| Infectious bronchitis(IB)is an acute and viral disease of chickens,which mainly affects the respiratory and genitourinary systems.This disease is widespread in the world,causing serious economic losses to the poultry industry.The genotype GVI-1(TC07-2 type)of infectious bronchitis virus(IBV)was first identified in Guangdong Province of China in 2007 and subsequently spread to other Asian countries.Epidemiological surveillance showed that the isolation rate of genotype GVI-1 strains continued to increase in recent years,especially in southern China.In this study,a RT-qPCR detection method for genotype GVI-1 IBV was established,the pathogenicity of this genotype of strain to chicks and laying hens was systematically evaluated,and a candidate attenuated vaccine strain was developed.1.Establishment of a RT-qPCR detection method for genotype GVI-1 IBVIn order to distinguish rapidly the genotype GVI-1 from other genotypes of IBV,specific primers and probe were designed based on the S1 gene sequence alignment analysis among different genotype strains to establish a RT-qPCR assay.The results demonstrated that this assay showed good linearity within the concentration range tested,with a correlation coefficient of 0.999.The limit of detection was 1.0×101 copies/?L.The assay is specific against GVI-1 strains and does not exhibit cross reactivity with other genotype strains such as the GI-1,GI-7,GI-13,GI-19,and GI-28.The variation coefficients of repeated test intra and inter group were both less than 2.5%.This assay and PCR were used to detect virus of oral and cloacal swabs,which were collected from infected chicks.The coincidence rate of the two methods was 96.7%,and the positive rate of RT-qPCR was higher.In conclusion,the assay for detection of GVI-1 genotype of IBV established in this study showed good specificity,sensitivity,and repeatability,which has important implications for GVI-1 genotype of IBV in epidemiological surveillance and rapid screening for clinical isolates.2.Evaluation the pathogenicity of genotype GVI-1 IBVExperiment 1:A total of 80 10-day-old female SPF chicks were randomly divided into the challenge group(n=40)and the control group(n=40).Each chick in the challenge group was inoculated with 0.1 mL genotype GVI-1 IBV of strain CK/CH/JX/2018/1(JX181)containing 106.5EID50 via the ocular-nasal route,and those in the control group were mock inoculated with PBS.The clinical symptoms of chicks were observed every day,performed regular necropsy,collected tissue samples for virus load detection and histopathological examination,and regularly collected oral and cloacal swabs for viral shedding detection.After the hens laid eggs,the daily egg number and egg quality parameters were recorded and determined.At the age of 180 days,all hens were euthanized and necropsied.After challenge,the chicks showed severe respiratory symptoms,hemorrhage in the larynx,trachea and bursa of Fabricius,pulmonary congestion,and cystic oviduct.The histopathological changes mainly occurred at 6?9 days post-infection(dpi),including cilia loss,sloughing of epithelial cells,and lymphocyte infiltration in the tracheal mucosa,pulmonary bronchial hemorrhage,increased number of macrophages in splenic sinus,swelling and necrosis of renal tubular epithelial cells,atrophy of lymphoid follicles and interstitial dilation of the bursa of Fabricius.Replication of the virus was detected in the trachea,lung,spleen,kidney,and bursa of Fabricius,with the highest viral load in the trachea.Viral shedding was detected in the oral and cloacal swabs,with higher levels and longer shedding duration of virus via oral cavity.At 180 days old,necropsy revealed that the hens in the challenge group had ovarian inflammation,the fluid yolk material or fibrin clots were observed in the coelomic,cystic oviduct,ovarian and oviduct growth retardation.The total egg production in the challenge group decreased by 15.1%compared with the control group.The results showed that the GVI-1 genotype IBV had extensive tissue tropism.Infection of hens could affect the development of ovary and oviduct,cause permanent damage to oviduct,and decrease egg production and egg quality.Experiment 2:Eight 200-day-old hens were randomly divided into the challenge group(n=4)and the control group(n=4).Each hen in the challenge group was inoculated with 0.1 mL genotype GVI-1 IBV of strain JX181 containing 106.5EID50 via the ocular-nasal route,and those in the control group were mock inoculated with PBS.The clinical symptoms of chickens were observed every day,and the daily egg number and egg quality parameters were recorded and determined.At 4 and 8 dpi,two hens in each group were randomly selected for autopsy,the infundibulum,magnum,isthmus,and uterus of oviduct were collected for virus load detection,histopathological examination,and immunohistochemical analysis.The results showed that the genotype GVI-1 IBV caused clinical symptoms such as dyspnea and tracheal rales in hens,resulting in a decline in egg quality(watery albumen,an increased proportion of blood spots,and thinning of eggshells).The autopsy showed laryngotracheal hemorrhage,lung congestion,flattening of mucosal folds of oviduct,thinning of oviduct wall,and foreign body in the lumen of infected hens.Histopathological examination showed that the tubular gland structure was destroyed,the tubular gland cells degenerated and the mucosal lamina propria was edema of hens in the challenge group.The virus replicated in all segments of the oviduct,and the replication ability from strong to weak was infundibulum,uterus,isthmus,and magnum,which was consistent with the staining intensity of the immunohistochemical test.The results showed that the genotype GVI-1 IBV had a strong tropism to the oviduct of laying hens,which resulted in serious damage to the oviduct and a decline in egg quality.3.Development of an attenuated vaccine strain for genotype GVI-1 IBVIn order to develop a novel attenuated vaccine against the genotype GVI-1 IBV,the strain JX181 was continuously passaged on SPF embryonated eggs.The strains passed to 5th,95th,and 115th generations were named JXP5 strain,JXP95 strain and JXP115 strain,respectively.The safety and immune protection of JXP95 and JXP115 were evaluated.The JXP5 strain caused typical clinical symptoms of IBV in chicks,causing damage to the trachea,lung,kidney,bursa of Fabricius,and oviduct.The chicks inoculated with JXP95 strain without any clinical signs,but there were slight laryngeal hemorrhage,thickening and tracheal mucosa lamina propria lymphocyte infiltration,pulmonary bronchial hemorrhage occasionally,and interstitial dilation of lymphoid follicles of Fabricius bursa.The chicks inoculated with JXP115 strain without any clinical signs or gross lesions,only a few lymphocytes infiltrated in the lamina propria of tracheal mucosa.Scanning electron microscope observation showed that the JXP5 strain caused large area cilia loss of trachea,the JXP95 strain caused mild tracheal damage,while the tracheal cilia of chicks inoculated with JXP115 strain integrity.The replication ability of JXP95 and JXP115 strains was significantly lower than that of JXP5 strains by RT-qPCR detection of viral load in tissues.The immune protective experiment showed that the chicks immunized with JXP95 and JXP115 strains had no clinical symptoms,the trachea cilia were intact and virulent,while those in the unvaccinated group had serious clinical symptoms,tracheal cilia loss and movement stagnated.The amount and duration of viral shedding in oral cavity and cloaca of JXP95 and JXP115 immunization groups were significantly lower than those of non-immunization groups.The above results showed that the IBV strain JX181 still caused slight damage to the tissues and organs of chicks after the 95th generation of SPF embryonated eggs,while the pathogenicity of the 115th generation strain was fully reduced and retained good immunogenicity.Therefore,the strain JXP115 is a good candidate strain of genotype GVI-1 IBV attenuated vaccine. |
PDF Full Text Request