大肠杆菌_O_78_菌毛油乳剂疫苗对鸡的免疫原性研究_英文_

大肠杆菌_O_78_菌毛油乳剂疫苗对鸡的免疫原性研究_英文_ 湖 北 农 学 院 学 报 第 20 卷 第 1 期年 2 月2000 Vol . 20 No . 1 Feb. 2000 Jo ur nal of Hubei Agricult ural College () Article ID :1004 - 3888 200001 - 0040 - 04 Ξ in Chickens Immunogen ic ity of an Esc he r ic hi a col i ( O) Pil i Vacc ine 78 1 1 2 3TAN G Ho u2kuan, DA I Ding2zhen, WAN G Yin2cai, L IAN G Si2jun ( 1 . H ubei A g ricul t u ral Col lege , J i n gz hou , H ubei 434103 , Chi n a ; 2 . J i a n gha n O i l2f iel d H usba n d ry Resea rch O f f ice , Q i a n j i a n g , H ubei 433123 , Chi n a ; )3 . Zhon gkou Fa r m , Q i a n j i a n g , H ubei 433104 , Chi n a () Abstract :Immunogenicit y of an oil2emulsified Esche richi a col i Opili vaccine was evaluat2 78 μμed in chickens. Chickens were vaccinated subcutaneo usly wit h 130g o r 65g of pili p rotein and challenged wit h ho mologo us E col i via t he po sterio r t ho racic air sac. U nvaccinated chickens were challenged to serve as po sitive co nt rols ,and unvaccinated and unchallenged chickens served as neg2 ative co nt rols. Vaccinated chickens were p rotected against acute respirato ry infectio n in t hat t hey ( ) ( ) agained bo dy weight s co mparable to t ho se of unvaccinated , unchallenged chickens ; bshowed () low o r no mo rtalit y ; chad mild gro ss lesio ns in air sacs , pericardial sacs , and livers , and t he ( ) ( ) sco red values were significantly P ?0 . 05lower t han t ho se in t he po sitive co nt rols ; and de2 liminated E col i f ro m tissues mo re efficiently t han t he po sitive co nt rols. Key words : Esche richi a col i ; pili ; oil2emulsified vaccine ; immunogenicit y ; chicken CL C number :S852 . 43 ; S858 . 31 Document code :A Colibacillo sis can be an eco no mically devastating disease of chickens and t ur keys. It is well established t hat Esche richi a col i pili , t he adhesio ns , play an essential role in t he pat hogenesis of chicken colibacillo2 1 ,2 sis. Adhesio n not o nly enables pat hogenic E col i to avoid eliminatio n by ho st defense mechanisms but also f avo rs colo nizatio n and invasio n of pat hogenic E col i in ho st tissues. St rains of E col i co mmo nly 1 ,2 pat hogenic fo r po ult ry —O,O,O,Oand Opo ssess so matic pili,w hich may serve as adhesio ns o r 1 2 50 78 88 2 virulence f acto rs. Adhesio n of infectio us agent s to ho st cell surf ace can be p revented by anti2pili antibo d2 3 ,4 ies. The p urpo se of t his st udy was to evaluate t he immunogenicit y of an oil2emulsified E col i Opili vac2 78 cine against colibacillo sis in chickens. 1 Materials and met ho ds 1 . 1 E col i stra in ( ) E col i serot ype Osupplied by Prof Li Kangran , Guangxi U niversit y ,Chinawas used fo r p reparatio n 78 of pili and fo r challenge . 1 . 2 Pil i preparat ion 5 Purified pili were p repared acco rding to t he met ho d of Brinto n et al ,wit h t he following mo dificatio n : () () aE col i grow n in 5 mL t ubes of nut rient brot h fo r 24 h at 37 ? was subcult ured 1 :100in 2 000 mL Ξ Recieved date :1999 - 11 - 18 ()Foundation item :Hubei Province Educatio n Co mmit te foundatio n item98A023 () Biogra phy :TAN G Hou2kuan 1947 - ,Born in Tianmen City of Hubei Province ,Associate Professor in Hubei Agricul2 t ural College ( ) ( ) of nut rient brot h total to 10 litersand incubated fo r 72 h at 37 ?; bbacteria were harvested by cen2 () t rif ugatio n at 11 000 g fo r 20 min at 4 ?; ct he pellet was resuspended in 100 mL of 0 . 05 mol/ L Tris - HCl buffer , p H 7 . 3 , and pili were seperated f ro m bacterial cells by magnetic stick stirring wit h high speed vo rtex in ice2bat h . The depilated bacteria were removed by cent rif ugatio n at 11 000 g at 4 ?fo r 20 min to ( ) remove any remaining bacteria ; dsufficient MgClwas added to t he super natant , and t he super natant 2 () was placed at 4 ?fo r 18 h ; et he p recipitate was collected by cent rif ugatio n at 24 000 gfo r 45 min at 4 ?, t he pili aggregate was resuspended in 5 mL of Tris - HCl buffer . The pellet o btained by t his p rocedure , as deter mined by elect ro n micro scop y , co nsisted almo st totally of pili . 1 . 3 Protein determinat ion The p rotein co ncent ratio n in t he pili p reparatio n was deter mined by ult raviolet abso rp tio n . 1 . 4 Vacc ine preparat ion 6 The met ho d described fo r p reparing and oil2emulsif ying vaccine was used. The p rotein co ncent ratio ns μμin t he vaccines used in experiment s 1 and 2 were 260g and 130 g per mL , respectively. 1 . 5 Immunogen ic ity experiment In t he first experiment ,o ne day2old co mmercial layer“579”chickens were divided into t hree gro up s of15 each and reared in isolatio n roo ms. Chickens in gro up 1 were vaccinated t wice at 1 day and 10 days old , each time wit h 0 . 25 mL of vaccine injected subcutaneo usly at t he base of necks. Each bird was injected μwit h a total of 130g of pili p rotein . At 21 days old , chickens in gro up s 1 and 2 were challenged via t he 7 po sterio r t ho racic air sac wit h 0 . 1 mL of a 24 h nut rient brot h cult ure co ntaining 5 . 2 ×10colo ny2fo r ming2 ( ) ) ( unit s CFU of E col i serot ype OGro up 2 served as po sitive co nt rol. U nvaccinated and unchallenged 78 chickens in gro up 3 served as negative co nt rols. In t he seco nd experiment , t he p rocedure followed was t he same as t hat in experiment 1 wit h t he fol2 μ() ( ) lowing changes : aeach bird was vaccinated wit h a total of 65g of pili p rotein , and bt he challenge in2 9 oculum was 1 . 12 ×10CFU of E col i per bird. Individual bo dy weight s were reco rded at 1 ,7 and 21 days of age . Af ter challenged , chickens were o b2 served daily fo r signs of illness such as ruffled feat hers , ano rexia , and st renuo us breat hing. Chickens died af ter challenge and at 27 days of age were sco red individually fo r gro ss lesio ns in t he air sacs , pericardial sacs , and livers o n a scale of 0 to 4 : 0 —no lesio ns ; 1 —clo udy air sacs o r pericardial sacs o r hepatitis ; 2 — mo derate airsacculitis o r pericarditis o r hepatitis ; 3 —bilateral airsacculitis o r pericarditis o r hepatitis ; and4 —severe and extensive fibrino us airsacculitis o r pericarditis o r hepatitis. Fo ur tissues f ro m each bird —heart bloo d , pericardial sacs , air sacs and livers —were cult ured fo r t he evaluatio n of E col i recovery. 1 . 6 Stat ist ical analysis Analysis of variance and Duncan’s multiple rang test were used to test fo r differences bet ween bo dy weight s and gro ss lesio n sco res. 2 Result s and discussio n Table 1 and table 2 give t he result s of t he average bo dy weight s , mo rtalit y , lesio n sco res , and persis2 tence of E col i in vaccinated and unvaccinated chickens in t wo experiment s. Table 1 showed t hat t he vacci2 nated chickens had an average bo dy weight t hat was co mparable to t ho se of t he po sitive and negative co n2 t rols. At 21 days of age , bo dy weight s of chickens in 3 gro up s were 199 g ,205 g and 206 g ,respectively. It suggested t hat t he vaccine had no adverse effect o n bo dy weight s of chickens. Mo rbidit y and mo rtalit y were not o bserved in t he negative co nt rol gro up . Fo r t he vaccinated chickens , ( mo rbidit y was very low . Af ter challenged , 2 of 15 vaccinated chickens developed mild signs of illness dull2 湖 北 农 学 院 学 报2000 年42 ) ness and ruffled plumage, w hereas unvaccinated chickens became ill —let hargy , ruffled plumage and dysp2 () ( ) nea —and five 33 . 3 %died wit hin 96 h of challenge Table 1. Ta ble 1 Ave ra ge bo dy weight s , mo rt ality , mo rbidity , le sio n sco re s ,a nd re co ve ry of E coli in t e st e d chic ke ns in the fir st e xp e rime nt AC No of No of Av. bo dy weight s / g No of ( ) lesio n scores scale 0 to 4 Recovery Treat ment 1 d 7 d 21 d Airsac Pericardium Liver chickens sick died of E col i aaaB 15 38 64 205 2 0 8/ 60 0 . 300 0 . 600 0 . 300Vac. Cha . U nvac. Cha bbb15 40 68 206 12 5 29/ 60 2 . 400 2 . 677 2 . 677 () positive co nt rol U nvac. U ncha aaa15 39 65 206 0 0 0/ 60 0 . 000 0 . 000 0 . 000 ( )negative co nt rol ( ) Notes :A : Values in each column followed by different let ters are significantly different P ?0 . 05;B : Vac . = Vaccinated ; U nvac . = U nvaccinated ; Cha . = Challenged ; U ncha . = U nchallenged ; C : Recovery of E coli means number po sitive / total number of tissues cult ured. The vaccinated chickens had very mild lesio ns in t he air sacs , pericardial sacs and livers , w hereas un2 vaccinated but challenged po sitive co nt rols had mo derate to severe lesio ns. Negative co nt rols were f ree of gro ss lesio ns. Average air sac , pericardial sac and liver lesio n sco res in vaccinated chickens were not statistically dif2 ( ) ferent P ?0 . 05 f ro m t ho se of t he negative co nt rols. On t he ot her hand , t he po sitive co nt rols had ( )serofibrino us airsacculitis , pericarditis , and perihepatitis , and lesio n sco res were significantly P ?0 . 05 higher t han t ho se of t he vaccinated and negative co nt rol chickens. Only in 8 of 60 tissues of vaccinated chickens E col i was detected , w hereas in 29 of 60 tissues of unvaccinated chickens E col i was detected. No E col i was isolated f ro m t he unchallenged chickens. Average bo dy weight s , mo rtalit y , lesio n sco res and efficiency in eliminating t he challenge E col i are show n in Table 2 . Vaccinated and unvaccinated chickens did not differ significantly in bo dy weight gain ( ) P ?0 . 05. Mo rbidit y was very low in t he vaccinated chickens. Only 5 birds f ro m t he vaccinated and challenged gro up showed mild signs of illness , and t wo birds died wit hin 96 h of challenge . Fo r t he unvacci2 nated but challenged chickens , 13 birds showed severe signs of illness. The signs o bserved were ruffled plumage , drooping eyelids , ano rexia , disinclinatio n to move and dysp nea . Nine of 14 unvaccinated but chal2 lenged chickens died wit hin 96 h of challenge . There was no mo rbidit y o r mo rtalit y in t he unvaccinated and unchallenged chickens. Ta ble 2 Ave ra ge bo dy weight s , mo rbidity , mo rt ality , le sio n sco re s , a nd re co ve ry of E coli in t e st e d chic ke ns in the se co nd e xp e rime nt CA No of Av. bo dy weight s / g No of No of Recovery ( ) lesio n scores scale 0 to 4 Treat ment 1 d 7 d 21 d Airsac Pericardium Liver chickens sick died of E col i B aaa15 39 74 203 5 2 10/ 60 Vac. Cha . U 1 . 133 1 . 100 0 . 933 nvac. Cha bbb14 38 76 206 13 9 29/ 60 3 . 067 2 . 667 3 . 067 ()positive co nt rol U nvac. U ncha a a a15 37 74 206 0 0 0/ 60 0 . 0000 . 0000 . 000 ( ) negative co nt rol Notes : Same as Table 1 . ( ) Gro ss lesio ns of vaccinated chickens were mild and significantly different P ?0 . 05f ro m t ho se ofunvaccinated and challenged chickens. Only in 8 of 60 tissues of vaccinated chickens E col i was detected. However , in 38 of 56 tissues of t he unvaccinated and challenged chickens E col i was fo und. There were no gro ss lesio ns in t he unchallenged co nt rol chickens , and fo und no E col i . U nder t he co nditio n of t he seco nd experiment , a few vaccinated and challenged chickens became ill and died. This might be at t ributed to t he higher do se of E col i used fo r challenge . Immunogenicit y of t he oil2emulsified E col i Opili vaccine was 78 demo nst rated again in t he seco nd experiment . ) (Gyimah et al repo rted o n t he immunogenicit y of a mo novalent Opili vaccine in p rotecting chickens 1 7 ,8 ( ) against ho mologo us challenge. Our result s also showed t hat an oil2emulsfied E col i Opili vaccine 78 ( ) co uld p rotect chickens against active respirato ry infectio n in t hat vaccinated chickens : a gained bo dy ( ) weight s co mparable to t ho se of unvaccinated , unchallenged chickens ; bexhibited low o r no mo rtality ; () chad mild gro ss lesio ns in air sacs , pericardial sacs , and livers and t he sco red values were significantly ( ) ( ) P ?0 . 05lower t han t ho se in t he po sitive co nt rols ; and deliminated E col i f ro m tissues mo re efficient2 ly t han t he po sitive co nt rols. Ref erences 1 Gyimah J E. Adhesin2recep tor interatio ns mediating t he at tachment of pat hogenic Escherichi a col i to chicken t racheal ep2 it helium J . Avian Dis ,1988 ,32 :74 ,78 . () Naveh M W. Adherence pili in avian st rains of Escherichi a col i : Effect o n pat hogenicityJ . Avian Dis , 1984 ,28 3:651 2 ,661 . 3 Acres S D , Isaacso n R E ,Babiuk L A , et al . Immunizatio n of calves against enteroto xigenic colibacillosis by vaccinating dams wit h p urified Kantigen and whole cell bacterinsJ . Infect Immun , 1979 ,25 :121 ,126 . 99 Moo n H W. Protectio n against enteric colibacillosis in pigs suckling orally vaccinated dams : Evidence of pili as p rotective 4 antigensJ . Ameri J Vet Res ,1981 ,42 :173 ,177 . 5 Brinto n C C , Buzzell A , lauffer M A. Elect rop horesis and p hage suscep tibilit y st udies o n a filament2p roducing variant of E col i bacterium J . BiochemBio p hys Acta ,1954 ,15 :533,542 . Sto ne H D. Preparatio n of inactivated oil2emulsio n vaccines wit h avian viral or mycoplasma antigensJ . Avian Dis ,1978 , 6 () 22 4:666,674 . ( ) 7 Gyimah J E , Panigrahy B . Immunogenicity of an oil2emulsified Escherichi a col i serot ype Opili vaccine in chickensJ . 1 Avian Dis , 1985 ,29 :1078,1088 . Gyimah J E , Panigrahy B , Williams J D. Immunogenicit y of Escherichi a col i multivalent pilus vaccine in chickensJ . A 8 2 vian Dis ,1986 ,30 :687,6891 大肠杆( O菌) 菌毛油乳剂疫苗对鸡的免疫原性研究 78 1 1 2 3汤厚宽,戴鼎震,汪银才,梁诗军 () 11 湖北农学院 ,湖北 荆州 434103 ;21 江汉油田畜牧研究所 ,湖北 潜江 433123 ;31 总口农场 ,湖北 潜江 433123 ( ) μ摘 要 :评价了大肠杆菌 Og菌毛油乳剂疫苗对鸡的免疫原性 。鸡经皮下接种 130 78 μ及 65 g 菌毛蛋白并用同源大肠杆菌经后胸气囊攻毒 ,未免疫鸡攻毒作为阳性对照 ,未免疫鸡 ( ) 不攻毒作为阴性对照 。免疫鸡可抵抗急性呼吸道感染 ,因为免疫鸡 : 1与未免疫未攻毒鸡在 () ( ) 增重上无显著区别 ; 2死亡率低甚至无死亡 ; 3在气囊 、心包 、肝仅有轻度病变 ,分值显著低 () 于阳性对照鸡 ; 4比阳性对照鸡更能清除体内的大肠杆菌 。 关键词 :大肠杆菌 ;菌毛 ;油乳剂疫苗 ;免疫原性 中图分类号 : S852 . 43 ; S858 . 31文献标识码 :A