Neospora Caninum in Vitro: Evidence That the Destiny of a Parasitophorous Vacuole Depends on the Phenotype of the Progenitor Zoite

Abstract

We previously reported that Neospora caninum can be induced to express BAG1, a bradyzoite antigen, within 3 days of culture under stress conditions. The main goals of the present experiment were to increase the expression of BAG1 in vitro (in part by extending cultures for 9 days), to observe parasitophorous vacuoles at various points of stage differentiation, and to test the ability of organisms produced in vitro to function like mature bradyzoites. Expression of BAG1 and of a tachyzoite antigen (NcSAG1) was monitored using a double-label immunofluorescence assay. For the purpose of this study, organisms expressing NcSAG1 were designated as tachyzoites, those expressing BAG1 were designated as bradyzoites, and those expressing both antigens were designated as intermediate zoites. The greatest percentage of intermediate zoites and bradyzoites (14%) occurred in bovine monocytes maintained for 9 days. These bradyzoites did not appear to be functionally mature; they did not induce patent infections in dogs, in contrast to bradyzoites that were produced in chronically infected mice. In vitro, large parasitophorous vacuoles contained either a pure population of tachyzoites or a mixture of tachyzoites and intermediate zoites, which is indicative of asynchronous stage conversion of organisms within a vacuole. Bradyzoites were first observed within small vacuoles on day 6, and bradyzoites never shared vacuoles with tachyzoites. This finding suggests that vacuoles containing bradyzoites may develop only if the cell is invaded by a zoite that has already begun bradyzoite differentiation. An alternative possibility is that cysts may develop if the establishing tachyzoite undergoes bradyzoite differentiation before multiplying. Cysts do not appear to arise from transformation of tachyzoites within large parasitophorous vacuoles.Infection with Neospora caninum, an apicomplexan protozoan, can cause abortions in cattle (Thilsted and Dubey, 1989) and goats (Barr et al., 1992). Dogs are a definitive host (McAllister et al., 1998), and ruminants are intermediate hosts. Wild canids and wild ruminants are suspected to be a natural reservoir for the infection (Dubey et al., 1999;Barling et al., 2000). NIH-PA Author Manuscript Lindsay et al., 1992) and induce patent infections in the definitive host by carnivorism.Bradyzoites are needed for scientific investigation. First, bradyzoites are needed for investigation of the factors that induce recrudescence of latent infection. Recrudescence is responsible for vertical propagation of N. caninum within infected lines of cows (Björkman et al., 1996) and may also be a cause of systemic neosporosis in dogs. Second, a supply of bradyzoites could enable the development of an oral vaccine to prevent farm dogs from shedding N. caninum oocysts and thus infecting cattle.We previously reported that N. caninum can be induced to express BAG1, a bradyzoite antigen, within 3 days of culture in human fibroblasts under stress conditions (Weiss et al., 1999). The main goals of the present experime...

Comparative Analysis of Stress Agents in a Simplified in Vitro System of Neospora Caninum Bradyzoite Production

“…Despite previous results by Tunev et al (2002), high bradyzoite formation rates would be linked to the balance between cell culture and parasite load, rather than to incubation time alone.…”

“…To standardize observations, parasites expressing only SAG1 were considered tachyzoites, those expressing both SAG1 and BAG1 antigens were designated intermediate bradyzoites (Tunev et al, 2002), and those expressing BAG1 only were considered pure bradyzoites.…”

“…Organotypic slice culture of neuronal tissue has been evaluated as an alternative method to study parasite behavior in the central nervous system (Vonlaufen, Gianinazzi et al, 2002), but bradyzoite conversion was not observed. Recent studies describe keratinocytes as good tools for bradyzoite conversion when used together with a continuous stress treatment (Vonlaufen, Muller et al, 2002), whereas discouraging results have been obtained with other cell lines (Tunev et al, 2002).…”

Stress-driven stage transformation of Neospora caninum

“…The biological role of these phenotypic differences is intriguing, but does parasite morphology dictate specific functions? So far, complex regulatory networks that can act as a molecular switch to permit the coordination of parasite progression and morphogenesis and achieve such morphological specialization are not fully documented (Kang et al 2008a, b;Tunev et al 2002;Vonlaufen et al 2004;Weiss et al 1999).…”

Gene Cloning and Characterization of the Protein Encoded by theNeospora caninumBradyzoite-Specific Antigen GeneBag1

“…NcBAG1 has been identified by its crossreactivity with a monoclonal antibody (Mab) against TgBAG1 . However, cloning of the NcBAG1 gene, which is orthologous to T. gondii TgBAG1, has not yet been reported, despite evidence that the NcBAG1 protein is expressed Tunev et al, 2002;Vonlaufen et al, 2002Vonlaufen et al, , 2004.…”