A Study Of Runt-like Symptom Causes、epidemiological Investigation And Comparative Transcriptome、comparative Proteomics In Raccoon Dog (Nyctereutes Procyonoides)

Raccoon dog(Nyctereutes Procyonoides)is one of the precious fur animals,which has been widely farmed in China.The runt-like raccoon dogs,also known as raccoon dog runt-like disease,occurring in recent years,has seriously affected the productivity of raccoon dog farming.in the present study,the clinical symptoms,infection source,blood physiological and biochemical parameters were investigated,and then,The prevalence of Microsporidia,eperythrozoon,and raccoon dog runt-like disease were examined to get a preliminary understand to mummification.Last,we used transcriptome and proteome to compare the normal and mummification animals to get deep into the mechanism :1)Using the runt-like raccoon dog samples in Hebei region,based on the pathologic examination,colloidal gold test paper,PCR,culturing bacteria,animal regression,and blood examination,in combine with the corresponding medical treatment to exclude the Coronavirus,Canine distemper virus,Parvovirus,Mink Aleutian disease virus,Raccoon dog blue fox source Aleutian disease virus,and the genetic reason,we comprehensively concluded that the infection of eperythrozoon was the main reason,while the combined infection of eperythrozoon and enterocytozoon bieneusi also resulted in the runt-like raccoon dog.2)The overall positive rate of enterocytozoon bieneusi in main productive regions is 22.30% based on the nested PCR,with the highest rate of 40.74% in Hebei region(Male = 27.10%,and Female = 17.33%).The six different ITS regions were identified through DNA sequencing,including four known genotype(D,CHN-DC1,NCF2 and CHN-F1),and two novel genotypes(NCR1 and NCR2).Phylogenetical analysis indicates that the six genotypes is defined as the groups 1-zoonotic groups,suggesting the possible infection source of human enterocytozoon bieneusi,which should be noticed in human public healthy.3)We examined the raccoon dog blood samples of the main region in China using the blood smear test method.The results showed that the overall positive rate of eperythrozoon was 62.29%,and Hebei Province had the highest positive rate of 68.64%,and the positive rates of female and male raccoon dogs were 61.16% and 63.38%,respectively.This finding suggested that the immunosuppression caused by eperythrozoon with large rate should be paid to attention.4)We investigated the overall positive rate of runt-like raccoon dog in 38 farms from 7 provinces.The results showed that there were no significant differences among these provinces,but the differences from farms with each province were significant.The difference between the high and low rate was 18.4 times.The rate of runt-like raccoon dog is conversely related to the management level.The farm with high management level had low rate of runt-like raccoon dog.5)We applied the transcriptome to examine and compare the liver and spleen from 14 samples.We obtained 1,571,229 transcripts and 290,568 unigenes.By blast against with Nr database,the results showed that raccoon dog had a similarity of 61.62% to dog,consisting with the known classification results.In comparison with healthy raccoon dog,there were 1,842 and 876 genes were up-regulated and down-regulated,respectively,in spleen of runt-like raccoon dog.There were 1,392 and 604 genes were up-regulated and down-regulated,respectively,in liver of raccoon dog.These findings provided candidate genes to the diagnosis and treatment and prevention of runt-like raccoon dog.6)The pathway enrichment of comparative transcriptome of spleen from runt-like raccoon dogs found 42 pathways from 6 categories.The clustering analysis showed the differently expressed genes in spleen between healthy and runt-like raccoon dogs obviously enriched in metabolism and PI3K/Akt signal pathway.The pathway enrichment analysis of comparative transcriptome of liver from runt-like raccoon dogs found 41 pathways involving in 6 categories.The clustering analysis showed the enrichment pathways of different genes in liver between healthy and runt-like raccoon dogs were mainly focused on ECM receptor interaction pathway,metabolism,and ECM receptor interaction pathway.7)A total of 2,469 proteins were identified in the comparative analysis of spleen proteome between healthy and runt-like raccoon dogs,of which 1,484 proteins were quantified.There were 23 proteins were up-regulated in runt-like raccoon dogs as compared with healthy raccoon dogs,which were cell skeleton components involving in the regulation of enzyme activity during the bioadhesion and cell adhesion processes.And there also were 33 proteins were down-regulated,which MHC protein components were involving in immune responses.KEGG analysis found the up-regulated and down-regulated genes participated in 25 and 2 pathways,respectively.8)A total of 2,025 proteins were identified in the comparative analysis of liver proteome between healthy and runt-like raccoon dogs,of which 1,282 proteins were quantified.There were 93 proteins in liver involving in 1 pathway,were up-regulated in runt-like raccoon dogs as compared with healthy raccoon dogs.And there also were 119 proteins were down-regulated,involving in 16 pathways.The first three pathways of the different proteins between healthy and runt-like raccoon dogs were oxidoreductase activity,iron ion binding,heme binding,which were closely related to some functional components such as the breath and oxygen delivery after the infection.