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In vitro invasion efficiency and intracellular proliferation rate comprise virulence-related phenotypic traits of Neospora caninum

By Javier Regidor-Cerrillo, Mercedes Gómez-Bautista, Itsaso Sodupe, Gorka Aduriz, Gema Álvarez-García, Itziar Del Pozo and Luis Miguel Ortega-Mora


In this study, we examined the in vitro invasion and proliferation capacities of the Nc-Liv and ten Spanish Neospora caninum isolates (Nc-Spain 1 H - Nc-Spain 10). The invasion rate was determined as the number of tachyzoites that completed their internalisation into MARC-145 cells at 2, 4, and 6 h post-inoculation (pi). The proliferation rate was evaluated by determining the doubling time during the exponential proliferation period. Significant differences in the invasion rates of these isolates were detected at 2 and 4 h pi (P < 0.0001, Kruskal-Wallis test). At 4 h pi, the Nc-Spain 4 H and Nc-Liv isolates displayed the highest, while the Nc-Spain 3 H and Nc-Spain 1 H isolates had the lowest invasion rates (by Dunn's test). Variations in the proliferation kinetics of these isolates were also observed. Between different isolates, the lag phase, which occurs before the exponential growth phase, ranged from 8 to 44 h, and the doubling time ranged from 9.8 to 14.1 h (P = 0.0016, ANOVA test). Tachyzoite yield, which combines invasion and proliferation data, was also assessed and confirmed marked differences between the highly and less prolific isolates. Interestingly, a direct correlation between the invasion rates and tachyzoite yields, and the severity of the disease that was exhibited by infected pregnant mice in previous works could be established for the isolates in this study (Spearman's coefficient > 0.62, P < 0.05). The results of this study may help us to explain the differences in the pathogenicity that are displayed by different isolates

Topics: Research
Publisher: BioMed Central
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  1. (2002). A: Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures.
  2. (2003). A: Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp Parasitol
  3. (2008). A: Quantitative analysis of parasite DNA in the blood of immunized and naive mice after infection with Neospora caninum. Parasitology
  4. (1996). Adhesion and invasion of bovine endothelial cells by Neospora caninum. Parasitology
  5. (2009). Apicomplexan cytoskeleton and motors: key regulators in morphogenesis, cell division, transport and motility.
  6. (1995). Blagburn BL: Mouse model for central nervous system Neospora caninum infections.
  7. (2008). C: Studies on synchronous egress of coccidian parasites (Neospora caninum, Toxoplasma gondii, Eimeria bovis) from bovine endothelial host cells mediated by calcium ionophore A23187. Vet Res Commun
  8. (1998). Comparison of intracerebral parasite load, lesion development, and systemic cytokines in mouse strains infected with Neospora caninum.
  9. (2011). Costas E: Adaptation of Neospora Regidor-Cerrillo et al. Veterinary Research
  10. (2005). Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol
  11. (2008). DJ: The extent of parasite-associated necrosis in the placenta and foetal tissues of cattle following Neospora caninum infection in early and late gestation correlates with foetal death.
  12. (2007). Dubremetz JF: ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii. PLoS Pathog
  13. (2008). El-Bahy MM: Toxoplasma gondii: virulence of tachyzoites in serum free media at different temperatures. Exp Parasitol
  14. (1999). Estridge BH: Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro.
  15. (2002). Genetic approaches to studying virulence and pathogenesis in Toxoplasma gondii.
  16. (2004). Hemphill A: 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
  17. (2001). HG: Attenuation of mouse-virulent Toxoplasma gondii parasites is associated with a decrease in interleukin-12-inducing tachyzoite activity and reduced expression of actin, catalase and excretory proteins. Microbes Infect
  18. (2006). Innes EA: Long-term passage of tachyzoites in tissue culture can attenuate virulence of Neospora caninum in vivo. Parasitology
  19. (2005). JP: Isolation of Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus). Vet Parasitol
  20. (2002). JT: Characterization of an outbred pregnant mouse model of Neospora caninum infection.
  21. (1999). JT: Comparison of the biological characteristics of two isolates of Neospora caninum. Parasitology
  22. (2009). JT: Genetic diversity amongst isolates of Neospora caninum, and the development of a multiplex assay for the detection of distinct strains. Mol Cell Probes
  23. (2006). Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii. Cytometry A
  24. (2006). LD: A secreted serinethreonine kinase determines virulence in the eukaryotic pathogen Toxoplasma gondii. Science
  25. (2009). Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers. Vet Parasitol
  26. (2006). Naguleswaran A: Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum. Parasitology
  27. (2004). Ortega-Mora LM: Characterization of pathology and parasite load in outbred and inbred mouse models of chronic Neospora caninum infection.
  28. (2006). Ortega-Mora LM: Comparative effect of Neospora caninum infection in BALB/c mice at three different gestation periods.
  29. (2007). Ortega-Mora LM: Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev
  30. (2009). Ortega-Mora LM: Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy. Vet Res
  31. (2010). Ortega-Mora LM: Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model. Vet Res
  32. (2006). Ortega-Mora LM: Influence of the stage of pregnancy on Neospora caninum distribution, parasite loads and lesions in aborted bovine foetuses. Theriogenology
  33. (2008). Ortega-Mora LM: Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain. Parasitology
  34. (2009). Ortega-Mora LM: Microsatellite markers for the molecular characterization of Neospora caninum: application to clinical samples. Vet Parasitol
  35. (2006). Ortega-Mora LM: Multilocus microsatellite analysis reveals extensive genetic diversity in Neospora caninum.
  36. (2002). Ortega-Mora LM: Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR.
  37. (2006). Ortega-Mora LM: Temporal distribution and parasite load kinetics in blood and tissues during Neospora caninum infection in mice. Infect Immun
  38. (2007). Ortega-Mora LM: The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum. Parasitology
  39. (2009). OrtegaMora LM: Isolation and characterization of a bovine isolate of Neospora caninum with low virulence. Vet Parasitol
  40. (2010). OrtegaMora LM: Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves. Parasitology
  41. (2006). Polymorphic secreted kinases are key virulence factors in toxoplasmosis. Science
  42. (2009). Saeij JP: Communication between Toxoplasma gondii and its host: impact on parasite growth, development, immune evasion, and virulence. APMIS
  43. (2008). Soldati-Favre D: Hijacking of host cellular functions by the Apicomplexa. Annu Rev Microbiol
  44. (2004). The cell-mediated immune response to Neospora caninum during pregnancy in the mouse is associated with a bias towards production of interleukin-4.
  45. (2005). The host-parasite relationship in bovine neosporosis. Vet Immunol Immunopathol
  46. (1995). Trees AJ: Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer, Topper and Uggla). Parasitology
  47. (2009). W: Molecular approaches to diversity of populations of apicomplexan parasites.
  48. (2001). Wastling JM: Genetic and biological diversity among isolates of Neospora caninum. Parasitology
  49. (2006). Wouda W: Pathogenesis of bovine neosporosis.
  50. (1999). Zhang YW: The in vitro development of Neospora caninum bradyzoites.