Epidemiological Investigation And S1 Gene Analysis Of Avian Infectious Bronchitis In Ningxia

The Infectious Bronchitis(IB)is a highly contact abd acute infectious diseases.The pathogen is Infectious Bronchitis Virus(IBV).The clinical manifestations of chickens infected with the virus are runny nose,cough,asthma,etc.Among them,the fallopian tubes of laying hens are seriously damaged and resulting in decreased egg laying quality.The disease has caused huge economic losses to the poultry industry.In order to understand the prevalence of IBV in Ningxia,a set of RT-PCR and RT-qPCR detection methods were established in this study,and the molecular epidemiological investigation was carried out on 1126 swabs from 5 prefecture-level cities in Ningxia.At the same time,virus isolation,identification and S1 gene analysis were carried out in chicken tissues from scale farms with epidemic history,followed by the animal regression tests and virulence for the pathogenicity of isolated strains.The results consist of the following three parts:1.The diagnostic method with RT-PCR was established for IB in chickens.RT-PCR primers,RT-qPCR primers and fluorescent probes were designed with N gene sequence of IBV standard strain,and RT-PCR and RT-qPCR detection methods were established.The specificity,sensitivity and reproducibility of the two methods were verified by tests,and the two methods were analyzed and compared.The results showed that the established RT-PCR and RT-qPCR methods have strong specificity,high sensitivity and good stability.And the reproducible test results showed that the coefficient of variation(CV)values of three repeated tests were all less than 1.0%,which proved that the RT-qPCR had good stability.The lowest detected concentration of RT-PCR could reach 1×10-4 ng/L,and the lowest detected concentration of RT-qPCR could reach 1×10-5 ng/L,which all met the national standards,can be used for clinical testing.2.Epidemiological investigation of IBV in Ningxia.In order to study the local IBV epidemic in Ningxia,a total of 1126 samples were collected in 15 districts(counties)of 5 prefecture-level cities in Ningxia from September 2021 to February 2022,and 152 positive samples were detected with the established RT-PCR method,with a positive detection rate of 13.50%.The results showed that the prevalence of this disease had obvious seasonal,with higher incidence in spring and winter.Chicks aged about 90 days were susceptible to this disease.At a mean time,it was found that the local epidemic pattern of IBV was basically the same as the main epidemic status in China.Mixed infection detection showed 32 mixed infections with IBV and AIV,7 with NDV,and 3 with triple infections.3.Virus isolation and culture and S1 gene analysis.In this experiment,a IBV strain was successfully isolated by chicken embryo culture and named NEWNXIBV.S1 gene of this strain was amplified and sequenced.Phylogenetic tree and homology comparison map were drawn by MEGA and DNA STAR software.The isolates were closest to CK/CH/GD/GDBL121 plants in Guangxi,China,belonging to TC07-2,with the sequence consistency to the local isolates CK/CH/GD/GDBL121 and TC07-2 in China of 98.37%and 98.91%,respectively,the sequence consistency to QX strains of the epidemic strains in China of 86.09%,the sequence consistency to LX4 strains of 83.44%,the sequence consistency to the sequences of serotypes LDT3-A,H52 and M41 of vaccines in China of 86.09%,75.38%,and 78.26%,respectively.Moreover,the results of animal regression test and toxicity test showed an infection rate of 100%,a mortality rate of 48%,and the isolated strain is renal type.