2005.08.001.CrossRefGoogle ScholarPubMed
Saeij, J. P., Boyle, J. P., Grigg, M. E., Arrizabalaga, G. and Boothroyd, J. C. (2005 b). Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains. Infection and Immunity 73, 695–702. doi: 10.1128/IAI.73.2.695-702.2005.CrossRefGoogle ScholarPubMed
Schock, A., Innes, E. A., Yamane, I., Latham, S. M. and Wastling, J. M. (2001). Genetic and biological diversity among isolates of Neospora caninum. Parasitology 123, 13–23.CrossRefGoogle ScholarPubMed
Sundermann, C. A. and Estridge, B. H. (1999). Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro. International Journal for Parasitology 29, 1725–1732.CrossRefGoogle ScholarPubMed
Teixeira, L., Marques, A., Meireles, C. S., Seabra, A. R., Rodrigues, D., Madureira, P., Faustino, A. M., Silva, C., Ribeiro, A., Ferreira, P., Correia da Costa, J. M., Canada, N. and Vilanova, M. (2005). Characterization of the B-cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites. Immunology 116, 38–52. doi: 10.1111/j.1365-2567.2005.02195.x.CrossRefGoogle ScholarPubMed
Whitten, M. K. (1957). Effect of exteroceptive factors on the oestrous cycle of mice. Nature, London 180, 1436.CrossRefGoogle ScholarPubMed
Williams, D. J., Guy, C. S., Smith, R. F., Ellis, J., Bjorkman, C., Reichel, M. P. and Trees, A. J. (2007). Immunization of cattle with live tachyzoites of Neospora caninum confers protection against fetal death. Infection and Immunity 75, 1343–1348. doi: 10.1128/IAI.00777-06.CrossRefGoogle ScholarPubMed