| Chicken coccidiosis is caused by Eimeria which is intracellular protozoan parasites，and result in large losses chicken-raising industries．Clinically, it generally causes by two ormore kinds of coccidian. In the study, a strain of Eimeria was sucessfully isolated, then itwas identified as the strain of E. maxima HF by morphology and molecular biologymethod, To make further observations on its pathological lesions on chickens,21-day-ldbroilers were infected with the sporulated oocysts of E. maxima, meanwhile some serumbiochemical values were determined post-infection．Firstly, the feces from classically symptomic coccidian chickens in hefei city werecollected, then the oocysts were primarily examined by microscope to make sure that itcontains the oocysts of E. maxima, then cultured with2.5%potassium dichromate at28℃.Cellophane was used as a carrier to separate single oocyst with the improved thesingle-occyst technology, and single oocyst were contained into capsule and orallyinoculated to5-day-old chicks.5days after it ensureed if they were infected. Secondly, theoffspring of the single oocyst were inoculated to12-day-old chickens for proliferation,120h after inoculation inoculated and the faces was checked every hour the faces per hour,and record the minimum sporulatic time and prepatent period. AgainThirdly, coccidia theDNA of isoalted oocysts was extracted, the specifitic primer was designed with thecomparation of the reported by Yao-Chi Su，the sequence was EM1：5’-TTG TGG GGCATA TTG TTG TG-3’; EM2：5’-CAA TGA GGC ACC ACA TGT CT-3’. ITS-1wasamplified by PCR and sequenced. Finally,E.maxima at5×10~4, heart blood was collected at0d,7d,11d,15d after post-infection to determine the index of serum plasma,serumenzymes and immune indicators．The results show that(1)six of twenty chickens were infected and the infected rate was30%;(2)The second generation of oocysts was27.51～36.68μm×20.96～31.44μm and32.18μm×25.84μm in size at average, the oocyst body exponent was1.25. The minimumsporulatic time and prepatent period was130h and35h, the first generation oocysts weremeasured through microscopic and the size was consistent with the standard E. maximaand also with either generation indicating that the second generation of coccidia oocyst ofisolation was E. maxima named HF strain;(3) PCR amplification showed that there was151bp fragment according to the expected, blast showed that the similarity was very highattained98.7with North Carolina Ⅱ;(4)Animal experiments showed that7th day afterinfection，the bursa of fabricius index decreased significantly (P<0.05) compared with that of the control, total protein, albumin also was very significantly decreased (P<0.01)compared with that of the control, Mg2+, IgA and IFN-γ were significantly increased(P<0.05) compared with that of the control, monocytes, leukocytes, lymphocytes, catalaseand NO were very significantly higher than the control (P<0.01);11th day after infection.in the average weight. the bursa of fabricius inde. alanine aminotransferase and alkalinephosphatase, it was very significantly decreased (P<0.01).In summary, we successfully established a single-oocyst-isolation technology andEimeria could be isolated by it, through morphological and molecular biologicalidentification. The coccidia can be identified as E. maxima (HF). The physiological andbiochemical indexes were measured. Tt initially proved the physiological and biochemicalindices at different stages. We offers a new approach to establish purebred oocysts. Theresults is very meaningful for preventing and curing chicken coccidian in Hefei andimproving economy benefit. |
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