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Attenuation of infectious Bursaldisease virus and vaccinationtrials under laboratory and fieldconditionsA. Rinaldi a , D. Cessi a , G. Cervio a & E. Lodetti aa Istituto Zooprofilattico Sperimentale della Lombardiaa dell'Emilia , Via Cremona 282, Brescia, 25100, ItaliaPublished online: 17 Sep 2008.

To cite this article: A. Rinaldi , D. Cessi , G. Cervio & E. Lodetti (1974) Attenuationof infectious Bursal disease virus and vaccination trials under laboratory and fieldconditions, Avian Pathology, 3:1, 51-57

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Avian Pathology Vol. 3, No. 1, 51—57.

ATTENUATION OF INFECTIOUS BURSAL DISEASE VIRUS AND VACCINATION

TRIALS UNDER LABORATORY AND FIELD CONDITIONS

A. RINALDI, D. CESSI, G. CERVIO, E. LODETTI

Istituto Zooprofilattico Sperimentale della Lombardia a dell'Emilia, Via

Cremona 282, 25100 BRESCIA, Italia

SUMMARY

Adaptation to the chicken embryo of a virus strain of infectiousbursal disease resulted in a reduction in virulence. After 60passages the strain was attenuated enough to be used as avaccine. The attenuation proved adequately stable. If givenorally in the drinking water, the strain induced the developmentof neutralising and precipiting antibodies and a strong resitenceagainst the disease. Promising results have also been obtainedin the field.

INTRODUCTION

The spread of infectious bursal disease (IBD) in chickens farms inItaly and in other countries (Badiola et ai. 1969; Chinn and Coomber,1966; Cosgrove, 1962; del Bono ef a/., 1969; Fabris ef a/., 1967;Faragher, 1972; Herceg 1971; Izzi ef a/., 1970; Landgraf ef a/., 1967;Lensing, 1968; Luethgen, 1969; Maire et al., 1969; Rinaldi étal., 1972),and the consequent economic losses have raised the problem ofcontrol of this infection (Cho, 1968; Dorn ef a/., 1968; Edgar andCho, 1964; Snedeker ef al., 1967; Winterfield and Hitchner, 1964;Winterfield, 1969}In Italy the prophylactic method which has given the most satisfac-tory results is the use of an attenuated vaccine. The developmentand use of this vaccine is described in this report.

MATERIALS AND METHODS

Embryos and chickens

Eight to 9 d old embryonated eggs and chicks 20 to 30 d old wereused. They originated from a flock held in isolation which wassusceptible to IBD virus (IBDV).

Received 9 November 1973

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52 A. Rinaldi, D. Cessi, G. Cervio, E. Lodetti

Virus

A field isolate of IBDV designated 1—65 PV was used. Adaptationand attenuation was achieved through serial embryo passages. Emb-ryos were infected via the allantoic cavity with a tenfold dilutionof allantoic fluid at each passage. Every 10th passage the embryolethal dose 50 (ELDSO) and mean survival time of embryos wascalculated. The residual pathogenicity for chicks, based on clinicalsigns of disease and post- mortem findings was also determined.

Stability of attenuation

The 60th embryo passage of 1—65 PV was inoculated into chicks20—30 d old by the oral route and then passaged 8 time usingextracts of the bursa of Fabrioius and the oral route of infection.

Virus neutralization and gel diffusion tests

Neutralization tests were conducted on sera inactivated for 30 mi-nutes in a water bath at 56° C. Each tenfold virus dilution was mixed1 : 1 with serum and incubated for 30 minutes at room temperature.For titration of virus and for neutralisation tests, 5 embryos wereinoculated per dilution using 0,2 ml. per embryo. Inoculated anduninoculated embryos were incubated for 8 d. Titres were calculatedfrom the embryonic mortality occuring after 24 hours postinoculationaccording to the methods of Reed and Mue.nch (1938). Logio virusneutralization indices were determined.

The gel diffusion precipiting technique was essentially that used byJordan and Chubb (1962). A homogenate of bursa of Fabricius fromexperimentally infected chickens was used for precipitating antigen.

Development of immunity after the administration of 60th passage IBDV

Groups of 10 chickens, 20—30 d old were given about 103 ELD50 of60th passage virus by the oral route. At intervals of 2 days, groupsof vaccinated chickens were infected by the oral route with about1045 ELDso of pathogenic IBDV, and iin each case were sacrificedafter a further 3 days.

Examination of chickens

All the chickens used for testing the attenuation, stability andimmunogenity of IBDV were examined for clinical signs of disease(diarrhoea, anorexia, depression, trembling) and gross lesions ofthe bursa of Fabricius (extensive swelling of the bursa of Fabriciuswith prominent striations and peribursal oedema).

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Infectious bursal disease virus vaccine 53

Use of attenuated IBDV in field conditions

Several trials were conducted on farms where disease was endemic.Groups of 10—20 000 were used in each trial. Each flock was divi-ded into 2 and housed in separate pens; half was given 60 th passagevirus in the drinking water, at 21 d old and the other half was keptas a control. In vaccinated and non vaccinated groups mortalityafter vaccination, feed conversion, average weight at 65 days (ageat slaughter), and number of fowls rejected at slaughter were re-corded.

RESULTS

Adaptation of virus

The ELD50 increased progresively from 1025 at the first to 1060 atthe 60th passage. The meansurvival time of embryos inoculated witha 10'2 dilution of allantoic fluid decreased progressively from 5 daysat the first to 2 days at the 60th passage (table 1).

Table 1. Adaption of IBDV virus to the chick embryo

Survival time in days, of embryosinoculated with 10-' virus dilution

No.

1

20

60

l_l_l_»50/ 1 1II

Log,o

2.5

5.0

6.0

shortest

3

2

2

longest

9

6

3

mean

5

3

2

Attenuation of IBDV

Clinical signs and lesions of the bursa of Fabricius in chicks infec-ted with IBDV decreased gradually with, the number of passagesthe virus had in embryos. Attenuation was complete at the 60thembryo passage (Table 2).

Table 2.

PassageNo.in

embryos

1

20

60

Pathogenicity of IBDVpassage of the virus in

Clinicalsigns

+ +

for chicken andchick embryos

Lesions inbursa ofFabricius

1111

stimulation of antibodies after

Day antibodiesappeared.

Neutralz- Precipitat-ing ing

CO

C

O

CO 11

8

10

Mean N. 1.at 21 st

day

4.5

5.1

5.2

N. I. = Neutralization index.

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54 A. Rinaldi, D. Cessi, G. Cervio, E. Lodetti

Stability of IBDV attenuation

After 8 consecutive passages in chickens of 60th embryo passageIBDV no clinical signs or lesions of the bursa of Fabiicius wereseen (Table 3).

Table 3. Stability of attenuated IBDV at 60th embryo passage

Passage inchickenClinical signs

Lesions ofthe bursaof Fabricius

Mean N. 1. 21days afterinfection

1

0/10a

0/10

5.0

a The denominator showsnumber of chickens witr

2

0/10

0/10

5.2

3

0/10

0/10

5.1

the number ofi clinical signs

4

0/10

0/10

5.0

5

0/10

0/10

5.3

treated chickensor lesions.

6

0/10

0/10

4.9

and the

7

0/10

0/10

5.3

8

0/10

0/10

5.0

numerator the

N. I. = Neutralisation index.

Development of immunity in chickens treated with attenuated virus

The appearence of neutralising and precipiting antibodies occurs?>—10 days after vaccination and is nearly simultaneous with thedevelopment of resistance against the disease (6—8 days postvaccination) (table 4).

Table 4. Onset of immunity in chickens treated with 60th passage virus

Interval in days between administration of 60th passages virus andinfection with pathogenic IBDVa

Lesionsof bursaof Fabricius + + + + + + -\— <+ + - -

8 10 12 14 16 18 20

Clinicalsigns + + + + + + +—N. I. <1.0 >1.0 1.0 1.8 2.0 2.7 3.0 3.6 4.5 5.4

<1.0 1.0 1.8 2.0 2.7 3.0 3.6 4.5 5.4

Precipit.antibody — — — — + + + + + +

a = Chicken were sacrificed 3 after challenge with pathogenic IBDV

N. I. = Neutralization index.

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Infectious bursal disease virus vaccine 55

Use of attenuated virus in the field

Field trials on farms where IBD was endemic have given promisingresults. A total of 1 000 000 chickens were vaccinated and the mor-tality, feed conversion and condemnation rate were reduced and theaverage weight at slaughter increased compared with the 1 000 000control chickens (table 5).

Table 5. Field trials with attenuated virus

No. of

chickens

1000000No. of controlchickens1000000

Mortalityafter day ofvaccination

No. %

9710 0.97

40620 4.06

Feedconversions

kg

2.37

2.89

Averageweight

at slaughter

kg

1940

1590

Condemnationat slaughter

No.

1450

21500

No. = number

a = kg. of feed eaten for the production of 1 kg of meat.

DISCUSSION AND CONCLUSIONS

Adaptation of a strain of IBDV to the chick- embryo resulted in arise of ELD50 in the allantöic fluid and shortening of the mean sur-vival time. In addition pathogenicity for chicks was reduced Withpassage. By the 60th passage pathogenicity was lost and thischaracteristic was apparently stable. The immunity stimulated bythe attenuated virus was demonstrated by the development ofneutralising and precipiting antibody, and by resistence againstthe disease.The appearance of neutralising and precipting antibodies was nearlysimultaneous with the developrtient of resistence against the disease.The gel diffusion test can be used for detecting the developmentof antibodies after vaccination because precipiting antibodiesappeared only one to 2 days after neutralising antibodies. Fieldtrials with attenuated Virus administered to 21 d old chickens onfarms where IBD was endemic have given promising results.Mortality, feed conversion and condemnation rates were reducedand the average weight at slaughter was increased. These are allimprovements of economic importance in the production of broi-lers. In other studies (unpublished data) it was found that mater-nal antibodies interfered with the immunological response of chick-ens vaccinated before they were 21 d old.

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56 A. Rinaldi, D, Cessi, G. Cervio, E. Lodctti

REFERENCES

Badiola, C., Oliver, F. and Parla, J., (1969). Enfermedad bursal infectiosa, VeterinaryBulletin, 39: abstract number 3926.

Chinn, W. T. and Coomber, D. H., (1966). Fatty liver and Kidney syndrome. Vete-rinary Record, 78: 219-221.

Cho, Y., (1968). A study of infectious bursal disease and its control by immuniza-tion. Dissertation Abstract, USA, B 29: 286-287.

Cosgrove, A. S., (1962). An apparently new disease of chickens. Avian Diseases,6: 385-389.

del Bono, G., Agrimi, P. and Braga, G., (1969). Malattia di Gumboro. Annali FacoltàMedicina Veterinaria di Pisa, 21: 13-45.

Dorn, P., Kronthaler, O. and Schindler, P., (1968). Erfahrungen ueber Verlauf undBekaempfung der Gumboro - Krankheit. Berliner und Muenchener TierarzlicheWochenschrift, 14: 272-275.

Edgar, S. A. and Cho, Y., (1964). Avian nephrosis (Gumboro disease) and its controlby immunization. Poultry Science, 44: 1336.

Fabris, G., (1967). Contributo alla casistica della malattia di Gumboro. Giornale delpollicoltori, 2: 1-6.

Faragher, J. T., (1972). infectious bursal disease of chickens. Veterinary Bulletin,42: 361-369.

Herceg, M., (1971). Istrazivanja Gumboro Bolesti v Paradi i Podaci o prvom usta-novljenju zaraze v jugoslaviji. Veterinarski Archiv, 41: 67-71.

Izzi, R., Capurso, A. and Urso, C. (1970). La malattia di Gumboro. Atti SocietàItaliana Scienze Veterinarie, 24: 673.

Jordan, F. T. W. and Chubb, R. C., (1962). The agar gel diffusion technique in thediagnosis of infectious Laryngo-tracheitis (I. L. T.) and its differentiationfrom fowl pox. Research in Veterinary Science, 3: 245-255.

Landgraf, H., Vielitz, E. and Kirsch, R., (1967). Untersuchungen ueber das Auftreteneiner Infektioesen Erkrankung mit Beteilung der Bursa Fabricii. DeutscheTieraerztliche Wochenschrift, 74: 6.

Lensing, H. H., (1968). Gumboro Disease. Tijdschrift Diergeneeskunde, 93: 1452-1460.Luethgen, W., (1969). Gumboro Disease. Information de Medecine Veterinaire,

1: 3-17.Maire, C., Renault, L, Alamagny, A. and de Breuille, M„ (1969). Existence de la

maladie de Gumboro en France. Recueil de Médecine Vétérinaire de l'Ecoled'Alfort, 145: 875-884.

Reed, L. J. and Muench, H. (1938). A simple method of estimating fifty percentendpoints. American Journal of Hygiene, 27: 493-497.

Rinaldi, A., Cessi, D., Valeri, A., Cervio, G. and Perini, S. (1972). Ricerche sullamalattia di Gumboro: diffusione dell'infezione e valore diagnostico di alcuneprove sierologiche. Clinica Veterinaria. 95: 39-45.

Snedeker, C., Wills, F. K. and Moulthrop, I. M., (1967). Some studies on the in-fectious bursal agent. Avian Diseases, 11: 519-528.

Winterfield, R. W. and Hitchner, S. B., (1964). Gumboro disease. Poultry Digest,23: 206-207.

Winterfield, R. W., (1969). Immunity response to the infectious bursal agent. AvianDiseases, 13: 548-557.

Résumé

ATTÉNUATION DU VIRUS DE LA BURSITE INFECTIEUSE ET ESSAISDE VACCINATION EXPÉRIMENTAUX ET PRATIQUES

Une souche de virus de la bursite infectieuse adapté á l'embryonde poulet présente une virulence réduite. Apres 60 passages lasouche est suffisamment atténuée pour pouvoir être utilisée commevaccin. L'atténuation est assez stable. Administré dans l'eau de boi-

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Infectious bursal disease virus vaccine 57

sson le vaccin stimule la formation d'anticorps neutralisants etprécipitants et provoque une résistance solide ä l'infection. Desrésulats satisfaisants ont également été obtenus dans la pratique.

Zusammenfassung

ABSCHWAECHUNG DES VIRUS DER INFEKTIOESEN BURSITISUND IMPFUNGVERSUCHE IM LABOR UND IN DER PRAXIS

Dia Adaption eines Virusstammes der infektioesen Bursitis an denHuehner embryo führt zu einer Abschwaechung seiner Virulenz. Nach60 Passagen ist der Stamm soweit modifiziert, dass er als Vakzineverwendet werden kann. Die Abschwaechung war stabil. Bei oralerVerabreichung mit Trinkwasser bewirkt der Stamm die Bildung vonneutralisierenden und praezipitierenden Antikoerpern und eine starkeResistenz gegen die Infektion. Vielversprechende Ergebnnisse wur-den auch im Feld erhalten.

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