| Diarrhea is a major disease that serious harms mink farming in China,with high mortality,high economic loss,complicated etiology,and lack of effective prevention and control technology.The aim of this study is to identify the main infectious pathogens of mink diarrhea and enhance the ability to prevent and control mink diarrheal diseases.We used descriptive epidemiological investigation and experimental epidemiological method to reveals temporal,spacial,and populational distributions of diarrhea in mink farms;sequenced mink intestinal samples with metagenomics to reveal bacterial and viral pathogens;and performed epidemiological investigation and molecular biological analysis on identified pathogens including canine distemper virus,pseudorabies virus,porcine circovirus type 2 and fever with thrombocytopenia syndrome virus,and established diagnosis and detection method of main pathogens to provide technical support for prevention and control minks diarrhea in mink farms.Below are the major results.1.Epidemiological analysis indicated that mink diarrhea diseases in Shandong Province are mainly distributed in Weihai,Qingdao,Weifang,Linyi cities.The onset time of mink diarrhea mainly occurred from June to October,which accounted for more than 90%of the total incidence.The incidence of diarrhea is high at the early stage of weaning and growing periods of minks,and a relatively low in the minks in mature period of winter hair and perinatal minks.2.Statistical analysis indicated that the incidence of the viruses in minks with diarrhea are:canine distemper virus 15.78%,mink enteritis virus 7.97%,astrovirus 9.33%,type 2 porcine circovirus 3.85%,pseudorabies virus 6.87%,the incidence of bacteria are:Escherichia coli 45.34%,Streptococcus 34.67%,Staphylococcus24.19%,Klebsiella pneumoniae 20.19%,Pseudomonas aeruginosa 4.99%,and other bacteria 8.39%.At the same time,there is a co-infection of canine distemper virus and other viruses.The co-infection rate of canine distemper virus and mink enteritis virus is 2.9%,the co-infection rate of porcine circovirus type 2 is 1.79%,the co-infection rate of astrovirus virus is 1.82%,the co-infection rate with the swine pseudo rabies virus is 1.10%.There were obvious regional differences in the detection rate of the viruses.Serological evidence indicated SFTSV infection in mink.3.Metagenomic sequencing revealed common mink viruses Aleutian disease virus and canine distemper virus,also detected mink coronavirus,feline infectious peritonitis virus,herpes simplex virus,reovirus.Statistical analysis show that 198 bacterial species existed in health minks and minks with diarrhea,of which 12 genera are common pathogenic bacteria including E.coli,K.pneumoniae,Pseudomonas,Acinetobacter,Proteus,Aeromonas,Citrobacter,Staphylococcus aureus,Shigella,Enterobacter sakazakii and Aerococcus.4.In minks fed with pork from pigs died of pseudorabies,the incidence of minks was 87%,the fatality rate was 100%.Two pseudorabies virus strains W-MPRV1 and W-MPRV2 were isolated from dead minks.W-MPRV1 had a 282bp deletion at the 3 end of the gD gene.The pseudorabies viral strains isolated from the minks are a strain of the pseudorabies virus.Moreover,the pseudorabies virus strains from minks are closely related to pseudorabies virus that are resistant to Bartha-K61 vaccine,suggesting that the vaccine resistance pseudorabies virus has been widely spread in China.5.Two porcine circovirus 2 strains were isolated from minks died of diarrhea,namely MiSD-1 and MiSD-2.MiSD-1 belong to PCV2b-1B group,MiSD-2 belongs to PCV2b-1C group.6.We established and standardized fluorescence PCR assay for detection 10 viruses and 7 bacteria in minks including mink canine distemper virus,parvovirus,coronavirus,rotavirus,astrovirus and calicivirus,Aleutian virus,Bocavirus,pseudorabies virus,porcine circovirus,and E.coli,P.aeruginosa,Klebsiella,Aeromonas hydrophila,Salmonella,Citrobacter,Acinetobacter.We also established duplex RT-PCR assay for detection of canine distemper virus and canine parainfluenza virus.The lowest detection limits are 1.2 × 10-3ng/and 1.5 × 10-3ng/?L for canine distemper virus and canine parainfluenza virus,respectively,which is much higher than the current minimum detection limit(1ng/?L).We developed a sensitive and specific triple fluorescent quantitative PCR method for detection of E.coli,K.pneumoniae and Acinetobacte with the lowest detection limits of the method as 2 ×10-1CFU/?L,9.2 x 10-1CFU/?L and 4.3 × 10-1 CFU/?L for three bacteria,respectively.7.We developed traditional Chinese medicine preparations for prevention and treatment of digestive diseases of mink including "Chinese medicine for treating digestive diseases of mink","a medicine for treating digestive tract diseases of mink caused by Escherichia coli" and "a medicine for treating Proteus mirabilis type digestive tract diseases of mink".Conclusion1.Mink diarrhea mainly occurs in minks in the weaning stage to the growing period from June to September.The incidence of mink diarrhea is correlated to the density of minks,the higher the density,the higher the incidence is.The poor awareness of biological safety and the relative lag of feeding and management are the main causes of epidemic diseases.2.Through epidemiological investigation,metagenomic and molecular biological analysis we found that the main pathogen of mink diarrhea is canine distemper virus.Under the infection of canine distemper virus,mink can co-infect with variety of viruses and bacteria including parvovirus enteritis and E.coli.Mink can also infect other pathogens such as circovirus,pseudorabies virus and SFTSV,even leading to outbreaks.3.Diagnostic and testing methods have been established and standardized for mink main pathogens. |
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