Genetic diversity of Theileria orientalis in tick vectors detected in Hokkaido and Okinawa, Japan.

In the present study, we investigated the possible tick vectors that can transmit Theileria orientalis in eastern Hokkaido, Japan. Questing ticks collected from three different districts, Taiki, Otofuke, and Shin-Hidaka, of Hokkaido included Ixodes persulcatus, Haemaphysalis megaspinosa, Haemaphysalis douglasi, and Ixodes ovatus, while all the ticks collected from Yonaguni island of Okinawa were identified as Haemaphysalis longicornis. When the ticks were screened by polymerase chain reaction (PCR) for T. orientalis, the parasite was commonly detected among all tick species. Genotype-specific PCR assays revealed that all tick species in Hokkaido were predominantly detected with type 2, while ticks collected from Okinawa (H. longicornis) were predominantly detected with type 1. Consistent with the genetic diversity of T. orientalis in ticks, genotyping PCR assays from cattle grazed in the same Hokkaido sampling locations identified type 2 as the most prevalent genotype. This study provides the first identification of I. persulcatus, H. megaspinosa, H. douglasi, and I. ovatus as possible tick vectors of T. orientalis, and finds that the variety of vectors apparently capable of transmitting T. orientalis is wider in Japan than expected. The authors suggest that tick control strategies should be modified in Hokkaido based on the seasonal activities of ticks identified in the present study.

Epidemiological survey of Theileria orientalis infection in grazing cattle in the eastern part of Hokkaido, Japan.

Theileria orientalis is one of the benign species of Theileria that is widely distributed in Japan and is sometimes responsible for serious economic losses in the livestock industry. In the present study, we surveyed the current status of T. orientalis infection in grazing cattle in the eastern areas of Hokkaido (Taiki, Otofuke, Shintoku, and Shin-Hidaka districts) using molecular methods, as well as traditional methods, of diagnosis. The genes encoding the major piroplasm surface protein (MPSP) and p23 of T. orientalis were identified using highly detectable polymerase chain reaction (PCR). Results of the MPSP-PCR assay indicated that grazing cattle in these districts, after about 1.5 months pasturage, showed high rates of infection, ranging from 10.0-64.8%. Although the main MPSP and p23 genotypes detected were the Ikeda- or Chitose-types, an MPSP gene closely relating to that found in Okinawa prefecture, and a p23 gene closely relating to the Australian (Warwick) Buffeli-type gene, were found in the cattle in Shintoku and Shin-Hidaka districts. The present survey indicated that there were at least five types of T. orientalis classified by their MPSP genes in Hokkaido, Japan, and that T. orientalis infection rates are still high in this region.

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.