Research On Cultivation Of Steinmann Eimeria In Vitro

Objectives:We obtained the pathogen of rabbit hepatic coccidiosis that is Steinmann Eimeria through culturing rabbit liver cells in vitro.To provide the necessary preconditions for further study on the life history and the pathogenicity of Eimeria, drugs and vaccine for prevention of this disease.Methods:Insecticide treated young rabbits was killed and remove its liver to conduct primary cell culture, and transferred them to 24-well culture plates after getting stable primary cells; at the same time, dealing with the rabbits'liver which was taken from those diagnosed coccidiosis in Changzhi rabbit farms, then getted the Steinmann Eimeria oocysts after separation and purification, we conducted sporulation until its hatching rate reached more than 85%, cell disruption with mechanical grinding methods; Isolated the spore, and inoculated which into liver primary cells, observed its proliferation and finally got the oocysts, then inoculate the oocysts into healthy young rabbits to validate it's virulence.The experiment was divided into four groups based on the quantity of Steinmann Eimeria oocyst,namely:control group, the experimental group 1, the test group 2, the test group 3, dissected the rabbits, observed liver's pathological changes and analyzed the results on 18d post inoculation.Results:(1) The results of rabbit liver primary cell cultures showed that:It was easier to get rabbit liver primary cells by killing it but not exsanguinated from neck.The cells showed sporadic adherent in culture process for about 16h, growth stably appeared in 4-5d.then got active rabbit liver primary cells through 20 days of culture.(2) Steinmann Eimeria oocysts sporulation experimental results showed that the optimum temperature for Steinmann Eimeria oocysts was 32? in 2.5% potassium dichromate solution, Steinmann Eimeria oocysts can be more than 85% after 5 days hatching.(3)The experiment of inoculation spore into liver primary cells showed that:the sporozoites showing slightly curved at central like a banana-shaped recessed under inverted microscope when just inoculation Steinmann Eimeria sporozoites, and the surface is gray and shiny, part of the structure had entered inside the cell after about 1h, sporozoites had totally entered the interior of the cell after 1.5h; there was no significant changes in the first few days, then found large amounts of gray shiny worm balls part of the cell shrinkage and cast-off cells under the microscope untill after 15d, observed tintact oocyst at 28td, rabbit liver cells and oocyst most shedding suspended in cell culture medium at 34td.(4)verified test results showed:Steinmann Eimeria oocysts are still pathogenic for young rabbits when cultivated in 2.5% potassium dichromate solution,oocysts can be found in feces just in one day after inoculated oocysts into the young rabbits, peaked at 9-13td,Meanwhile,the young rabbits inoculated with coccidia oocysts developed clinical symptoms in varying degrees. Pathological changes weremainly found in the liver, found varying degrees of liver volume increases,the substance and surface of the liver appeared a large number of visible circular white or yellowish nodules, other organs had no significant pathological changes.Conclusions:we obtained the rabbit liver primary cell in vitro successfully whose survival rate was high and improved the Eimeria stiedae purification method, so that more suitable for the sporulation and purification of Steinmann Eimeria oocysts, we also acquired Steinmann Eimeria oocysts and sporozoite with less impurity. We finally obtained invasive Steinmann Eimeria oocysts in vitro, provided the conditions for the obtainess of coccidia oocysts in large quantities in vitro.