Influence Of Genotype Ⅴ Chicken Reovirus To Immune Organs In Broilers

Infecting chicken reovirus also named avian viral arthritis.It is an infection that can lead chickens to dwarf,arthrocele and lameness by avian reovirus（ARV）.This virus is particularly serious for commercial broilers.The symptoms are depression,loss of appetite,malabsorption,etc.And the autopsy showed that there was a large number mucus in arthrosis.In recent years,the infection rate of chicken reovirus in chickens has been increasing,which has seriously endangered the normal development of chicken industry in China.To better understand the amount of expelling of toxin in chickens,viral load and tissue damage in immune organs at different time,in this study,we carried out the experiments on chicken pathogenesis by this virus（NMG）that preserved in the laboratory.The specific research contents are as follows:Took this virus which stored in the laboratory and purified it.The typical pathological changes of syncytium appeared after virus inoculated for 36 h was inculated in chicken embryonic hepatocytes.Primers were designed according to the ARVσC gene sequence.By RT-PCR,the result was consistent with the target strip detection.Sequenced the NMG ARVσC gene and analyed the homology,the results showed that the virus belongs to genotypeⅤand had high homology with classical ARV FJ793534,FJ793535 and FJ793542,up to 98.9%.And the homology between this virus and classical ARV KR856987,AF354226 and FJ793532 is relatively low,the lowest is only 50.9%.120 one-day-old white feather broilers（Ross-308）were randomly divided into three groups,each group of forty.Among them,group A was the oral group and Group B was the inoculation of theweb-footed group.Each chicken injected with 0.4 m L of this virus(TCID₅₀=10^-5.5/0.1 m L).Group C is the control group,each chicken injected with 0.4 m L of 0.9%Na Cl.Continuous observed for 30 days in 1 dpi,3 dpi,5 dpi,7 dpi,9 dpi,12 dpi,15 dpi,18dpi,21 dpi,24 dpi,27 dpi,30 dpi.Recorded the weight and counted the incidence rate and death.Three chickens were killed randomly in each group at the same time as above.Collected cotton swab of chicken cloaca,blood and took their liver,spleen,thymus,bursa of Fabricius,cecal tonsil and other immune organs.Recorded the pathological changes.Used qPCR and ELISA to detect whether the virus is detoxed,the time of suppressive infection and the changes of the antibody level.Some materials fixed with 4%paraformaldehyde by 24 h.Prepared of sections（HE）to observe the histopathology.In addition,hybridoma cell that preserved in the laboratory were resuscitated,which can secret ARVσC protein.Inoculated it into healthy BALB/C female mice and lead it product ascites.WB showed that this ascites can specifically bound to ARVσC protein.Collected immune organs in 1 dpi,3 dpi,5 dpi,7dpi,9 dpi,12 dpi,15 dpi,18 dpi,21 dpi,24 dpi,27 dpi,30 dpi for immunohistochemistry by used this ascites.The results showed that the weight of chickens in group A and B was significantly lower than that in group C.In group A,few chicks were mental fatigue,cold and occasional arthrocele.In group B,symptoms such as mental fatigue,anorexia and arthrocele.And the autopsy showed that the typical diseases of ARV such as hepatomegaly,hepatorrhagia,splenomegaly,megalothymus,bursa of Fabricius swelling and cecal tonsil hemorrhage.And there was mucus in articular cavity.Both groups had severe pathological changes in 1-9 dpi.But the symptom in group A was slighter than group B.In terms of expelling of toxin,chickens in Group A began in 1 dpi.Group B began in 3 dpi,slightly later than Group A.However,the expell of toxin time can be as long as 30 dpi,far higher than that of Group A.Furthermore,the immune organs of chickens in group A and B were infected in 1dpi.The viral load of group B is slightly higher than that of group A.And both groups had higher viral load level in the early time（1-9 dpi）.The results of ELISA showed that,in 1-9dpi,the antibodies in immune organs of chickens in group A and B were significantly higher than those in group C,and then gradually decreased and tended to be stable.The pathological section（HE）showed that the pathological changes in group A and B were basically the same.They all have the pathological changes of bursa of Fabricius edema,enlargement of hepatic sinusoid,hepatic cord arranged mussily and loss of splenic lymphocytes.And the most serious problem is in 3-9 dpi.The changes in Group A were relatively slight,and the changes in Group B were particularly obvious.The results of IHC showed that positive signals were detected in spleen and bursa of Fabricius in both group A and B in 1 dpi.The immune organs of the two groups of chickens were also seriously damaged in the early time（1-9 dpi）.Among them,the positive signal of group A is weak,but that of group B is relatively obvious.It can be seen from the above results that the age of chickens which were infected by ARV is low.The most susceptible age was 1 day.It will not only leaded to dwarf syndrome and arthrocele,but also caused serious impact on immune organs.Among them,spleen and bursa of Fabricius were the most seriously injured.Immune organs were infected in 1-9 dpi,while symptoms such as arthrocele appear relatively late,which indicates that the main target organs of ARV attacked maybe spleen and bursa of Fabricius.In addition,the time of expelling of toxin in theweb-footed group was longer,while the time of suppressive infection in the oral group was longer.That is to say,different modes of infection will lead to different duration of expelling of toxin and suppressive infection.Comprehensive infection and lession in different age,it can provide reliable theoretical support for the prevention.