| Avian trichomonosis, a parasitic disease caused by the flagellated protozoan Trichomonas gallinae(T. gallinae) is one of the most serious diseases against pigeons, leads to considerable economic losses to the pigeon industry worldwide. T. gallinae parasitizes live mainly in the upper digestive tract of different avian species, where they can cause mucosal inflammation, ulcers and granulomatous lesions that occlude the oesophagic lumen, leading to the death of birds as a result of severe starvation. In clinical applications, several nitroimidazole drugs including metronidazole, dimetridazole and tinidazole have been considered the main drugs for the prevention and treatment of T. gallinae. However, the use of nitroimidazole drugs has been greatly restricted with the continual emergence of drug resistant organisms, the toxic side effects, the drug residues and the public concern of safety in animal foodstuff. So, it has become one of the hot topics that select new drugs which effective and low toxicity against T. gallinae to replace and reduce the use of synthetic drugs.In current pharmaceutical market, the plant and animal based naturally produced agents have reached more than 30%. Among them, traditional medical based naturally produced agents is becoming more popular with the advantages of variety, low prices, low toxicity, no residue,no resistance and no pollution. Trollius chinensis Bunge is a traditional Chinese tea, as well as an endemic medicine herb, have been used to treat upper respiratory infections, bacillary dysentery, pharyngitis and urinary tract infection. It had been well proven that the aqueous extract of Flos trollii possesses antimicrobial, antiviral and anti-T. vaginalis activities. Celosia cristata L is beneficial for liver injury underlies disease like hepatic fibrosis, livercirrhosis and hepatocellular carcinoma as a traditional medicine herb. It also has a relatively potent effect on T. vaginalis and the improvement of animal performance. In addition, significant effect was confirmed that compound group consists of various single herbs used to treat avian trichomonosis. Firstly, methods including separation, identification and culture of T. gallinae were established in this study. Then, several traditional Chinese medicines with the anti-protozoan activities on T. gallinae in vitro were selected. The IC50 on T. gallinae was determined by counting the live trophozoites, the TC50 on CEF was evaluated by using a MTT method and the SI of the drugs was obtained by the analysis of IC50 and TC50. Finally, the mechanism of drugs-induce T. gallinae death was detected. This experiment includes next parts:1. The separation, identification and culture of T. gallinae in vitroT. gallinae was isolated from oral swabs of a pigeon and identified by clinical symptoms,pathological changes, etiological diagnosis and molecular biology diagnosis in this study.Then the growth curve of sterile T. gallinae was established in the medium of HF. The result showed that the initial density of was 1.5×105 /ml T. gallinae cells were able to well grow in HF and the number of T. gallinae cells were straight up during 36 hours to 60 hours.2. The screening of traditional Chinese medicines which has the anti-protozoan activities on T.gallinae in vitroThe experiment showed that the water decoction of Trollius chinensis Bunge, Celosia cristata L, Zanthoxylum bungeanum Maxim and Elsholtzia ciliata(Thunb.) Hyland have a higher activity on T. gallinae in test medicines(MLCs=100mg/mL). The water decoction of Artemisia carvifolia and Cnidium monnieri(L.)Cuss have a moderate efficacy on T. gallinae(MLCs=200mg/mL). But the others drugs like the water decoction of Agrimonia pilosa Ledeb, Nepeta cataria L and Senecio scandens Buch-Ham ex D Don have low efficacy on T.gallinae(MLCs≧400mg/mL).3. Sensitivity determination of T. gallinae in vitro and the evaluated of SIBased on our preliminary experiments, different drugs including metronidazole,dimetridazole, Trollius chinensis Bunge, Celosia criatata L, Comound A, Compound B were chosen to against T. gallinae cells in vitro. The relative inhibitory rates at different hour were determined by counting the live trophozoites. The results showed that the T. gallinae strain was resistant to metronidazole and dimetridazole. The IC50 of the water extracts of Trollius chinensis Bunge, Celosia criatata L, Comound A and Compound B were 0.91、0.34、0.26 and0.39mg/mL respectively. Our results reveal that the potencies in descending order for the herbs examined was Comound A﹥Celosia criatata L﹥Compound B﹥Trollius chinensis Bunge.After 48 hours’ incubation in vitro, the TC50 was evaluated on CEF by using a MTT method. The TC50 of the water extracts of Trollius chinensis Bunge, Celosia criatata L,Comound A and Compound B were 1.91、1.80、7.06 and 9.09mg/m L respectively. The results showed that the water extracts of Trollius chinensis Bunge has a low selectivity index, the water extracts of Celosia criatata L has a significant selectivity index, the water extracts of compound A and B are no toxicity. These traditional Chinese medicines are then a potential source of a new anti-T. gallinae drug.4. The mechanism of drugs-induce T. gallinae cells deathThe methods including morphology aspects, nuclear staining by DAPI, FSC/SSC change, JC-1 staining and FITC&PI staining were used to detect the mechanism of drugs induce the T. gallinae death. The results showed that the shape and nuclear of T. gallinae which incubated with metronidazole and the water extracts of Trollius chinensis Bunge were destroyed, the membrane remains intact, the hydrogenase membrane potential of a lage number of T. gallinae cells were changed in a shot time and most of apoptosis T. gallinae cells were detected among the death parasites. The above results suggested that the two drugs induce apoptosis on T. gallinae. However, the T. gallinae which incubated with the other three drugs were breakdown rapidly, the nuclear bocome round and larger, the hydrogenase membrane potential of part T. gallinae cells were changed at 24 h and majority of apoptosis T. gallinae cells were detected among the death parasites. The above results suggested that the other three dyugs lead to apoptosis, oncosis and necrosis. |
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