| Background: A mink is a kind of small carnivorous mammal that has soft and sick fur that can be manufactured into various popoular fur products.The mink industry has achieved a fast development recent years in China.At the same time,many infectious diseases,especially varial infectious diseases make great trouble to the mink industry.Mink refractory diarrhea is a seasonal infectious disease caused by mink circovirus(MiCV).The disease usually occurs during the period between autumn and winter,which can cause red and grey diaherra,accompanied by anorexia and unkempt fur.Most sick minks recover but suffer poor quality of fur and the fecundity of female minks declines.About 7-8% minks get worse,showed black bloody stools and pale muzzle and died as a result.The disease has been epidemic seasonally in mink farms in China since 1970 s and has made a significant influence to the mink industry.However,the prevention and control technology of mink refractory diarrhea is deficient and the research on the epidemiology of MiCV is relatively poor,too.Object: Firstly,the aim of the study is to investigate the prevalence of MiCV in the main mink farming districts in China in order to get the epidemic law and to provide theoretical bases for the prevention and control of the disease.Secondly,the aim of the study is to establish a recombinase polymerase amplification(RPA)method which can provide an advanced detection technique for the disease.Lastly,the aim of the study is to express the capsid protein(Cap)of MiCV by baculovirus expression vector system and to develop a subunit vaccine against MiCV which can provide a technical support for the prevention,control and elimination of the disesase.In summary,the aim of the study is to provide a comprehensive solution of mink refractory diarrhea including the epidemiological study,detecting techniques and vaccine development.Methods:1.In 2019 and 2020,we went to the mink farms in Zhuanghe(Liaoning Province),Changli(Hebei Province)and Zhucheng(Shandong Province)to investigate the prevalence of the mink refractory diarrhea and collected fecal samples for a further detection.We compared the prevalence in different years and districts.We also compared the morbidities,fatality rates and infection rates of male and female minks and one-year old and multi-year old minks.In addition,some healthy and sick minks were dissected.Various tissues and organs were observed for pathological changes and were collected and further processed for a pathological section examination.We detected MiCV in various tissues and organs of the sick minks.We also analysed the mutations of Cap gene of the MiCV strains in 2020 compared to the ones in 2013.2.We established the basic RPA and real-time fluorescence RPA based on the RPA kits of Twist Dx(UK)with the design of probes and the screen of several candidate primer pairs according to the ampliction performances.We evaluated the sensitivities of the two RPA methods using standard plasmids as templates for reaction.We evaluated the specificities of the two RPA methods by detecting several positive samples of other kinds of mink viruses and comparing the results.We also evaluated the repeatabilities of the two RPA methods by detecting the same samples repeatedly and comparing the results.Moreover,we optimized the reaction conditions and determined the best reaction tempreture,time,etc.We detected 46 clinical samples by the two methods and compared the results with that from the PCR method.3.We constructed the transfer vectors containing Cap gene and transferred them into DH10 Bac competent cells that have the genomes of baculoviruses in them.The DH10 Bac competent cells were cultured for an amplication and the recombinant bacmids were extracted using alkaline lysis.Sf9 cells were transfected with recombinant bacmids and the recombinant baculoviruses generated.The recombinant baculoviruses were cultured for amplication and the titer was measured.The genetics stability of the recombinant baculoviruses was evaluated via the comprision of Cap gene sequence of the recombinant baculoviruses of 1,5,10,15 and 20 generations.Whether Cap was successfully expressed or not was confirmed by indirect immunofluorescence assay,SDS-PAGE and Western blotting.High Five cells were infected by recombinant baculoviruses and cultured in suspension.The optimal dosage of seeds and culture time were explored in order to get the highest yield of Cap.The subunit vaccines were constructed with Cap and appropriate adjuvant.Then we performed a field vaccination trial in two mink farms in Zhuanghe(Liaoning Province)and Zhucheng(Shandong Province).Each mink in vaccinated group and control group received one dose of the subunit vaccine and control preparation respectively.We continuely observed the appearance of the feces of the two groups and collected and detected fecal samples for MiCV from about the end of September when mink refractory usually occurs.The morbidities and positive rates of the fecal samples of the two groups were compared in order to evaluate the efficacy of the vaccine.In addition,the average litter sizes of the two groups were also counted.Results:1.The epidemiological investigation of mink refractory diarrhea in Zhuanghe(Liaoning Province),Changli(Hebei Province)and Zhucheng(Shandong Province)indicated that the infection rate,morbidity and fatality in Zhuanghe in 2019 were 42.0%,36.6% and 6.1% respectively.And those in Zhuanghe in 2020 were 36.0%,30.3% and4.8%;those in Changli in 2020 were 31.0%,14.3% and 3.3% and those in Zhucheng in2020 were 26.0%,1.8% and 0%.Compared to 2013,the infection rate,morbidity and fatality all declined.From the perspective of different districts,the prevalence in Zhuanghe was most serious,that in Changli comed second and that in Zhucheng was most slight and the portion of the red diaherra was also lowest,there is almost no dead case despite typical symptoms in Zhucheng.As to sex,there is no significance between males and females;as to different ages,the infection rate and morbidity of the multi-year old minks were lower than those of the one-year old minks.We discovered the pathological changes in the spleen,kidney and liver of the sick mink.Pathological section examination indicated that inflammatory cells accumulated around the glomeruli and central vein of liver lobules of the sick mink.More goblet cells could be observed in the epithelium of intestine of the sick mink than that of the healthy mink.The result of the detection of MiCV in different tissues and organs indicated that MiCV existed in vairious tissues and organs including the large intestine,liver,spleen and kidney.The result of the comparision between Cap gene of MiCV in 2020 and 2013 in Zhuanghe,Changli and Zhucheng indicated that several base mutations occured.Among them,14 mutations of gene leaded to 4 mutations in amino acid sequence in MiCV-DL20;11mutations of gene leaded to 3 mutations in amino acid sequence in MiCV-Hebei20;3mutations of gene didn't lead to any mutations in amino acid sequence in MiCV-Shandong20.2.The basic RPA and real-time fluorescence RPA methods for MiCV detection were successfully established.The sensitivity of the basic RPA reached 5.4×102copies/?L while that of the PCR was 5.4×103 copies/?L,the sensitivity of the basic RPA was a little higher.We used the basic RPA method to detect the genome of mink enteritis virus,canine distemper virus and Aleutian mink disease virus and didn't find any amplification bands,which indicated that the basic RPA method was of good specificity.We used the basic RPA to detect standard plasmids of different concentrations for three times and the sensitity could reach the maximum in each detection,which indicated that the basic RPA was of good repeatability.We optimized the reaction conditions and determined 39? and 20 min as the best reaction conditions.The basic RPA needs about 40 min in total to finish detection,which is shorter than the time that PCR needs.Moreover,we established the real-time fluorescence RPA method,the sensitivity of which reached 5.4×101 copies/?L,which was a further enhancement.And the method was also of good specificity.The repeatability experiments within-run and between-runs indicated that the sensitity could reach the maximum in each detection and the average coefficient of variations were both in the reasonable range,which means the repeatability was relatively good.We optimized the reaction conditions and determined 39? and 20 min as the best reaction tempreture and time respectively and 140 m M as the best concentration of Mg OAc.The method further shortened the detection period compared to the basic RPA.The entire detection period is merely 20 min and the method can achieve on-spot detection with a portable fluorometer.We used the two RPA methods and PCR to detect a batch of clinical samples.The positive rates of the detection result from the two RPA methods were higher,but the statistical analysis indicated that there was no significant difference compared to that from the PCR.3.We constructed the recombinant transfer vectors,recombinant bacmids and rescued the recombinant baculoviruses,the titer of which reached 108 TCID50/m L,with good genetics stability(No mutations occurred at least in 20 generations).Cap was demonstrated to be successfully expressed according to indirect immunofluorescence assay,SDS-PAGE and Western blotting.Then the High Five cells were seeded by the recombinant baculoviruses and were cultured in suspension.We estimated that the maximum yield of Cap reached 200 ?g/m L.We prepared the MiCV subunit vaccines based on Cap.The result of the field vaccination trial indicated that only 36 minks presented the typical diaherra among 2000 minks in vaccinated group(the morbidity was 1.8%)while 745 minks presented the typical diaherra among 1000 minks in the control group(the morbidity was 74.5%).The morbidity of the vaccinated group was significantly lower than that of the control group.The fecal sample detection results indicated that the positive rate of the fecal samples of the vaccinated group was 2.1%while that of the control group was 70.0%.No adeverse effects were found during the field trial.In addition,through counting the previous pregnant minks and new-born minks in the following spring,we found that the average litter size of the vaccinated group was higher than that of the control group.Conclusions:1.Compared to 2013,the infection rate,morbidity and fatality of mink refractory diaherra all declined.In different districts,the prevalence differed.The susceptibility of male and female minks was same,while multi-year old minks were less susceptible than one-year old indivisuals.Pathological changes occurred in the kidney,spleen,liver and intestinal epithelium of the sick minks.MiCV existed in various tissues and organs.Several base mutations occurred in Cap genes of the MiCV strains in 2020 in Zhuanghe,Changli and Zhucheng compared to the MiCV strains in 2020,which also caused some amino acid mutations.2.The basic RPA and real-time fluorescence RPA were successfully established,whose sensitivities reached 5.4×102 copies/?L and 5.4×101 copies/?L respectively,both with good specificity and repeatability.These two detection methods were of higher sensitivity and higher detection speed and can be used in normal temperature compared to the PCR.And the real-time fluorescence RPA is suitable to on-spot detection.3.Cap was successfully expressed and the subunit vaccine against MiCV was developed based on it.The field trial indicated that the vaccination could reduce the morbidity and infection rate of refractory diarrhea among mink groups without any adeverse effects,which means the vaccine was of good efficiency and safety.In addition,the average litter size of the vaccinated group was higher than that of the control group.The study provided the theoretical foundation and technical support to the prevention and control of MiCV. |
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