Identification of Histomonas meleagridis by in vitro microculture and polymerase chain reaction
Wei Liu1,*, Jun-yu Peng2,*, Fen Li1, Hong-yan Sun3, Ying Ding1, Jing He1, Yi Liu1
1College of Veterinary Medicine, Hunan Agricultural University, Changsha; 2NeX Eco-Agriculture Technologies, Inc, Chenzhou, Hunan; 3Key Laboratory of Applied Marine Biology of Guangdong Province and Chinese Academy of Sciences, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, People's Republic of China
*These authors contributed equally to this work
Background: In this study, methods for the in vitro microculture of Histomonas meleagridis were defined to lay the foundation for further study of its diagnosis, life cycle, and pathogenic mechanism.
Methods: After serial dilutions of H. meleagridis, a single parasite was taken and cultured at 40°C. Its proliferation was checked by microscopy, and an 18S rRNA gene fragment of H. meleagridis was amplified using specific primers (Hmf/Hmr) and sequenced. The sequences were assembled manually and aligned using the Clustal X 1.81 program.
Results: Two tubes showed proliferation of parasites and, after infection with parasites from these tubes, acute histomoniasis was rapidly induced in turkeys. 18S rRNA gene fragments of the two strains were 492 and 495 base pairs in length, respectively, and the sequence homologies with known H. meleagridis sequences were both 99.4%. Sequence homology between these two strains was 99.6%, and the variation between the two sequences and Trichomonas was more than 20%.
Conclusion: We conclude that microculture can produce high-purity and highly pathogenic H. meleagridis.
Keywords: Histomonas meleagridis, microculture technology, polymerase chain reaction, identification
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