| China's pig industry is booming towards large-scale and intelligent farming mode.Due to the asymmetry of farming scale and management,there are more and more kinds of pig diseases.The complexity of disease course has intensified,and the mixed infection of various diseases which has brought difficulties to clinical diagnosis and treatment,also has seriously restricted the development of China's pig industry.Many small and medium-sized pig farms,especially those that feed on the kitchen waste produced by life,are facing more and more difficulties in disease prevention and control due to they are inferior in their feeding management and poor awareness of disease prevention and control.In addition,factors such as the transportation of pigs and the lack of biosafety prevention and control in the current situation of raising pigs in China have aggravated the spread of various pathogens among pig farms.The infection of various diseases seriously affects the health level of the swinery and the bio-safety system,and increases the threat of new animal diseases and major foreign diseases to the farm.In this study,we established two high-efficiency,sensitive and automated high-throughput detection methods to conduct epidemiological investigation of viral pathogens in different farms,and analyzed the effective prevention and control ability of different pig farms on diseases,and the new risk of infection with foreign animal diseases.Further more,new and exotic animal diseases such as porcine reproductive and respiratory syndrome NADC30-like strains(NADC30-like PRRSV)and African swine fever(ASF)were detected and monitored real-time,the main results are as follows:(1)A high-throughput detection method for swine viral disease with capillary electrophoresis was established,which can effectively detect 9 kinds of pig pathogens simultaneously,including PRV,CSFV,FMDV,JEV,PCV2,PRRSV,PEDV,TGEV and PPV.The minimum detection limit of the nine viruses is 10~4 copies/?L,which has high detection sensitivity,good specificity and repeatability.By testing the clinical samples collected between 2015 and 2017,it was found that the pathogen infection was more serious,especially the multiple mixed infections were more complicated.The positive detection rate of the sample was 49.44%.Among the detected viral diseases,the proportion of PRV,PRRSV,PCV2,PEDV was high,and the mixed infection of PCV2 and PRRSV was extremely serious.Therefore,it is particularly important to improve the management level of pig farms and establish effective disease prevention and control measures.(2)Based on the target sequence enrichment multiplex PCR(TEM-PCR)technology and liquid phase chip system,a high-throughput detection method for simultaneous detection of nine viral diseases in pigs was established.Through the optimization and verification of the detection method,the application and comparison of clinical samples,it was found that the method has higher sensitivity than the capillary electrophoresis detection method,and the minimum detection limit of 9 viruses can be detected was 10~3 copies/?L.Specificity and repeatability were good.The results of clinical samples were highly consistent with the results of capillary electrophoresis,which provided a powerful technical support for the high-throughput clinical differential diagnosis of pig diseases,and provided a reference for the application of advanced detection techniques to veterinary clinical diagnosis.(3)Using the established two high-throughput detection methods,341 clinical samples collected from three different types of pig farms were tested and pathogenic molecular epidemiological investigations were conducted.The effective prevention and control ability of different farms on diseases were preliminarily analyzed,and infection susceptibility of new and foreign animal diseases were inferred.The results showed that the positive detection rate of pathogens in pig farms in our province was high,and the mixed infection of multiple pathogens was common and serious.In particular,the miniature pig farms had the most serious pathogen infection,which indicated that the effective prevention and control ability in the miniature pig farms were the worst.Further,through clinical monitoring of NAD30-like PRRSV in recent years,the positive detection rate of NADC30-like PRRSV in clinical samples collected was 21.41%.The positive detection rate of the miniature pig farms was the highest,which was 45.90%.Followed by small and medium-sized pig farms,the positive detection rate was 22.03%.The lowest was large-scale pig farms,the positive detection rate was 11.73%.In order to further improve biosafety measures,pig farms should be well managed and managed,combined with existing vaccine control to reduce the possibility of pig farm infection.(4)By constructing recombinant plasmids and optimizing expression conditions,the prokaryotic expression of ASFV pK205R recombinant protein was successfully performed,and the protein were purified and validated.Using pK205R as the coating antigen,a rapid ASFV indirect ELISA antibody detection method was established by optimizing the reaction conditions,and ASFV ddPCR nucleic acid detection method with ultra-high detection sensitivity was established.This method was used to test 474 samples collected between2015 and 2017,and to carry out clinical demonstrations in China's border and port entry animal inspection and quarantine.At the same time,real-time monitoring of ASFV was conducted in different pig farms,and the test results were all negative,indicating that no infection of ASFV was found in this study.This study provided data reference for domestic ASFV real-time monitoring and provided reliable technical reserves for ASFV inspection and quarantine. |
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