| Avian infectious bronchitis(IB)is characterized by Avian infectious bronchitis virus,IBV is a series of multi-symptom disease syndrome caused by respiratory tract,digestive tract and reproductive tract.This disease has a wide distribution range and strong infectivity.It belongs to the second class disease which can bring great economic losses to poultry breeding industry.In terms of viral taxonomy,avian infectious bronchitis belongs to the genus Nidovirales,Coronaviridae,and gamma-coronaviruses,and was first identified in the United States in 1930.Due to its special single helix RNA structure,this virus is prone to base replacement,deletion and insertion in the replication process,resulting in the characteristics of high mutagenicity of this virus,and the cross protection between different serotypes and genotypes is relatively weak,etc.Vaccine prevention always has certain limitations when taking into account the cost of breeding.And for the treatment of the disease of poultry according to the clinical symptoms of the disease,it is passive.The purpose of this study was to carry out epidemiological investigation of IBV in chicken farms,slaughterhouses and other places in Shandong Province,in order to grasp the latest epidemic trends of the disease in recent years.In addition,this experiment also established the detection method of IBV microdrop digital PCR to assist the investigation.In this study,a large number of samples were collected from chicken farms and slaughtering houses in different cities of Shandong Province from 2021 to 2023.A total of407 samples(including tissues,organs,cloacal and throat swabs)were collected for IBV molecular biology detection.Through the analysis of space,time and test results,the epidemic situation of IBV in Shandong Province has been preliminarily grasp.Among the 407 samples from 11 cities in Shandong province,the positive rate was 27.27%(111 samples).In terms of time distribution,the positive infection rates were mainly in cold seasons such as early spring,late autumn and winter.In terms of spatial distribution,the positive infection rates were highest in Jinan(53%),Tai ’an(33%)and Dongying(28.8%).At the same time,34 IBV strains were successfully isolated and sequenced in this study,and the homology between the34 sequenced strains was 76.5% to 100%.Among them,the homology between X613C01 and NKY-2S1 strains was slightly lower than that with the other 32 sequenced strains.At the same time,some of the sequenced strains were highly similar to the foreign reference strains,and it is not difficult to exclude the transmission caused by livestock product trade or migratory birds.In addition,the 34 sequenced strains in this trial included 5 genotypes,including 2strains of GI-22 genotype,1 strain of GI-13 genotype,1 strain of GI-7 genotype,1 strain of GI-1 genotype and 29 strains of GI-19 genotype.The representative strain of GI-19 genotype was QX genotype,which was also the most dominant strain in various provinces of China.It is widely distributed in all parts of China,and some studies have shown that the QX strains of the same genotype show antigenic variation and differences in pathogenicity.Meanwhile,three QX genotype strains(DY566,Zhang Qiu3-18 and Dong Yingjk15)were successfully used in animal experiments and pathological tissue sections to explore the differences in pathogenicity and tissue tropism among the same genotype IBV.Because IBV and other avian diseases have many similarities in clinical symptoms,infected chickens will also cause secondary and mixed infection due to low immunity,so it is necessary to establish an accurate,rapid detection method,dd-PCR,which can directly detect the tissue tropism of infected strains.In this experiment,universal primers and probes were set up according to the M protein,one of the conserved proteins of IBV virus.The conditions of the detection method were explored and optimized by using the constructed positive plasmid as the standard.The test results are as follows: The optimal primer concentration(800n M/L),probe concentration(4800n M/L),rising and cooling rate(1.5 ℃ /s),annealing temperature(56.5 ℃)and sensitivity(1.5copies/ μ L)of this method were tested,and the results of specificity test,repeatability test and linear relationship were analyzed.The results show that the Taqman probe droplet digital PCR detection method established in this study has the advantages of high sensitivity and specificity,and can achieve absolute quantitative detection of nucleotide in positive samples.Therefore,using this characteristic,the tissue tropism of IBV in chicks challenged with DY566,Zhang Qiu3-18 and Dong Yingjk15 strains was determined.Finally,combined with the results of pathological tissue sections,the following results were obtained: Zhang Qiu3-18 was a strong renal strain,DY566 and Dong Ying JK15 were adenogastric strains.It is proved that this method has high clinical practicability. |
PDF Full Text Request