The Post-embryonic Development Of Blood-spleen Barrier In Duck And Its Pathological Characteristics In Duck At Different Ages During DTMUV Infecting

The avian spleen is located on an important pathway of blood circulation and has lymphatic tissue structure,which is a unique secondary immune organ of the body.The particularity of the spleen allows it to filter aging and necrotic red blood cells,blood-borne microorganisms,and cell debris in the blood,as well as regulate cellular and humoral immunity.These functions cannot be effectively performed without the support of the bloodspleen barrier(BSB).The blood-spleen barrier is a new kind of immune barrier proposed in recent years.In mammals,the barrier is an effective filtration bed located between the arteries and veins in the marginal region of the spleen.The avian spleen has a special structure.without marginal zone but added with the structure of periellipsoidal lymphatic sheaths(PELS)in the white pulp.The previous research of our laboratory showed that the BSB of chickens and ducks were located in ellipsoid structure and the PELS region of spleen white pulp,and there was related research about the development of BSB in post-embryonic chicken.But compared with chicken,the structure of BSB in ducks lack ellipsoid-associated cells(EAC).Furthermore,ducks are waterfowls and have two pairs of lymph nodes.The immune mechanisms between chickens and ducks are very different.The orderly progress of duck spleen immune function is closely related to the post-embryonic development of BSB,but there is no related resear-ch at present.Duck Tembusu virus(DTMUV)is a new flavivirus that has been widely spread in China since the spring and summer of 2010.This virus can cause symptoms such as decreased appetite,delayed growth,and ataxia in ducks.Because DTMUV can cause sudden laying down,follicles bleeding and rupture,this disease was originally called duck hemorrhagic ovarian inflammation.DTMUV is not only pathogenic to laying ducks and meat ducks,but also infects chickens,geese,sparrows,and mice.It is a potentially zoonotic disease.Previous studies have found that the spleen is one of the main target organs of the virus,and that younger animals are more likely to be infected.However,no detailed study on the characteristic lesions of BSB caused by ducks at different ages.This study firstly researches the development of BSB structure and function at different ages after embryo to determine when the B SB was well developed.Then DTMUV was used to research the detailed pathological changes of the BSB on ducklings with immature BSB and adult ducks with mature BSB,to explore the important role of well-developed BSB in virus resistance.These studies aim to provide some theoretical supports for the research on avian immune mechanism and the pathogenesis and prevention of DTMUV.Experiment I Study on the blood-spleen barrier in the duck spleen during postnatal development.The spleen is the largest peripheral lymphoid organ and an important site of immune activity,in which the blood-spleen barrier(BSB)plays a significant role to resist various pathogens.The BSB structure of duck spleen is different from that of the chicken and mammals.However,no information on BSB development after postembryonic age has been reported in ducks.The current study investigated the spleen of ducks at 1,7,14.21,35,60 and 180 days old,by light and electron microscopy,to provide the development of BSB.The results showed:the spleen index of duck was increased from 1 to 14-day-old ducks.During the early age of ducks,the spleen showed no definite zone of white and red pulp.The ellipsoid diameter was continuously increased from 1 to 35-day-old duck spleen,while periellipsoidal lymphatic sheaths(PELS)and periarterial lymphatic sheaths(PALS)continuously developed.The reticular fibers of BSB developing with age,up to 14 days old these fibers show a developed framework in duck BSB.After 7 days old,the endothelial cell of the sheathed capillary showed a typical cuboidal shape;there were distinct gaps between endothelial cells as age advanced,while thick basement membrane and collagen fibers were also increased with the age.The results of ink injection experiments showed one ring layer of carbon particle was restricted in the white pulp in day-old duck spleen.however,with the development of BSB,in 14～180 days,these particles were restricted in the ellipsoid and PELS,forming two ring layers of carbon particle.Collectively,the structure and function of duck BSB basically developed up to 35 days post-embryonic age to perform their immunity role.Experiment II Pathological characteristics of BSB in DTMUV infecting ducks at different ages.The aim of this study was to compare the pathological changes of the blood-spleen barrier(BSB)and associated tissues caused by duck Tembusu virus(DTMUV)infecting 7day-old duckling with unmatured BSB and 180-day-old adult duck with matured BSB.105 TCID50 dose of DTMUV XZ-2012 strain was intramuscularly injected into ducks.Spleens were collected at 3,7 and 11 days post-injection,and the spleen samples were analyzed by light and transmission electron microscopy.The results of HE staining showed:at 3 dpi,the boundaries of red and white pulp were unclear and there was necrocytosis in the ellipsoids and periellipsoidal lymphatic sheaths(PELS)in both age groups,but the lesions of ducklings were more serious.At 7 dpi,the spleen of adult ducks begun to recover,while vacuolar degeneration was observed around the ellipsoids at ducklings’ spleens.At 11 dpi,the structure of adult duck spleen was basically normal while the structure of ellipsoids and PELS at ducklings’ spleens were still unclear.The results of IHC showed:the positive reaction of DTMUV at duckling spleens was stronger than those of adult ducks during 3～11 dpi.The results of reticular fiber staining showed:at 3 dpi,DTMUV caused a large amount of decrease of reticular fibers in the area of the ellipsoid wall and PELS in the spleen of ducklings,which were more serious than those in adult ducks.During 7～11 dpi,the recovery of BSB reticular fiber of duckling was slower than that of adult duck.The ultrastructural results showed:at 3 dpi,cell nuclear membrane shrinkage and cell edema in the spleens at ducklings were more serious than those in adult ducks.At 7 dpi,DTMUV induced apoptosis of splenic cells in both age groups.At 11 dpi,there was lipid in cells of spleen at duckling.In conclusion,compared with the adult ducks,the damages of BSB and surrounding tissues caused by DTMUV infecting at ducklings with immature BSB were more serious and the recovery of spleens at ducklings were slower DTMUV induce apoptosis of splenic cells in vivo,which provides support for the study of pathogenesis mechanism of DTMUV.