| Animal acariasis is a kind of chronic dermatosis, it is mainly caused by the mites belonged to the family Sarcoptidae, Psoroptidae and Demodicidae through parasitizing the body surface, epidermis and hair follicles of the hosts. It is pathogenic characterized by intense pruritus, exudative dermatitis, depilation and hyperinfection. This disease distributes all over the world and spreads rapidly. Furthermore, it could infect many kinds of domestic animals and wild animals. At present, chemical synthetic drugs such as macrolides, formamidines and organophosphates are still widely used to prevent and treat the psoroptic mange. However, the mite resistance, drug residues and environmental pollution have already caused people’s widespread concern because of the long-term or incorrect usage of chemicals. Thus, the research on the acaricidal activity of Chinese herbal medicine and its components has become a hot topic in this field. Eugenol, existing in many plant oils such as the clove oil, camphor oil and nutmeg oil, is a commonly used acaricidal drug, but its molecular mechanism on killing mites has not been reported. Based on the acaricidal activity of eugenol on P. cuniculi in vitro, the present study measured the effect of eugenol on the mRNA transcriptional level of glutathione-S-transferase(GST) in P. cuniculi, the aim was to elucidate the acaricidal mechanism of eugenol. The results were as follows:1. Determination of the acaricidal potency of eugenol on P. cuniculi in vitro: After P. cuniculi were treated with eugenol solution at 4, 2, 1, 0.5, 0.25 and 0.125 mg/mL, the mortality of mites was observed and recorded under the microscope. In addition, the regression analysis of mortality was proceeded with probit, then the median lethal concentration(LC50) and median lethal time(LT50) of eugenol-exposed adult mites of P. cuniculi was calculated. Results showed that the LC50 of P. cuniculi treated with eugenol was 3.219, 2.289, 0.872, 0.695, 0.507, 0.319 mg/mL at 2, 4, 8, 12, 18, 24 h respectively, and the LT50 at 4, 2, 1, 0.5, 0.25, 0.125 mg/mL was 1.6, 3.5, 8.0, 19.8, 29.4, 52.1 h respectively, which indicated that the acaricidal activity of eugeol was significantly time-dependent and dose-dependent.2. The cDNA fragment cloning of glutathione s-transferase gene in P. cuniculi: Psoroptes ovis and P. cuniculi all belong to the Psoroptidae of Acariformes, the phylogenetic relationship of them is close, thus the primers were designed according to the the gst gene sequence of P. ovis(GenBank accession number: AF078684). The results showed that the cDNA fragment of gst gene of P. cuniculi was successfully cloned, the fragment size was 194 bp, and the similarity of nucleotide sequence was 99% compared with P. ovis. DNAMAN 6.0 speculated that it could code 64 amino acids, and the similarity of 64 amino acids sequence was more than 68% compared with P. ovis, Sarcoptes scabiei type suis and Sarcoptes scabiei type canis. It indicated that fragment of gst gene of P. cuniculi could be cloned successfully by designing primers according to the gst gene of P. ovis.3. Effect of eugenol on the mRNA expression of gst gene in P. cuniculi: LC10, LC30 and LC50 of eugenol(1.682, 2.468 and 3.219 mg/mL) at 2 h were used to treat P. cuniculi, liquid paraffin was set as negative control. After treated with 2, 4, 6 h, the RNA of mites was extracted, and the gst gene was measured by RT-PCR. Results showed that eugenol could significantly reduce the relative expression of gst gene after treated 4 h and 6 h at 2.468 mg/mL. At 3.219 mg/mL, eugenol in three time points all could significantly down regulated the relative expression of gst gene in P. cuniculi. It indicated that eugenol had the function of down-regulating the relative expression of gst gene in P. cuniculi. |
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