Interconversion between tachyzoites and bradyzoites of Neospora caninum plays a pivotal role in transmission of the parasite. Although significant efforts have been made toward understanding the mechanisms that trigger and control stage conversion of the parasite, little is known about this process. We used annealing control primer (ACP)-based polymerase chain reaction (PCR) technique to characterize the differences in transcription between tachyzoites and bradyzoites of N. caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by treatment of infected cultures with 70 μM sodium nitroprusside. Subsequently, the gene expression profiles of the tachyzoites and bradyzoites were analyzed through comparison of the level of messenger RNA expression. ACP-based PCR revealed 85 amplicons that were consistently differentially expressed between tachyzoite and bradyzoite stages. Of the 85 differentially expressed transcripts identified, ten were cloned into Topo TA cloning vector, sequenced, and further analyzed by the Basic Local Alignment Search Tool. These differentially expressed transcripts include a combination of known genes and as yet unidentified genes. The present work provides candidate genes for further investigation on molecular basis of stage conversion from tachyzoites to bradyzoites of N. caninum.
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This study was financed by a grant from the National Veterinary Research and Quarantine Service, Korean Ministry of Agriculture and Forestry. The authors wish to thank Khac-Minh Thai and Dung Tien Le for their helpful discussions.
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Differential expression of transcripts between two developmental stages. ACP-based PCR is applied to identify DEGs between tachyzoites and bradyzoites using GP, a combination of 120 arbitrary primers and two anchored oligo(dT) primers of ACP-based fishing kit (See-Gene, Seoul, Korea). Gels are photographed to show differential banding patterns between tachyzoites (2) and bradyzoites (3), and Vero cells (1) produced in vitro after ACP-based PCR. The amplified cDNA products are separated on 1.5% standard agarose gels stained with ethidium bromide (DOC 7.13 MB)
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Kang, S., Kweon, C., Lee, E. et al. The differentiation of transcription between tachyzoites and bradyzoites of in vitro cultured Neospora caninum . Parasitol Res 103, 1011–1018 (2008). https://doi.org/10.1007/s00436-008-1082-5
- Vero Cell
- Tissue Cyst
- Amplify Polymerase Chain Reaction Product
- Stage Conversion
- Specific Polymerase Chain Reaction Product