The in vitro development of Neospora caninum bradyzoites

Weiss, Louis M.; Fen, Yan; Halonen, Sandra K.; McAllister, Milton M.; Zhang, Yi Wei

doi:10.1016/s0020-7519(99)00130-7

Cited by 52 publications

(34 citation statements)

References 34 publications

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“…The mean number of organisms per 9.2-cm 2 well and the percentages of bradyzoites and intermediate zoites are shown in Table I. Previously reported values for bradyzoite production (Weiss et al, 1999) should be compared with bradyzoites plus intermediate zoites in the present experiment. The greatest percentages of bradyzoites (7.7%) and intermediate zoites (6.4%) (total 14.1%) occurred on day 9 in bovine monocytes, without the addition of sodium nitroprusside (superior to all other protocols, P < 0.001).…”

Section: Examination Of Individual Organismsmentioning

confidence: 97%

“…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 experiment were to increase the proportion of organisms that express BAG1 in vitro, to observe expression of BAG1 and also of a tachyzoite antigen (SAG1) within parasitophorous vacuoles over a longer period (9 days), and to determine if the BAG1-expressing organisms have the ability to function like mature bradyzoites.…”

Section: Introductionmentioning

confidence: 99%

“…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 experiment were to increase the proportion of organisms that express BAG1 in vitro, to observe expression of BAG1 and also of a tachyzoite antigen (SAG1) within parasitophorous vacuoles over a longer period (9 days), and to determine if the BAG1-expressing organisms have the ability to function like mature bradyzoites.…”

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confidence: 99%

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

Tunev

McAllister

Anderson-Sprecher

et al. 2002

Journal of Parasitology

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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...

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Section: Examination Of Individual Organismsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Tunev

McAllister

Anderson-Sprecher

et al. 2002

Journal of Parasitology

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“…NcSAG4 is expressed early during the process of stage conversion (Fernández-García et al 2006) and has been shown to be a reliable marker for chronic infection (Aguado-Martínez et al 2008, 2009b. Moreover, antibodies against the T. gondii bradyzoite antigen 1 (TgBAG1), specifically expressed in this stage (Bohne et al 1995), cross-react with N. caninum tissue cysts and bradyzoites obtained in vivo and in vitro, respectively (Weiss et al 1999) suggesting that an orthologous protein exists in this parasite.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

Aguado‐Martínez

Álvarez‐García

Schares

et al. 2010

Acta Parasitologica

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Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite's molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite's surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein's bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4 c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4 c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

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“…Another desirable tool would be a diagnostic technique to detect specific antibodies found only in naturally infected animals. For instance, antibodies against a specific protein of 28kDa (homolog to BAG5 of Toxoplasma gondii), which is only present in bradyzoites 36 , should be found in naturally infected cattle independently whether is vaccinated or not.…”

Section: Control Strategiesmentioning

confidence: 99%