| The intestinal disease known as coccidiosis caused by protozoan parasites of genus Eimeria is one of the most economically devastating diseases in the poultry industry world wide. Of the seven species of Eimeria, which infect the chicken , Eimeria tenella is one of the most ubiquitous and the most pathogenic.This study mainly establish a new single-oocyst isolation technique and get HeFei strain of E.tenella from hefei around. Furthermore ,we identify HF strain through morphology and molecular biology method. Finally we made study on it's pathogenicity.First, the oocysts of fecal of cecum, from classically symptomic coccidian chicken in hefei city , were collected by centrifugation and floatage, then cultrued with certain 2.5% potassium dichromate and 27℃in incubator. We improved the single-occyst technology on previous experiment, and separate technology of single oocyst were improved that 2% agar block was used to separated single oocyst , and made single oocyst into capsule .The test on using single oocyst infect chicken indicated the character in sizes and oval index and parasite place are similar after challging of continuious generation, so it can be identified as Eimeria. tenella . Of eight chickens there are three chickens were infected, and the victory rate is 37.5%.Then, the oocyst were identified in morphology aspect. The oocyst were propagated twice through 12-day-old chickens, then 150 ooccysts of either generation were measured. The result of the first generation is: the oocysts are 16.8～24.6μm×16.1～20.7μm and 21.24μm×18.14μm in average size。The oocyst body exponent is 1.17.The sporanges are 10.10～12.95μm×4.40～7.25μm and 11.60μm×5.99μm in average size.The result of the second generation is:the oocysts are 17.6～23.3μm×15.5～20.5μm and 21.11μm×18.04μm in average size,The oocyst body exponent is 1.17. The sporanges are 10.10～12.95μm×4.60～7.25μm and 11.59μm×6.01μm in average size。The first generation Oocysts were measured through microscopic and the result of measure is comparatived with standard E.tenella and also with either generation .The result showed that the oocysts of isolation is E.tenella, named HF strain.Furthermore the oocyst were identified in molecular biology aspect. After the oocysts were purified in several steps, the oocysts were cracked and DNA was extracted with a DNA Extraction Kit. The primer was designed according to Schnitzler, B.E.: ETF: 5'-AAT TTA GTC CAT CGC AAC CCT-3';ETR: 5'-CGA GCG CTC TGC ATA CGA CA- 3'. IST1 was amplified by PCR and sequenced. Predicted 278 bp were amplified and were comparatived on blast. It shows that the similarity is very high. By far this method is firstly used in identity of Eimeria tenella in our province.Finally, HF strain's pathogenicity was analysed in clinic symptom, pathological changes integral , pathology changes and so on. It could cause death when the chickens was inoculated 10×10~4 oocysts. The mortality rate rose along with more oocysts. When the chicken was inoculated 4.0×10~4 oocysts, the mortality rate is 30%,when the inoculation is 8.0×10~4,the mortality is 70%,when the inoculation is 1.6×10~5,the mortality is 90%. The result shows that the pathogenicity of Eimeria tenella is very high.Above all, we succed in building a new single-oocyst isolation technique and isolated Eimeria tenella HF strain. The traditional morphology method and molecular method was used in identity. We also anlysed it's pathogenicity . The single-oocyst-capsule isolation technique provide a new method to establish pure oocysts; The isolation,identity and pathogenicity of Eimeria tenella HF strain would be very meaningful for preventing and curing chicken coccidian in HeFei and improving economy benefit. |
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