| Blue fox,as an important fur economic animal,is widely bred in northern China.In recent years,the cases of blue foxes infected with Encephalitozoon cuniculi have occurred in several main breeding areas in China.After infection,blue fox pups were mainly characterized by growth stagnation,diarrhea,emaciation and death.Even if they are not dead,their growth is affected and they form"stiff foxes".In the later stage of disease in blue fox,neurological symptoms and kidney enlargement were the main symptoms.Some adult blue foxes do not show clinical symptoms after infection,but they can be a source of infection to spread pathogens.After the disease,not only the quality of blue fox fur is seriously reduced,but also the recessive infection of the breeding fox would be vertically transmitted to the next generation,thus bringing huge economic losses to the blue fox breeding industry.Encephalitozoon cuniculi is a kind of Microsporidia,which is a single-celled eukaryote parasitic in cells and belongs to the fungus family.Mature spores can be excreted through the urinary tract of blue foxes,thus spreading and infecting other animals.When female animals are occult infected with microsporidia,they can also infect their offspring through vertical transmission,resulting in the elimination of a large number of breeding animals.The recessive infection rate of this disease is high,which can not only spread widely among animals,but also cause zoonotic diseases.In addition,there is currently a lack of effective immunization and treatment methods.Therefore,it is worthy of our in-depth study to establish a detection method that can detect whether the blue fox is infected with Encephalitozoon cuniculi.In this study,Encephalitozoon cuniculi was isolated and purified by inoculating dog kidney(MDCK)cells,and verified by PCR and Gram staining.When the ITS1 gene sequence was analyzed,it was found that it belonged to the Encephalitozoon cuniculi type III,and the phenomenon of heavy staining was found in the Gram staining.Polyclonal antibodies and blue fox antibodies against Encephalitozoon cuniculi were prepared and purified by making immunization program.The titer of polyclonal antibody was 1:5.112 x 10~5 and the Ig G content was 1.77 mg/ml.The titer of positive fox serum was 1:64 and the Ig G content was 0.15mg/ml.Through the continuous optimization of reaction conditions,a sandwich ELISA method for detecting Encephalitozoon cuniculi in blue fox was established,and finally the best concentration of coated antibody was 5μg/ml,and the best concentration of detected antibody was 37.5μg/ml;The antigen was incubated for 2 hours,and the antibody was incubated for 1.5hours.The best coating condition was 4℃overnight;Enzyme-labeled second antibody was diluted at a ratio of 1:5000 and then reacted for 1 h;Choose 5%skim milk as sealing solution and seal for 1 h.The sensitivity,reproducibility,specificity and clinical sample detection effect of this method were evaluated.The lowest detection sensitivity for purified Encephalitozoon cuniculi was 6.25 x10~5 spores/ml;The lowest detection sensitivity for artificial addition was 1.25×10~6spores/ml;Repeatability was less than 10%;There was no cross reaction with Streptococcus agalactis and Pseudomonas aeruginosa.In preliminary application,the coincidence rate with PCR detection method was 79.97%.The results showed that the sandwich ELISA of Encephalitozoon cuniculi in blue fox established in this experiment had good performance in detecting purified spores and artificially added spores,and also had certain applicability in practice.The sandwich ELISA method established in this experiment can provide a more simple and rapid detection method for large-scale screening of blue foxes infected with Encephalitozoon cuniculi,and also provide a theoretical basis for the study of immunological detection methods of Encephalitozoon cuniculi. |
