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Epidemiology of infection with bovine viral diarrhoea virus in a dairy herd : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Virology at Massey University

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(1)Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author..

(2) EPI D E MIOLOGY O F INFECTION WITH B OVI N E VIRAL DIARRHOEA V I R US I N A DAIRY H ER D.. A B R AHAM GOPILO G OLO. A THESIS P R E S E NTED I N PARTIAL FU LFI LMENT OF THE REQU I RE M E NTS FO R THE D E G R E E OF MASTER OF P H I LOSOPHY I N VETE R I N ARY V I ROLOGY A T MASSEYU N IVE RS TY I. DECEMBER 1 989.

(3) i. Abstract. This thesis descri bes studies on epidemio logica l a spects of infection of a da iry herd with bovi n e vi ra l dia rrhoea virus. Seru m neutra lizing a ntibodies w e re m ea s u red a n d i s o la ti o n of vi ru s wa s attempted f ro m a n i ma l s in o n e commercia l da i ry fa rm a nd fro m bobby ca lves collected fro m da i ry fa rms i n t h e Ma nawatu Reg ion for sla ug hte r at a loca l a battoi r. The work is prese nted i n six cha pte rs. The fi rst cha pte r is a review of the literature releva nt to the p resent wo rk. The second is a description of the g e n e ra l mate ria ls a nd m ethods. The t h i rd , fourth a nd fifth give t h e res u lts of the i nvestigatio n s ca rried out a nd the sixth is summa ry , a g e n e ra l discussion a nd conclusio n of the major findi ngs. Refe rences a re cited at the e nd of the thesis. Appe n dices I a nd. 11. d e sc ribe the prepa ration of the m edia for cell. c u lture , reagents a nd so lut i o n s , a nd publicatio ns rela ted to the work. All of the repla ce ment heifers a nd 97. 1 % of the adu lt mi lking herd had s e ru m neutra l i zi ng a nti bodi e s to bovine vira l dia rrhoea vi rus. There wa s no significa nt difference (p. >. 0 . 05) i n the geometric mea n titres of each a g e g roup. of the a nima ls i n the herd. '. The serum a nti body titres of the cohort of one to fou r day o ld ca lves reflected those of their da m s a nd decli ned steadily, with a ha lf life of 25 days, s o t ha t by e i g h t m o n t h s o f a g e , a l l ca lves tested nega t ive .. The s e ru m. n eutra lizing a nti body titres i n a g roup of bobby ca lves at a loca l a battoir were significa ntly lowe r (p. <. 0 . 00 1 ) tha n those of the cohort of ca lves o n the fa rm .. Bovi n e vi ra l dia rrhoea vi rus wa s isola ted from blood sa m p les fro m 2 1 o f 340 (6. 1 % ) anima ls a nd five ( 1 .4%) of these a nima ls were sti ll i nfected when retested 1 2 m o nths la te r. The distri butio n of serum neutra lizing a ntibodies i n i nfected a ni m als ra nged from. <. 0 . 6 to 2 . 7.. Th ree o f the persiste ntly i nfected. a ni ma l s had n o detecta b l e n e u t ra lizing a nti bodies (tit res. <. 0.6) but two had. titres of 0.9 a nd 1 . 8. The age o f the infected a nima ls ra nged fro m one to more t ha n eight yea rs. Persistent ly i n fected a n i ma ls p rovided an adeq u ate mecha n ism for the ma i ntenance of bovi ne vi ra l diarrhoea virus in the herd. lt is postu la ted tha t.

(4) ii. animals , i n contact with persiste ntly i nfected ani mals, may b e rei nfected when thei r anti body titres decline below p rotective levels. The vi ral interfe rence assay and fluorescent antibody test were u sed to d etect v i ru s i n cu l t u re s o f b l o o d s a m p l e s . T h ese two m et h o d s were evaluated fo r sensitivity and s pecificity and appeared to be equally s u itable for vi rus i solatio n . In the present wo rk t h e re was 88.4% ag reement between the two tests, w h i l e the viral i nte rfe re nce assay was found to be relatively more sensitive but less specific than the fluorescent antibody test. Th e resu lts of t h i s wo rk i n d icat e t h at i n fecti o n w i t h bovi n e vi ra l diarrhoea virus i s endemic o n t h e farm studied and that most ani mals beco me i nfected by the time they are twe lve m o nths old. However, depe nding on the particu lar m anagement practices used, so me g roups of heife r calves may not be e x posed to virus befo re t h ey reach b reeding age. lt also app ears that i n fection with bovine viral di arrh oea virus i s co mmon on many dai ry farms i n the Manawatu Region. If ide ntification and elimi nation of persiste ntly i nfected a n i m a l s i s a n option fo r t h e c o n t ro l o f bovi n e vi ral d i a r r h o e a vi ru s i n an e nde m ically i n fected popu l ati o n , a labo rato ry test with h i g h s pecifi city and sensitivity and wh ich is easy to perform wi l l be required. I f e r ad i c at i o n o f B V D V f r o m t h e f a r m i s n o t p ra ct i c a l t h e n i m m u n i zati o n o f heifers aro u n d t h e age o f 1 2 mo nths, either by exposure to persistent ly i nfected animals i n the h e rd o r by using i nactivated vacci nes,, may protect them against i nfection during the fi rst breeding period thus m i n i mizi ng foetal i nfecti o n ..

(5) iii. STATEMENT. This i s to certify the work o n which this thesis is based has not been accepted in whole o r i n part fo r any other deg ree or diploma, and was carried o ut by the u nd e rsigned. Assi stance rece ived is specif ically recorded i n the Acknowledgements section bou nd with the thesis.. Abraham G op ilo Golo 1 5 Dece m be r, 1 989..

(6) iv. ACKNOWLEDG EM ENTS. Th a n ks are due to P rofes sor B .W. Manktelow w h o n ot o n ly m ade avai lable the faci lities of t h e D e part m e nt of Vete ri nary Patho logy and Public H ealth , Massey University , but also provided valuable sugg estions duri n g this study. M a n y thanks to my s u pervisors D r. C . R . Wi lks and P rofessor D . K . B l ackm ore f o r thei r co nti n u o u s e ncou rag e m e nt , const ru ctive critici s m a n d u nfo rg ettab l e friendshi p t h ro u g h o ut t h i s project. I wou ld particu larly l i ke to thank Dr. C . R . Wi lks for ve ry valuable co mmunications, w hich he made with t h e s p o n s o rs of my sc h o l a rs h i p , i n E t h i o p i a , w h e n eve r t h e n e ed a rose t h roug hout m y stay in New Zealand. Thanks are due to M r. M . Rice for his excel lent tech nical support , Mr. P. N . Wildbo re fo r ad mini strative assistance, Mr. T. Law for preparation of the p h otog rap h s and Mr. A. H a rd i e and h i s st aff at No. 1 dai ry farm fo r t h e i r assistance d u ring the co llecti o n of sampl es. My s pecial thanks t o m y wife Fantaye Abamegal and my daug� te rs D e bo rah a n d Lily for their love , suppo rt and the joy they broug ht du ri ng the time of this p roject..

(7) V. TAB L E OF C O NTENTS ABSTRACT STATEMENT. iii. ACKNOWLEDG EMENTS. iv. TAB L E O F CONTENTS. V. viii. LIST OF TAB L ES. ix. LIST OF FIG U R ES Chapter one. R EVIEW OF THE LIT ERAT U R E. 1. History. 1 2. The vi ru s The disease Bovi ne vi ral d iarrhoea ( BVD). 5. Muc osal disease (MD) I nfec tion of t h e foetus. 8 10. Reproduc tive disease. 12. I nfec tion of other animals with pestivirus. 12. Border disease. 13. I nfec tion of pigs. 14. Epidemiology of i nfec ti o n of c attle with BVDV. 15. Transmission. 15. Distribution of S N antibodies i nc attle populat i o n. 17. 6. The role of persiste ntly infec ted i m munoto l e rant a ni mals. Chapter two. 18. Contro l of i nfec tion with BVDV. 22. Gene ral c onc epts ofc o ntro l. 22. The possi ble options. 23. Aim and sc ope of the present study. 26. G ENERAL MATERIALS AN D M ETHODS. 27. 2. 1. G e n e ral materials and methods. 27. 2.2. The farm. 27. 2.3. Ani mals sampled. 29. 2.4. Collecti o n of blood samples from bobby c alves. 29. 2.5. Collect i o n and proc essi ng of blood samples. 29. 2.6. Cell c ultu re tec h n iques. 30.

(8) vi. 2.6. 1. P ropagati on and maintenanc e ofc el l monolayer. 2.6.2. Trypsinization o fc o nfluent monolaye rs. 2.6.3. Preparation of p ri mary c e l l c u ltures of bovi ne foetal lung. Chapter th ree. 30 31. 2.7. T h e seru m ne utralization (SN) test. 33. 2 . 7. 1. The c ell c u ltu re. 33. 2.7.2. Propagati on and sto rag e o f the stoc k virus. 33. 2.7.3. Titration of the stoc k vi rus. 33. 2.7.4. The SN test proc edure. 34. 2.8. Isolation of BVDV fro m bloodc lots. 34. 2.8. 1. The c ells. 34. 2.8.2. I n oc u latio n of the sample. 35. 2.8.3. Viral i nte rfe re nc e assay proc edure. 35. 2.8.4. Direc t fluoresc e nt anti body test (FAT). 35. S E R O L O G I C A L C O M P A R ISONS OF N EW Z EA L A N D ISOLATES OF B V D V WITH TH E NADL STRAIN. 37. I ntroduc tion. 37. Materials and methods. 38. The c ell c u ltu re. 38 38. The viruses The sera SN te�. Chapter fou r. 30. 38. ResuH s. 38 39. Disc ussion. 41. Summary. 42. S E R O LO G I C A L ST U D I ES ON TH E E P I D E M I O L OG Y O F I N F ECTION WITH B V DV I N A DAI R Y H E R D. 43. I ntroduc tion. 43. Materi als and methods. 44. Resuh s. 45. Cross sec tional observation. 45. Cohort ofc alves. 50. Bobby c alves. 57 60. Disc ussion Sum mary. 63.

(9) vii. Chapter five. Chapter six. VIRO LOG I CAL STU D I ES ON T H E EPI D E MI O L O G Y O F INFECTIO N WITH BVDV I N A DAIRY H E R D. 64. I nt roduc tion. 64. Materials and methods. 65. Resu lts. 65. Disc ussion. 69. Summary. 72. G EN ERAL DISCUSSION AND CONCLUSION. 73. REFE R E NC ES. 80. AP PEN DIX. 96. AP P E N D IX 11. 96.

(10) viii. L IST OF TAB L ES. Table 3- 1 . Neutralization of strains of NADL and BVD- MDB K by rabbit and pig antise ru m raised agai nst NADL, 1 09, 5 1 0 and 709 strai ns of BVDV, and also by bovi ne sera c ollec ted from the dai ry farm.. 40. Table 4- 1 . Se ru m neutralizi ng anti body to BVDV in heifers and c ows fro m the same farm.. 46. Table 4-2. Se ru m neutralizing antibody to BVDV in a g ro up of heife rs between 1 2 and 24 months of age.. 49. Table 4-3. Seru m neutralizing antibody to BVDV in the c o hort of 1 -4 day oldc alves and their dams.. 51. Table 5- 1 . Detec tion of vi ru s and neutralizing anti body in blood ofc attle.. 67. Table 5-2 . C o mpari son of the vi ral i nte rfere nc e assay and fluo re sc e nt antibody test i n detec ting viraem ia.. 68.

(11) ix. LIST OF FIG U R ES.. Fig . 1 - 1 . Pathogenesis of m ucosal disease .. 20-2 1. Fig . 2-1 . Diag ram of Massey dai ry farm N o . 1 . S hed and paddocks.. 28-29. Fig. 4- 1 . Distribution of seru m neutralizi ng a nti body ti tres ag ai nst BVDV in heife rs and cows o n o n e farm.. 47-48. Fig. 4-2 . Distri bution of seru m neutralizing a nti body titres ag ai nst BVDV in a co hort of 21 calves.. 52-53. Fig . 4-3. Serum neutralizi ng antibody to BV DV i n a cohort of 21 calves.. 54-55. Fig . 4-4. Dist ribution of seru m neutralizing antibody titres to BVDV in g roups of cattle on the farm and bobby calves at an abattoi r.. 58-59.

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