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Tissue Culture and Explant Approaches to Studying and Visualizing Neospora caninum and Its Interactions with the Host Cell

  • Andrew Hemphill (a1), Nathalie Vonlaufen (a1), Arunasalam Naguleswaran (a1), Nadine Keller (a1), Michele Riesen (a1), Nicole Guetg (a1), Sangeetha Srinivasan (a1) and Ferial Alaeddine (a1)...


Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.


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Beckers, C.J., Wakefield, T., & Joiner, K.A. (1997). The expression of Toxoplasma proteins in Neospora caninum and the identification of a gene encoding a novel rhoptry protein. Mol Biochem Parasitol 89, 209223.
Bjerkås, I., Mohn, S.F., & Presthus, J. (1984). Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Zeitschr Parasitenkunde 70, 271274.
Björkman, C. & Hemphill, A. (1998). Characterization of Neospora caninum iscom antigens using monoclonal antibodies. Parasite Immunol 20, 7380.
Björkman, C. & Uggla, A. (1999). Serological diagnosis of Neospora caninum infection. Int J Parasitol 29, 14971507.
Bohne, W., Heesemann, J., & Gross, U. (1994). Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: A possible role for nitric oxide in triggering stage conversion. Infect Immun 62, 17611767.
Buxton, D., McAllister, M.M., & Dubey, J.P. (2002). The comparative pathogenesis of neosporosis. Trends Parasitol 18, 546551.
Caldelari, R., Suter, M.M., Bauman, D., DeBruin, A., & Müller, E. (2000). Long term culture of murine epidermal keratinocytes. J Inv Dermatol 114, 10641065.
Cannas, A., Naguleswaran, A., Müller, N., Eperon, S., Gottstein, B., & Hemphill, A. (2003a). Vaccination of mice against experimental Neospora caninum infection using NcSAG1- and NcSRS2-based recombinant antigens and DNA-vaccines. Parasitology 126, 303312.
Cannas, A., Naguleswaran, A., Müller, N., Gottstein, B., & Hemphill, A. (2003b). Protective effect of vaccination with recombinant microneme protein NcMIC3 in mice following challenge infection with Neospora caninum tachyzoites. J Parasitol 89, 4450.
Carruthers, V.B. (2002). Host cell invasion by the opportunistic pathogen Toxoplasma gondii. Acta Tropica 81, 111122.
Carruthers, V.B., Hakansson, S., Giddings, O.K., & Sibley, L.D. (2000). Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment. Infect Immun 68, 40054011.
Cooper, C.E. (1999). Nitric oxide and iron proteins. Biochem Biophys Acta 1411, 290309.
Dobrowolski, J.M., Niesman, I.R., & Sibley, L.D. (1997). Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form. Cell Mot Cytoskel 37, 253262.
Dobrowolski, J. & Sibley, L.D. (1996). Toxoplasma invasion of mammalian cells is powered by the actin skeleton of the parasite. Cell 84, 933939.
Dobrowolski, J. & Sibley, L.D. (1997). The role of the cytoskeleton in host cell invasion by Toxoplasma gondii. Behring Institut Mitteilungen 99, 9096.
Dubey, J.P. (1999). Recent advances in Neospora and neosporosis. Vet Parasitol 84, 349367.
Dubey, J.P., Barr, B.C., Barta, J.R., Bjerkas, I., Björkman, C., Blagburn, B.L., Bowman, D.D., Buxton, D., Ellis, J.T., Gottstein, B., Hemphill, A., Hill, D.E., Howe, D.K., Jenkins, M.C., Kobayashi, Y., Koudela, B., Marsh, A.E., Mattsson, J.G., McAllister, M.M., Modry, D., Omata, Y., Sibley, L.D., Speer, C.A., Trees, A.J., Uggla, A., Upton, S.J., Wiliams, D.J.L., & Lindsay, D.S. (2002a). Redescription of Neospora caninum and its differentiation from related coccidia. Int J Parasitol 32, 929946.
Dubey, J.P. & Beattie, C.P. (1988). Toxoplasmosis of Animals and Man. Boca Raton, FL: CRC Press.
Dubey, J.P., Dorough, K.R., Jenkins, M.C., Liddell, S., Speer, C.A., Kwok, O.C.H., & Shen, S.K. (1998). Canine neosporosis: Clinical signs, diagnosis, treatment and isolation of Neospora caninum in mice and cell culture. Int J Parasitol 28, 12931304.
Dubey, J.P., Hattel, A.L., Lindsay, D.S., & Topper, M.J. (1988). Neonatal Neospora caninum infection in dogs: Isolation of the causative agent and experimental transmission. J Am Vet Med Assoc 193, 12591263.
Dubey, J.P., Hill, D.E., Lindsay, D.S., Jenkins, M.C., Uggla, A., & Speer, C.A. (2002b). Neospora caninum and Hammondia heydorni are separate species. Trends Parasitol 18, 6669.
Dubey, J.P. & Lindsay, D.S. (1996). A review of Nespora caninum and neosporosis. Vet Parasitol 67, 159
Fagard, R., Van Tan, H., Creuzet, C., & Pelloux, H. (1999). Differential development of Toxoplasma gondii in neural cells. Parasitol Today 15, 504507.
Fuchs, N., Ingold, K., Sonda, S., Bütikofer, P., & Hemphill, A. (1999). Detection of surface-associated glycoconjugates and glycoproteins in Neospora caninum tachyzoites. Int J Parasitol 29, 15971612.
Fuchs, N., Sonda, S., Gottstein, B., & Hemphill, A. (1998). Differential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites. J Parasitol 84, 753758.
Gottstein, B., Eperon, S., Dai, W.J., Hemphill, A., & Greif, G. (2001). Efficacy of toltrazuril and ponazuril against experimental Neospora caninum infection in mice. Parasitol Res 87, 4348.
Gross, U., Bormuth, H., Gaissmaier, C., Dittrich, C., Krenn, V., Bohne, W., & Ferguson, D.J. (1995). Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo. Clin Diagn Lab Immunol 2, 542548.
Halonen, S.K. & Weidner, E. (1994). Overcoating of Toxoplasma parasitophorous vacuoles with host cell vimentin-type intermediate filaments. J Eukariotic Microbiol 41, 6571.
Halonen, S.K., Weiss, L.M., & Chiu, F.C. (1998). Association of host cell intermediate filaments with Toxoplasma gondii cysts in murine astrocytes in vitro. Int J Parasitol 28, 815823.
Hemphill, A. (1996). Subcellular localization and functional characterisation of Nc-p43, a major Neospora caninum tachyzoite surface protein. Infect Immun 64, 42794287.
Hemphill, A. (1999). The host–parasite relationship in neosporosis. Adv Parasitol 43, 47104.
Hemphill, A. & Croft, S.L. (1997). Electron microscopy in parasitology. In Analytical Parasitology, Rogan, M. (Ed.), pp. 227268. Heidelberg: Springer Verlag.
Hemphill, A., Felleisen, R., Connolly, B., Gottstein, B., Hentrich, B., & Müller, N. (1997a). Characterization of a cDNA clone encoding Nc-p43, a major Neospora caninum surface protein. Parasitol 115, 581590.
Hemphill, A., Fuchs, N., Sonda, S., Gottstein, B., & Hentrich, B. (1997b). Identification and partial characterization of a 36 kD surface protein on Neospora caninum tachyzoites. Parasitology 115, 371380.
Hemphill, A., Gajendran, N., Sonda, S., Fuchs, N., Gottstein, B., Hentrich, B., & Jenkins, M. (1998). Neospora caninum tachyzoites: Identification and partial characterization of a novel dense granule-associated protein. Int J Parasitol 28, 429438.
Hemphill, A. & Gottstein, B. (1995). Immunology and morphology studies on the proliferation of in vitro cultivated Echinococcus multilocularis metacestodes. Parasitol Res 81, 605614.
Hemphill, A. & Gottstein, B. (2000). A European perspective on Neospora caninum. Int J Parasitol 30, 877924.
Hemphill, A., Gottstein, B., & Kaufmann, H. (1996). Adhesion and invasion of bovine endothelial cells by Neospora caninum tachyzoites. Parasitology 112, 183197.
Hoppe, H.C., Ngo, H.M., Yang, M., & Joiner, K. (2000). Targeting rhoptry organelles of Toxoplasma gondii involves evolutionary conserved mechanisms. Nat Cell Biol 2, 449456.
Howe, D.K., Crawford, A.C., Lindsay, D.L., & Sibley, L.D. (1998). The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. Infect Immun 66, 53225338.
Howe, D.K., Mercier, C., Messina, M., & Sibley, L.D. (1997). Expression of Toxoplasma gondii genes in the closely related apicomplexan parasite Neospora caninum. Mol Biochem Parasitol 86, 2936.
Howe, D.K. & Sibley, L.D. (1997). Development of molecular genetics for Neospora caninum: A complementary system to Toxoplasma gondii. Methods 13, 123133.
Howe, D.K. & Sibley, L.D. (1999). Comparison of the major antigens of Neospora caninum and Toxoplasma gondii. Int J Paraitol 29, 14891496.
Innes, E.A., Andrianarivo, A.G., Bjorkman, C., Williams, D.J., & Conrad, P.A. (2002). Immune responses to Neospora caninum and prospects for vaccination. Trends Parasitol 18, 497504.
Jardine, J.E. (1996). The ultrastructure of bradyzoites and tissue cysts of Neospora caninum in dogs: Absence of distinguishable morphological features between parasites from canine and bovine origin. Vet Parasitol 62, 231240.
Keller, N., Naguleswaran, A., Cannas, A., Vonlaufen, N., Bienz, M., Björkman, C., Bohne, W., & Hemphill, A. (2002). Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans. Inf Immun 70, 31873198.
Khan, I.A., Schwartzman, J.D., Fonseka, S., & Kasper, L.H. (1997). Neospora caninum: Role for immune cytokines in host immunity. Exp Parasitol 85, 2434.
Kirkman, L.A., Weiss, L.M., & Kim, K. (2001). Cyclic nucleotide signaling in Toxoplasma gondii bradyzoite differentiation. Infect Immun 69, 148153.
Kritzner, S., Sager, H., Blum, J., Krebber, R., Greif, G., & Gottstein, B. (2002). An explorative study to assess the efficacy of toltrazuril-sulfone (ponazuril) in calves experimentally infected with Neospora caninum. Ann Clin Microbiol Antimicrob 1, 4.
Lindsay, D.S., Dubey, J.P., & Duncan, R.B. (1999). Confirmation that the dog is a definitive host for Neospora caninum. Vet Parasitol 82, 327333.
Lindsay, D.S., Speer, C.A., Toivio-Kinnucan, M.A., Dubey, J.P., & Blagburn, B.L. (1993). Use of infected cultured cells to compare ultrastructural features of Neopsora caninum and Toxoplasma gondii. Am J Vet Res 54, 11031106.
Lovett, J.L., Howe, D.K., & Sibley, L.D. (2000). Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum. Mol Biochem Parasitol 107, 3343.
Luft, J.H. (1971). Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec 171, 347368.
McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., & McGuire, A.M. (1998). Dogs are definitive hosts of Neospora caninum. Int J Parasitol 28, 14731478.
McAllister, M.M., Parmley, S.F., Weiss, L.M., Welch, V.J., & McGuire, A.M. (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, 354355.
McGuire, A.M., McAllister, M.M., Jolley, W.R., & Anderson-Sprecher, R.C. (1997). A protocol for the production of Neospora caninum tissue cysts in mice. J Parasitol 83, 647651.
Mehlhorn, H. & Heydorn, A.O. (2000). Neospora caninum: Is it really different from Hammondia heydorni or is it a strain of Toxoplasma gondii? An opinion. Parasitol Res 86, 169178.
Meissner, M., Schlüter, D., & Soldati, D. (2002). Role of Toxoplasma gondii myosin in powering parasite gliding and host cell invasion. Science 298, 837840.
Mugridge, N.B., Morrison, D.A., Heckeroth, A.R., Johnson, A.M., & Tenter, A.M. (1999). Phylogenenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii. Int J Parasitol 29, 15451556.
Müller, N., Vonlaufen, N., Gianinazzi, C., Leib, S.L., & Hemphill, A. (2002). Application of real time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous tissue. J Clin Microbiol 40, 252255.
Müller, N., Zimmermann, V., Hentrich, B., & Gottstein, B. (1996). Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay. J Clin Microbiol 34, 28502852.
Naguleswaran, A., Cannas, A., Keller, N., Vonlaufen, N., Björkman, C., & Hemphill, A. (2002). Vero cell surface proteoglycan interaction with the microneme protein NcMIC3 mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii. Int J Parasitol 32, 695704.
Naguleswaran, A., Cannas, A., Keller, N., Vonlaufen, N., Schares, G., Conraths, F.J., Björkman, C., & Hemphill, A. (2001). Neospora caninum microneme protein NcMIC3: Secretion, subcellular localization and functional involvement in host cell interaction. Inf Immun 69, 64836494.
Naguleswaran, A., Müller, N., & Hemphill, A. (2003). A novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp Parasitol 104, 149158.
Nishikawa, Y., Inoue, N., Xuan, X., Nagasawa, H., Igarashi, I., Fijisaki, K., Otsuka, H., & Mikami, T. (2001). Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection. Vaccine 19, 13811390.
Nishikawa, Y., Xuan, X., Nagasawa, H., Igarashi, I., Fujisaka, K., Otsuka, H., & Mikami, T. (2000). Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells. Int J Parasitol 30, 5158.
Opitz, C. & Soldati, D. (2002). The glideosome: A dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii. Mol Microbiol 45, 597604.
Peters, M., Lütkefels, E., Heckeroth, A.R., & Schares, G. (2001). Immunohistochemical and ultrastructural evidence for Neospora caninum tissue cysts in skeletal muscles of naturally infected dogs and cattle. Int J Parasitol 31, 11441148.
Powell, H.C., Gibbs, C.J., Lorenzo, A.M., Lambert, P.W., & Gadjusek, D.C. (1978). Toxoplasmosis of the central nervous system in the adult. Electron microscopic observations. Acta Neuropathol 41, 211216.
Quinn, H.E., Ellis, J.T., & Smith, N.C. (2002). Neospora caninum: A cause of immune-mediated failure of pregnancy? Trends Parasitol 18, 391394.
Sam-Yellowe, T.Y. (1996). Rhoptry organelles of the Apicomplexa: Their role in host cell invasion and intracellular survival. Parasitol Today 12, 308316.
Schock, A., Innes, E.A., Yamane, I., Latham, S.M., & Wastling, J.M. (2001). Genetic and biological diversity among isolates of Neospora caninum. Parasitology 123, 1323.
Soete, M., Camus, D., & Dubremetz, J.F. (1994). Experimental induction of bradyzoite-specific antigen expression and cyst formation by the RH strain of Toxoplasma gondii in vitro. Exp Parasitol 78, 361370.
Soldati, D., Dubremetz, J.F., & Lebrun, M. (2001). Microneme proteins: Structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int J Parasitol 31, 12931302.
Sonda, S., Fuchs, N., Gottstein, B., & Hemphill, A. (2000). Molecular characterization of a novel microneme antigen in Neospora caninum. Mol Biochem Parasitol 108, 3951.
Speer, C.A., Dubey, J.P., McAllister, M.M., & Blixt, J.A. (1999). Comparative ultrastructure of tachyzoites, bradyzoites and tissue cysts of Neospora caninum and Toxoplasma gondii. Int J Parasitol 29, 15091519.
Stoppini, L., Buchs, P.A., Brun, R., Muller, D., Duport, S., Parisi, L., & Seebeck, T. (2000). Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei. Int J Med Microbiol 290, 105113.
Stoppini, L., Buchs, P.A., & Mueller, D. (1991). A simple method for organotypoic cultures of nervous tissue. J Neurosci Methods 37, 173182.
Tenter, A.M. & Johnson, A.M. (1997). Phylogeny of the tissue-cyst forming coccidia. Adv Parasitol 39, 70141.
Thurmond, M.C. & Hietala, S.K. (1997). Effect of Neospora caninum infection on milk production in first-lactation dairy cows. J Am Vet Med Assoc 210, 672674.
Tomley, F.M. & Soldati, D. (2001). Mix and match modules: Structure and function of microneme proteins in apicomplexan parasites. Trends Parasitol 17, 8188.
Tunev, S.S., McAllister, M.M., Anderson-Sprecher, R.C., & Weiss, L.M. (2002). Neospora caninum in vitro: Evidence that the destiny of a parasitophorous vacuole depends on the phenotype of the progenitor zoite. J Parasitol 88, 10951099.
Vonlaufen, N., Gianinazzi, C., Müller, N., Simon, F., Björkman, C., Jungi, T.W.S., Leib, S.L., & Hemphill, A. (2002a). Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: An alternative approach to study host–parasite interactions. Int J Parasitol 32, 533542.
Vonlaufen, N., Guetg, N., Naguleswaran, A., Müller, N., Björkman, C., Schorrs, G., von Blumioeder, D., Ellis, J., & Hemphill, A. (2004). In vitro induction of Neospora caninum bradyzoites in vivo cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites. Inf Immun 72, 576583.
Vonlaufen, N., Müller, N., Keller, N., Naguleswaran, A., Bohne, W., McAllister, M.M., Björkman, C., Müller, E., Caldelari, R., & Hemphill, A. (2002b). Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures. Int J Parasitol 32, 12531265.
Weiss, L.M., Ma, Y.F., Halonen, S., McAllister, M.M., & Zhang, Y.W. (1999). The in vitro development of Neospora caninum bradyzoites. Int J Parasitol 29, 17131723.


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Tissue Culture and Explant Approaches to Studying and Visualizing Neospora caninum and Its Interactions with the Host Cell

  • Andrew Hemphill (a1), Nathalie Vonlaufen (a1), Arunasalam Naguleswaran (a1), Nadine Keller (a1), Michele Riesen (a1), Nicole Guetg (a1), Sangeetha Srinivasan (a1) and Ferial Alaeddine (a1)...


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