| Encephalitozoon cuniculi(E.cuniculi),belongs to the genus Encephalitozoon.It can cause opportunistic infections in rabbits,foxes,humans and dogs,and is an important zoonotic parasite.E.cuniculi is highly susceptible to blue foxes.In 2013,Liaoning Province appeared in China’s first blue fox Encephalitozoonosis.The disease mainly parasitizes the kidneys and brain tissues of young foxes and spreads through the placenta and urine,causing the blue fox to be largely eliminated,bring about huge economic losses to the blue fox breeding industry.At present,there are very few reports on the disease in China,and there is no relevant immune prevention measures.A suspected blue fox Encephalitozoonosis outbreak occurred in some blue fox farms in Jiutai,Baicheng and Mohejiang provinces of Jilin Province.The experiment was tested by clinical symptoms,pathological examination and PCR.Then,the strains were purified,stained,isolated,and observed by electron microscopy,and the nucleotide sequences of the isolates PTP1,PTP2,and PTP4 were analyzed,which laid a theoretical foundation for immunodiagnosis.This study investigated the outbreaks of blue fox farms in Jiutai,Baicheng and Mohe,Heilongjiang Province.The diseased fox showed different degrees of dehydration,growth stagnation,and blindness of the eyes.The necropsy showed that the kidney was pale and swollen.The kidney,liver and other diseases were fixed to prepare paraffin sections and subjected to HE staining,modified Masson trichrome staining and Gram staining.Microsporidia were detected in major organs such as kidney and liver.According to the E.cuniculi ITS1 gene sequence registered by Genebank,the specific primers were designed.The 762 bp fragment was cloned by extracting the kidney tissue DNA.After sequencing analysis,12 cases of blue fox E.cuniculi were diagnosed.The pathogen is E.cuniculi gene type III.In this study,the kidneys were ground and centrifuged to purify the worms.The purified parasite was inoculated with MDCK cells,and the cells were observed to have a certain degree of swelling after continuous blind transmission to the sixth generation.The parasite accumulates on the inner side of the cell.The isolated parasites were observed under a 20,000-fold transmission electron microscope and have various shapes,mainly with short rods with obtuse ends at both ends.The middle part of the parasite has a filament extending,and the isolated parasite is determined to be a E.cuniculi,and the JL strain is named.This study was based on the PTP1,PTP2,and PTP4 gene sequences that Genbank landed.Using software to design nested primers,DNA was extracted and PCR amplified to obtain fragments of 1128 bp,834 bp and 831 bp,respectively.The threesegment genes were ligated to the PMD-T vector,and after PCR and double enzyme digestion,three sets of recombinant plasmids were successfully constructed.The sequence was compared with the domestic and foreign classical strains,and the highest of the three protein genes were 98.7%,99.9% and 98.8%,respectively.The genetic phylogenetic tree showed that the PTP1 gene belongs to the same branch as the domestic fox source isolate and the American source isolate,and the 26 amino acid sequence deletions existed in the same locus as the fox source isolate.The PTP2 gene is more conserved and only one base is deleted,laying the foundation for the next step of tube protein application in diagnosis and immunotherapy... |
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