Isolation And Identification Of Neospora Caninum And Establishment Of Immunological Detection Methods

Neosporosis is a protozoal disease,which is caused by the Neospora caninum parasites in vitro of multiple animals,and it can cause the death of livestock.Now,it has been proved that canine is the final host of the Neospora caninum,which is a protozoan parasite with a great variety of intermediate hosts such as cow,horse,sheep,spotted dear,wild ox,pig,guinea pig,gerbil and domestic cat.This disease that can cause the abortion of pregnant domestic animal and dead fetus and bring huge economic loss to the animal husbandry,is not treated and prevented by efficacious drugs.So,it is still the most effective way of preventing and stamping out this disease through diagnosing,isolating and eliminating sick animals.Currently,detection technologies of Neospora caninum have been researched by many domestic and foreign scholars.The methods of detection through seperating Neospora caninum from the brain tissue of infected animal based on vero cells and HE staining and observing pathological changes and estamblishing immunohistochemistry and indirect immunofluorescence to artificially infected experimental animals.1.In the study,cellular pathogen isolation method of neospora caninum was established.Firstly,cattle's fetal brain tissue homogenate in abortion caused by suspiciously infecting with neospora caninum was inoculated to evenly growing single-layer Vero cells.Meanwhile,it was inoculated to mice through intraperitoneal injection.Cellular morphology was examined under a microscope.Meanwhile,mental state and clinical symptoms of mice were observed.After inoculating cells,there was falcate polypide under the microscope.Polypide DNA in cells and DNA in primary solid organs of mice with symptoms were extracted.According to NcSRS2 gene in GenBank,the complex primers were designed to amplify and test sequences.The homology was 99%.Neospora caninum was successfully separated.2.In order to better observe the clinical symptoms after the host was infected by the neospora and the degree of injury of tissue organs,injected larvas via abdomen to artificially infect rats and observed the clinical symptoms that they occurred.Meanwhile taking the tissue organs to make paraffin section and using HE stain to stain the section,to observe the degree ofinjury of the tissue organs.After 3 days when the rats were inoculated neospora,they were depressed,appetite decreased,drinking appetite enhanced in the earlier stage,afterwards symptoms like afraid of seeing people and huddle up,then the drinking appetite also decreased and seldomly ate in later stage.At necropsy kidney swelling,meningeal congestion and there were red lesions on the lungs were seen.Tissue organs were taken to make paraffin section.After HE dyeing,pathological changes were observed,showing extensive bleeding in visceral organs,necrosis of cardiac muscle fibers,uneven myocardial cell arrangement,disorder hepatocyte arrangement,marginalization of cell nucleus,swelling hepatocyte,congestion of sinus lienis,tube wall thickening of central artery,broad pulmonary alveoli mesenchyme,disappear of morphology,inflammatory cell infiltration,gradual disappear of glomerular morphology,kidney extravasated blood,small kidney tubule cavity,extravasated blood,swelling degeneration of cerebral neuron,and brain tissue bleeding,etc.3.Immunohistochemical method was established to detect the neospora.The neospora immunohistochemical test method was successfully established in the study by optimizing conditions to the working concentration of the primary antibody and secondary antibody,and the incubation time and the repair of antigens.The result was best when diluting the primary antibody 400 times and secondary antibody 100 times,with background staining light and positive cells staining dark.Cysts can be detected in the brain tissues of the infected rats with this method.Immunohistochemistry examining the rat tissues with the artificially infected toxoplasma and B.bigemina,the result proved negative,which proved that this method had better specificity.4.Optimal screening antibody's working concentration by using the neospora tachyzoite as the known antigen.When diluting the primary antibody 400 times and 200 times dilution of the FITC-labeled secondary antibody,the non-specific fluorescence is weakest with the most clear polypide morphology,emitting bright fluorescence with the greenish yellow color,which successfully established the indirect immunofluorescence in testing the neospora.Conducted test to the positive serum of the toxoplasma and B.bigemina,the result proved negative,without fluorescence,showing the method had good specificity.Repeatedly tested 3 times by randomly extracting serum,with no significant difference between the 3 results,showing the method had good repetitiveness and stability.Tested the 236 cow serum samples collected from multiple dairy farms in Jilin Province with this established method,there were 38 serum samples proved positive,with the positive rate of 16.1%.