| Chicken infectious anemia(CIA)is an immunosuppressive disease that severely affects poultry production.This disease mainly affects 1-3 week old chicks.The infected chickens mainly showed obvious growth inhibition,chick aplastic anemia,yellowing of bone marrow,and early systemic lymphocyte atrophy.Chicken infectious anemia virus(CIAV)is the pathogen of CIA,and the clinical mortality rate of CIA is relatively low.As a result,farmers often neglect to prevent and treat the disease.Since 1992,CIA has been widespread in many provinces in China,causing great economic losses to China’s poultry industry.This study combines the epidemiology of CIA in China and the situation in Henan to conduct a systematic analysis.The disease cases were collected from the Poultry Disease Research Institute of Henan Agricultural University,and the virus was isolated and identified,and the genome sequence was determined and analyzed;A cross-section survey of live attenuated live attenuated vaccines for poultry in the market and collection of disease data from chicken farms in various regions of Henan conducted an epidemiological survey of CIA in Henan.The specific research content is as follows:1.Meta analysis of CIA infection rate in ChinaIn order to understand the prevalence of CIAV in China,the relevant documents of the CIAV infection rate survey were downloaded from various Chinese and English literature platforms,and a meta-analysis of the data in the literature was performed using the Meta software package in version 3.5.3 R software.The analysis showed that the overall positive rate of CIAV in China was 0.48(95%CI:0.36-0.61),suggesting that CIAV is commonly infected in domestic flocks.Due to the heterogeneity of the literature,this study further performed a subgroup analysis of the literature.The results showed that geographically,the overall positive rate of 0.56(95%CI:0.43-0.68)in East China was significantly higher than 0.38(0.24-0.52)in other regions;The overall positive rate of literature before 2010 was 0.52(95%CI:0.38-0.66)and the overall positive rate of literature published after 2010 was 0.45(95%CI:0.31-0.60);The overall positive rate of virus detection by ELISA method was 0.66(95%CI:0.54-0.77),which was significantly higher than that of PCR method 0.26(95%CI:0.15-0.42)and 0.30(95%CI:0.14-0.53)of other methods;The overall infection rate of chickens at the brooding stage of younger than 60 days is 0.37(95%CI:0.28-0.47).The infection rate of adult chickens older than 250 days is 0.80(95%CI:0.49-0.98).It is suggested that CIAV infection is widespread in adult chickens.2.Epidemiological study of CIA in HenanIn order to understand the prevalence of CIA in Henan,this study collected clinical data from 2017-2019 in Henan and randomly purchased attenuated vaccines sold in the Henan market.Epidemiological investigation of tissues and vaccines using PCR..A total of 437 tissue disease materials were collected,of which 41 were positive for CIAV nucleic acid,with a positive rate of 9.38%(95%CI:6.8-12.5);A total of 120 lyophilized vaccines were collected from 24 manufacturers,5 different types and different batches.of which 53 were Newcastle disease and bronchitis live vaccines,31 were chickenpox live vaccines,34 were Newcastle disease live vaccines,I were Mycoplasma live vaccines,1 were laryngeal and fowlpox virus genetically engineered vaccines.In the end,4 CIAV-positive vaccines were detected,and the overall CIAV nucleic acid positive rate was 3.33%(95%CI:0.9-8.3).The 4 positive vaccines came from 3 biological product factories.The positive rate of the vaccine manufacturers was 3/24,and the overall positive rate was 12.5%(95%CI:2.7-32.4).The above results show that there is currently CIAV contamination of live vaccines for chickens.In addition to natural infections.chickens infected with this disease may also be associated with CIAV contamination in vaccines.3.Isolation and identification of CIAV Henan strain and whole genome analysisIn this study.diseased chicken tissues were collected from the Institute of Poultry Diseases of Henan Agricultural University,and the 12 virus strains were isolated from the tissues and their entire genome sequences were determined and analyzed.The results showed that the similarity between the 12 Henan isolates was high,ranging from 97.1%to 99.3%,and the similarity to the vaccine strains Cux-1 and Del-Ros,ranging from 97.8%to 98.6%.The 12 isolates of Henan are divided into three small subgroups on the whole gene phylogenetic tree.It may have evolved from the transmission of this weak strain.The three viruses HN-2,HN-4,and HN-6 are on the same branch as the previous Henan HN1405 strain,and HN-1,HN-8,HN-9,and HN-12 are on the same branch as the Henan HN1504 strain.The strains on these two branches are more concentrated and are in the same branch as the previous isolates from Henan,and the amino acid loci are not much different,indicating that these strains may be the dominant strains that are prevalent in the native area of Henan.After analyzing three complete proteins encoded by 12 isolated viruses,it was found that the amino acid locus of the capsid protein VP1 protein has the most variation,and the overall variation is 0-3.4%.Among them,the VP1 proteins of HN-1,HN-8,HN-9,and HN-12 all carried Q139 and Q144,and the other eight Henan isolates carried residues K139 and E144.HN-4,HN-6,and HN-8 have A290P mutations at position 290,which has never been reported before.VP2 and VP3 proteins have no obvious changes compared with other strains.Recombinant detection of 12 isolated CIAV strains using RDP4 software revealed that HN-4 and HN-8 strains had genetic recombination.In general,Henan strains have undergone recombination and evolution with other strains of South Korea,Japan,and the United States,and there have been certain genetic recombination events.It is speculated that the recombined strains will have higher levels of adaptability,prevalence and spread.4.Biological characteristics of CIAV Henan isolate HN-4In order to understand the biological characteristics of CIAV Henan isolate HN-4 strain,SYBR Green I real-time quantitative PCR and q-PCR were used to determine the virus titer.The virus was studied in Marek’s disease lymphoblast cell line(MDCC-MSB1)growth characteristics and proliferation rules.The results show that the HN-4 strain can proliferate well in the MSB1 cell line,produce very obvious CPE 48 hours after infection with the virus,and the genomic copy number in the cell lysate reaches a peak 72 hours after infection(1×107.215 copies/μL).stable.One-day-old SPF chickens were intramuscularly injected with 1 mL of the HN-4 strain(1 × 107 copies/μL)isolated from the cell culture inoculum to replicate the typical symptoms on the chicken body.7 days after infection,the vaccinated strains can be isolated from the infected chickens,and the final mortality of the chickens is 50%.Compared with the control group,the weight of chickens in the infected group was significantly reduced at 7 days after infection,and the body weight of the chickens in the two groups was significantly different at 21 days after infection(P<0.05).Compared with the control group,the thymus,spleen,and bursa of chickens in the infected group were atrophied to varying degrees at 16 days after infection(P<0.05).Renal atrophy is not obvious,and the liver is slightly swollen.The bone marrow of the infected group turned pale yellow and the bone was brittle.Tissues and organs were collected to prepare pathological sections for HE staining.The results showed that the tissues and organs of infected chickens had typical CIA-induced pathological damage.The above results show that the HN-4 strain is highly pathogenic. |
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