Research group of the standing technical committee at the Istituto Zooprofilattico Sperimentale-1969

EwRC?2AN COMMISSION FOR THE CONTROL OF FOOT-AND-MOUTH DISEASE

REPORT OF THE MEETING of the

::::::�:;c��J

G�CU? OF THE STANDING TECHNICAL COMMITTEE at the �3-�ituto Zooprofilattico Sperimentale BRESCIA, ITAL'f 26-28 September 1969

FOOD AND AGRICULTURE ORGANIZAT:ON o·F THE UNITED NATIONS

- 16 -

APPE.'fDIX I

.220:)UC'l'ION OF FOOT-AND-MOUTH DISEASE (FMD) VACCHf.E at the

:STI�UTO ZOOPROFILATTICO SPERil'.E.NTALE AT BRESCIA by CJartini B., Nardelli L., Bareis., Panina

a.,

Lodetti E.

?oo�-2.=.d.-�ou-:..b. disease (F>::D) vaccine produc1tion started at the Istituto Zoopra :'ilat-;ico Sper:.:::antala at Brescia in 1938, immed.iately attar 'Waldmann described hie :athod at tho International Congress of Veterinary Medicine held at Zlirich.

Virus �aJ cbt�ined froo the tongue epithelium ot cattle that had been infected .ntrader�o-lin��all7 prior to slaughter. ?ro� t�e outaot, the vaccine gave excellent results in the field but with the :as�a.;c o� ti:.o a nu.::iber of problems arose. Production proved very costly and tifficultioa �o:.o �con encountered in obtaining sufficient cattle to produce emergei n:p;,lioa o: vi:.::.a for vaccino when a new variant appeared.

The greatest drawback, .o�ever, vaa tbo danger ot spread ot virus.

3et;;ee.:. :91..�7 and. 1951, Frankel developed for the first time the cultivation or �:.':.:) vi::uu :;,.n v::.. -�::o on a large scale.

Frenkel' a 'Work 'Was recognized aa a great tch1Gvo:::or.-; ove;::-:,.1-Jb.ara.

The Brescia Institute (Ubertini et al., 1956) established ,n 1ndu3t=1al p�ant similar in type.

FY.I> virus was cultivated on cattle tongue 1pitheliu.:n, first in 40 litre stainless steel tanks and later in 200 litre tanks.

W1 th t.;1i;:; ;.:.c·;.hod, affective vaccines could be produced inside a laboratory, thu ,vo1tling the 8p::o�d of virus.

The difficulty, however, of the scarcity ot cattle :oraisted.

In Italy, �hare the production o� meat is limited, large industrial lau&htorhouses do not exist.

Slaughter is carried out in many small abattoirs and ,he collection of large qu�ntities of epithelium presents many difficulties •

•

?rod.uc·;io.;"l of .?:.m virus by the Frenkel method oontinued in Brescia using epi the � fro� It�ly a�d �broad (Yugoslavia) until 1966 vhen it became possible to replace hia ma��od ontiroly by cell monolayer culture techniques.

Such cell cultures had �en produced o� a large scale since 1958, three years after Bachrach and Sellers :rat aho�ed t�o possibility of multiplying FMD virus on calf and pig kidney cell onolayera in vit�o.

Our first industrisl scale plant (Ubertini!!.! al., 1960; Nardelli!!.! !!1•, 1960) ermittad a koeklJ production of 4,368 kidney cell monolayers in static Rou.x bottleB nd functioned froc 1958 to 1962.

The ne�d for highly concentrated FMD virus led tc ta davalop�ont of a method of cell culture in l litre roller-bottles (Ubertini!.!� 963) yioldin0 largor nu.cbera of cells with a reduced volume of culture medium. The t1l1zation of co��on one litre cylindrical plasma bottle■ in place of Roux plates ompenaatad for the higher cost of the rotating apparatus.

Du.r:n� 1962-1963 a pilot plant capable of producing 600 calf kidney monolayers n roller-bottles per �ee� vae brought into operation.

In 1964 the plant was uv=:ented �o a �eekly potential of 11,880 roller-bottles.

Special mechanical qui�rnant wao alao introduced to overcome the problem of handling bottle•� Baskets,

-17 -

�:- �s:.i��c :3 c'..:�u:e bottles, •hich fitted easily into a rotating apparatus in .::::...; ::.J. :o:-3 .:..t•.i c.;i-..;.:..i be co��d on a conveyor systam !or filling and discharg,9 gT1i1atl. '.i'.01.a plant functioned vith cal:f' kidney monol11yers in 1964.

J:..'-.:: 0:. L-�,:, ·.. o.:-:: :c�;;;.. i) 1 6 S ;�•::.;--�-.� ot tho l:lonolayora produced were !rom oalf k".. Y.,1eya vhila the r•-­ a:.r.:.:-.; 25 .:-.:::-cJ.::� ·,i33 obtained from baby hamster kidnt'y (.Bl:i.K) cells and the pro­ o.:-�:.c� o� ;.:;� �cr.olayars waa increased to 45 percent in 1966.

:.

: .. 1

0::. :. .I'1::.u.::.:-�- :.967 the prese.1.t plant waa inaugurated which baa an incubation apacit.7 o: �3,3co bottles (�bertini !.! .ftl•, 1967a).

This plant i� housed in a ;;,:,.;;.. al l.7 cor..:; �:.:--..c ted. building vhi ch meets all security requirements to prevent vir1 aca.;>e.

Ju:::-:.ng t::.c �-oar tl:e transition to BHK cells continued vith 50 percent calf' i�r.ay ar.� 50 �orcent EID: cell cultures.

In 1968 the calf kidne1 cultures were c=pletely replaced by those of BHK cells • • I� orde� to e��and the production of cell cultures in roller-bottles to an ndustrial sc�le, it was necessary to modify the technique described by Ubertini ! �1., in 1563. In this report we shall draw attention to the moat important ,oints and to tne results obtained in cell culture and multiplication of' FMD virus.

(1) In o.:-d.e::.- to obtain from each calf kidney the greatest possible number of cell :ultures i� �i�ro, both the cortex and the medulla of th9 kidney are trypsinized • . n tnia �ay tfia 3ield of cells is increased by at least 60 percent (Panina, 1965).

Zzpar�=enta carried out in 1965 aho�ed that with the cortex alone from a kidneJ ,f a 150 :��- c�lf it �aa possible to prepare an average of' 84 cultures in roller­ )ottleo uhile by trypsinizing both the cortex and the medulla the number or bottle• :o�ld be increased to 138. Both systems gave cells of the same sensitivity to FMD rirua.

Tiss�e f�o= the cortex and medulla of calf kidneys, cut into large pieces :�bout 4-5 picc�a por kidney) ia sliced with a special machine, the essential parts if �tich are t:;o �OU?S of 15 circular parallel blades (diameter 10 cm.) rotating )n t.o axlea at 1000 r.p.m.

The thickness of the slices is 3 mm. and the working :a�acity ia 10 kidneys per �inute.

(2)

':'rr:::iinization

�ryys1�1=ation ia effected in 10 litre vessels which are mechanically agitated �t J�n � at j�0°�"'• • -.,1v l'•r•r::•, J��

Bach ve3sol conta1no about 1.5 kg. kidney tissue and the digestion is carried for six hours.

Every two hours the trypsin solution is replaced.

�ta cells are recovered by centrifugation, weighed and then diluted in the cu::ure medium.

C;,,;.l:uro r.-.edium for the

cells: preparation, filtration

and

distribution

T�a c�l:uro mediu.m 1s yrapared 1n two 250 litre vessels alternately filtered �,�r-:)uct :::.:L:. So::. tz fi l tors and tranaf erred, together vith filtered cattle serum The final cell suspension ia obtained :��:) ��o :oo li:�o ata1nleos steel tanks� ·�.:,- .:..le.in,;- "t.t.t:i co::contrated calla to the filtered medium.

Under pressure thia s�1j;�n3ion is distributed into culture bottle■ uaing a simple device which permitn

;�e d1s:ri���1c� o: the au.s�ension into the 18 bottles in a basket aimultaneouslye In th1J A�� JSJ litres of culture medium per hour ar• filtered and 1800 bottle :er hou: can ba filled. �to bottlo3 ara sealed in the basket by a thin sterile rubber sheet fixed to a :et.al covar.

I

\

\.4)

I�cubat1on is effected in four incubators e ach holding 7,200 roller-bottles. las�ats a�a �ransported on conveyor belts directly to the different levels ot the .ncubators and incubation lasts six days, during which the culture medium i■ not :hang.ad.

(5)

r�oc�lation of the monolayar

Tho c�lturo �ediwn is poured off the batches ot 18 bottles by inverting each >ask.et on a rota.ting stand. i.:aintonanco medium and virus inoculum are then added in the same vq aa the .�itial cell su.spenaion.

Each bottle receives 80 ml. ot medium, containing virus per cell. :alculated to give 0.2-0.5 TCD 50

The virus strains are adapted by 3-4 passages in calf kidney cells and then ,tored in a freezer at -30 ° c vith 10 percent glycerin added.

�he quant��Y of virus needed to infect the first batch of cells ia prepared; rom this �atoria¼ but for subsequent batches, part of the virus produced the week ,efore, harvested after a short incubation period, filtered and stored at 4 ° c, ia ,add as the :rou ti:ie inoculwn.

At the 10th passage, fresh inoculum is prepared from the 3rd-4th passage s eed • ·irus kept in the freezer. ( 6)

Harvesting the virua

The virus is harvested mechanically in a tunnel washing machine, the bottles ieing automatically inverted at the point of entry.

Tha c.ea1\..:.Ill containing virus is funnelled into a collection vessel from which it .a �.ansfarred icmediately to a 1000 litre tank through a coaxial tube, in the o uter Thus refrigeration or virua :.ham'L.)er of wi.ach cold -water is circulated at +4 ° c. :ommences a fe� aaconda after harvesting.

The bottlea advance automatically through the tunnel undergoing successive rash1n� and three rinses, at a speed of 1, 800 bottles per hour. ( 7)

tc�alo��ent of cell cultures

?l:.a d..ava :..o ;,:..en t of calf kidney ce 11 cultu.res in roller-bottlea ia considered tc :e oat1afactory. �ithout changin& the culture medium a complete monolayer of not .eJs than 100 x 10 6 cells per bottle is usually obtained after six days of incuba­ ;ion. �ha parcenta8e ot gro�th failure is not more than one percent.

Soce of t�d reasons which may influence the failure of growth have been .d.,nt1.f1ed:

(a)

�-.:.�::..::.. -:.y and unif'ormi ty of kidneys is very important.

The �::..d�a:a u3cd by un all coffle from three to four month old c.'.l.boo, veighing 150-180 �g. and produced at industrial The J�oe�:ng centres which use reliable tee d mixtures. �idn0ys are collected at small private abattoirs so that the origin of the calves can always be ascertained.

Our experience indicates that it is impossible to prepare oa.t:..ofactor;r cell �onolayera with kidneys from certain aourceo, possibly due to the quality of the feed or particular additives that the producers use.

(b )

':'.r.0 cattle se1·um employed for culture media is collected from industrial slaughterhouses by bleeding the animals fron the jugular vein. The blood is detibririated cechani­ cul::..y in the abattoir and centrifuged in a separator.

It i3 then collected in 50 litre plastic containers and ia immediately frozen to -2ooc.

On the day that the serum is to be used it ie tr.awed and sterilized by filtration.

Several batches of this serum proved toxic for calf kidney cella; vith some there was only a el�w growth, while others af�ectively provented growth.

We attribute these diffi­ culties to contamination of the serum by urine or gastric contents in the serum, which may happen during bleeding if special care ia not exerted.

In recent years all batches of serum have been systematically tested for effects on cell 6ro�th bafora industrial scale use since a simple check by bringing the serum into contact with formed cell mono­ layers may tail to reveal toxicity which affects cell growth.

(c)

As far aa the glass of the culture have noted that the bottles we uoe bes� raaulta after repeated use a.DB, gro� at al� in new bottles, and only first four to five times a.:t; use.

bottle, is concerned, we (Neutro Murano) give washing. The cells do not with difficulty for the

Tho percentage of contamination which can be attributed to tha industrial process is not more than one percent; never­ thele::rn throe to four batches per yeal' are :found to be heavily contaminated.

In order to determine the origin ot th12 contamination we carried out culture tests at each stage of the process and found that very often they were duo to bacteria present in the first lot of liquid used to �asn the c�lf kidneys. It is obvious that the need to work with a mixture of kidneys in order to obtain more homogenic culture growth brings a higher risk of contamin3tion.

Ir. order to prevent contamination ample uae must be made ot antlbiotics and periodic checks of the sensitivity of the orzanlshlo concerned are required so that new antibiotics =.ay be introduced 1f bacterial agents threaten to c�uae difficulty.

- 20-·

(3)

In t� �:.o :. �=a oho�n the infective titres ot three typea ot F:MD virus o, A, c, :cC..t,;.coC. d1..:.:in,; the trienniw:l 1965 /66/67 on calt kidney cell monolayers in roller ottles.

TABLE 1

.F�:,�D virus culture on monolayers of calf kidney cells in roller bottle�.

Average infective titres in suckling mice.

Number

Year I of infected bottles

l'.967

01

I

C

Brescia 1964

7.7 7.8

7.3

265 ,161

I

A7

7.3

298,602

I

LD5 ofml

Switzerland 1965 Parma 1962

239,329

1965

1966

LoglO

7.4

7.6

7.8 ·, .8

7.6

The best t:trea �era obtained with virus types A and C while the titres of ·irua O (Switzerland 1965), were consistently lower.

Table 2 s�o�a the range of infective titre which can be obtained within a year 1f comrr.er.. cing F:rn virus production in cell cultures.

TABLE 2

Variability of mouse infective titres of the various FMD virus batches produced in 1967 on calf kidney cell monolayers in roller bottles.

i

I

Type of i ?lumber ot

virus I !

I

01

A7 ,,..

I.,

I

batches

Average Number of batches with titre volume or (LoglO LD5ofml)s each batch 7.0-141!5 Ll!5-8 .. 0 )8.o in litres ( 7.0

19

260

l

6

2

28

10

320

1

1

13

1

20

360

0

l

11

8

�=-3

�n tto �0t�ot �0acribed the monolayers are not washed &fter the removal ot tbe :.�o-.. :.:: c;;.l:. ,:-.c;:.i i.:...""J ::troo tho bottles.

Since all the bovine •�rum uaed comes rtur �1���a v�cci�u:�d aGain�t foot-and-mouth disease and therefore contains FJI.D anti­ od1e�, �o :.�3toJ tne influence vhicn residues or aeru.m might h.ive on the develop�e1 'f' tne virua.

To this end we inoculated parallel groups or monolayers: a) b)

�c�ediately after discarding the growth medium and, after having washed the monolayers repeatedly with butter solution.

�te exparicent has been repeated five times and the results reproduced in Table • indicated tr.a� washing the monolayers, after removal ot the growth medium, had nc eneficial effect on subsequent virus multiplication.

TABLE i

l

!

3

FKD virus cultures type Con calf kidney cell monolayers in roller bottles washed or unwashed before inoculation.

Infec-:.i �.,e titres in suckling mice.

XD50 for type Cbovine se=u...:1 present at a conoglO LD5Jml L centration of 10 percent in t.i:e cell culture Washed monolayers Unwashed monolayers mediu..":l

l

:

32

7.8

7.7

7.7 7.8

7.3

7.5

a.o

l • .1..

8.3

a.o

8.4

?RODi.JCTION OF FOOT-AND-2'!0UTH DISEASE VIRUS ON BHK CELLS

In 1962 ;,:o-wat and Chapman reported on the sensitivity of BHK 21 cell lines to ;-�'.D virus.

As a result i t became possible for laboratories to cultivate FJ.ID virus ·'" v�tro in unlimited quantities without having to rely on a flov of material trom :.-.e abattoir.

During 1962 the Institute of Brescia began a series of investigations aimed at .iapting existing installations to the large-seal& production of BHK cells (Ubertini !t �., 1957b).

It �as noted that such cells can be cultivated in roller bottles and that the 1ivant�Ge of this method, over Roux bottles, was considerably greater than that )':served. �i th calf kidney eella.. In four to five days of incubation, vi thout :�an�ing the cul�ure mediwn, monolayers composed of 300 million to 400 million cellr :0uld be obta1nod in roller bottles, while in the Roux bottles the number of cell• i1� not exceed the 60 �illion to 100 million range.

- 22 -

: n:. � ::.. :.::. ::.,· , �::.a ::;::.: c e l l a v e r e obtained by thtt d i ge a t i on o f o the r mono laye rs . ·" :. : .::. � •• e cu :.. :.. .J .·::;;:::. o.no b o t t le , f i v e nev bot t l e s c oulci be prepared .

However , t hi s �u ��o� � 3 0 3 o on : o und t o b e uno u i t able for l arge-s cal e app l i c at i on .

F o r i nd u s tr i a l pro:i u c t i o .n of : J , 0C0 b o t t l e s pe r veek di ge s t i on o f 2 , 000 bot t l e • v ould have bee n reG u i rad and the nu..T.a r o us mani pu lat i ons w ou ld be accompanied by a h i gh c ont ami n a t i o n rata • • .n t te =.eant :. � e , Caps t i ck e t a l . , ( 1 96 2 ) re porte� the cul t iv a t i on of e ome Our s ubsequent i nve s t i ga t i ons , w h i c h s t ra i ns o f ��< ce l ls i n a gi tate?suspens i o n . � e r o conducted a l o.ng t he s ace l i nes a s t h o s e o f the Ins t i tu te a t Pirbri gh t , v e r e s u c c e s s f u l a nd i n 196 3 � a s ta r t ed w i th a smal l 4 0- l i tre s t a i n l e s s s te e l contai n e r '- l t h w t. :. ch l t ·.i as ;,o s s i b l e t o o bt &. i n 30•- l i tree o f c e l l sus pe ns i on avery 48 hours i n s e� 1 - con t 1 nuous c u l t ur e .

From thi s c e l l suspe ns i on , mono l ayers i n r o l le r bot t l e& cou l d be prepared d i re c t ly , w i thout cent r i fu ga t i on , f o l l ow i n g d i l ut i on of the s us­ p e ns i o n w i th c ul t ur e medi um .

A n a w ce t h od co u l d , t h e r e fo r e , b e p l anned w h i ch could eas i ly be appl i e d o n an i nciu s t ri � l s ca l e : sem i -co n t i nu o us cul ture of BHK c e l ls in s uspens i o n , use or the s u s � e ns i o n f or t h e pr e pa r a t i o n of mono l aye rs in r o l l e r bot t l e s , and product i on o f Fr� v i rus i n the mono l aye rs thus e s t ab l i shed .

! n 1 96 5 w o rk e r s a t the P i r b r i ght Ins t i t ut e ( Caps t i ck e t a l . , ) reported o n t he ;,ot:; s i b i l i ty of cul t i va t i ng FKD v i ru s ciire c t ly i n a gi tated auspene i cns of BHK ce l l s .

Cur f 1 nd i n £S c o n f 1 r�ed thi s , b o t h i n sma l l cont a i ne rs ( 4 l i tre s ) and i n large ones ( �O , 200 and 6 CO l i t r e s ) . How e ve r , d i f f i cul t i e s such as tho sa of obtaining bovi n e s e r um fre e f rom Q n ti bod i e s have hampe red the deve l opment of large-s cale v irus c u l ture d 1 re c t ly o� c e l l s i n s us pe ns i on.

Fur the rmore , a s repor ted re cent ly by t he Fed e ra l 6epub l 1 c o f Gar�a ny , t h e r e was a s e r i ous inc id&nce of de l ayed anaphylaxis i n ca t t l e a t t r i but ab l e t o :::,:::> va c ci ne w i th v irus cul t ivated o n BHX ce l l s i n s us pe ns i on. Thi a has i nduc e d us t o continue , f o r t he pre s e n t , o ur t echnique combini ng ce l l produc t i o n W i th the n� vaccine prepar e d :. n s u s pens i on w :. t .t. vi rus produc ti o n i n mono l aye rs . w i t h t t 1 s v 1 r uo , � e have n e v e r o bs e rved an.r d e l ayed L_ypersens i t i vi ty ana lo gous t o t ha t r e p o r t e d i n Ge rmany .

(1)

C u l : �r e o f BHK c a l l a i n suspens i o n

F l. eur e 1,0 . 1 s hows t he s ta ge s used for the preparat i on of the a us pendec. ce l l cul t u r e . �he s t o ck o f Bffi< ce l l s i s cons e rved i n v i a l s o f 1 m l . i n l iqui d ni trogen ( ?dn i n a e Du l ?ra to , 1 96 5 ) .

C e l l a frozen five y e ars ago are s t i l l v iab le .

A f t e r t he tw o paa s aee s i n mon o l ayers ( four ounce b o t t l e s , Roux p l aque s ) re q ui re d t o o u t a i n a s uf f i ci e nt quan t i ty o f c e l l s , c u l t ur e in suspensi on begi n s w i th a v o l ume o f 400 ml . in a 1 . 5 l itr e gl a s s c o n t a i ne r , B e l l co typ e .

A f t e r 7 2 hours , w he n tr.e d e v e l o pment o f the c e l l c u l tu r e i s s uf f i ci e n t ly � d v � n c ed the c e l l s a re tr ans fe rred t o a f i ve l i t r9 contai ner o f the New Brunsw i c k t y p e , 0av 1 n � a c � l t u r e v o l ume o f four l i t re s .

In 48 hours a c o nc e n t ra t i on of 3 x 1 0 ° c e l l s/m l ie u s ua l ly o b t a i ned .

Fi eure ?io . 1 .

ii I{

- 0 - €:::::3> - (,._-P A

f'I ,;:::

I!...

....., GJ_:, r�-�L...;, ,� ii 'I iI !\ I 'I :j ll• I

i I!

�

,, :: I' °'7"::l

p ii !1 d \1:, ,,i II ., I, 'l 1 : l1 0-:0 ,, 1· .,!' ,, I'

1,

·I I

.,

;1 I

,I

!I

o.;o

'i I

"

-

II ii" ,,:I 11

I,

O:;;CJ

I I

ck 11 �

I

�l

1r� _�r

)< Ill 4 ,-4 ,-J (l) 0

1, -

hours

·· ­

� 1 t h 8-10 l i t r e s o f t hi s s us pe ns i on , care ful ly t e s t e d for s te r i l i ty , cul ture i n s ta i n l e s s s te e l c o nt ai n e r s of 1 50 l i t r e capa c i ty i� i ni ti ated .

The pres ent i ns t a l l a t i o n c o ns i s ts o f 3 i x s uc h c o nt a i ne rs , w i t h automat i c t e mpe r ature and pH cont r o l .

In 43 h o u r s , a ce l l c on c e n t r a t i o n o f 2-2 . 5 x 1 0 ce l l s/m l i s reached.

One p a r t o f t he s us p e n s i o n ( ab o u t 100 l i t r e s ) i s t he n removed for the pre par at i o n o f rr. o n o l a�· e r s , w h 1 l e t h e rem a i n i ng 2 0 li t r e s are u s e d tor anothe r cyc l e o ! c u l t u r e , v 1 t h addi t i o n a l c u l t ur e m e d i um .

By e x t e r.d i n� t he i n cub a t i o n p e r i o d , i t i s p os s i b l e t o o b t a i n a h i ehe r concen­ t ra t i on o f c e l l s , b u t t o p r e p a r e o the r cul tur e s ( b o th monol ayers and sus pens i on ) be t t e r v i a b i l i ty 1 s o b t a i ne d by removing the c e l l s when they w e re s ti l l � n the l o �� r 1 t h� 1 9 p ta s e of m u l t i p l i c a t i o n.

I n 7 2 hours , c oncentra t i o ns o f the order o t 3 - 5-4 x l C 0 ce l l s/ml c a n b e o b t a i ne d , b u t i n o u r o p i n i on s uch c e l l s are n o t sui t a b l e f e r o t te r c u l t u r e s .

� n t � b l a 4 a r o gr o up e d t o ge �he r t h e r e s u l t s o f 3 00 c u l ture s or BHK c e l l s i n � �B � b n s i o n i n 1 5 0 l i t r e t ar..i-: a , � h i c h w e re p r e p a r e d i n the b ie nn i um 1 96 7 - 6 8 . S i x r,e r c e n t o f t h e � u � t u r e s � e r e c o � t dm i n a t ed ; t w o pe r c e nt w e r e c o ns i de red as undev e l oped ( l e s � t han l x lG p� r m l � ) ; 1 2 pe r c e n t w e g e i n s uf f i c i e n t ly d ev e l o pe d ( numbe r at � d l l g ; e r � l : b e t� e e n l x 10 6 and 1 . 5 x 1 0 ) , w h i l e in t he rema i n i ng 8 0 p e r c e n t d � v � l o p�& n t w � a s a t i s f a c to ry ( m o r e t h 3 n 1 . 5 x 1 0 c e l l s/ml . ) .

- 24 TABLE 4 I

i

I

I

!;Z( cul tures in suspension prepared i n 150-li tre tanlca . . c o i tl .10 ' b ienni um 1 967/68 �uri:.i

Dovelopment or culture after 48-60 hours .

Number of g ulturea with nwnber of vinble ce l l s x 10 /ml : 1- 1 . 5 1 . 5-2 2-2 . 5 2.5-3 ') 3 1

1h.:.•':'lber of

;;c..:Jbo:-

o f cul-

cul tures contaminated

tuxoa

I

<

!

3co

20

114

43

35

6

40

42

ln an ea�liar c ommuni cati on (Ube rti ni il 1967b ) , we reported di ffi cul ty i �01 ng b eyond t� o cycl e s i n aeries or culture i n s uspens i on , in large containers , �e c aus e 0£ boavy c ontami nati on.

The appli cation ot aatety measures has now ensure :ond i t i ons of cooplete s teri l i ty. It is now possible to keep 1 50-litre container• Ln cont inuous pro�ucti on for s everal months (20-40 cycles of culture) .

I n Fi gure �o . 2 , fo r o=ample , va show the results obtained vith 24 consecutive cell cultures i n a 1 50-li tre tank ove r a peri od ot tvo m�ntha .

!l• ,

Figure No. 2

cu l t u res corr i e d ou t I n . . s e r i e s i n 1 50 l i t r e f ermen t�rs·iOu r o t i on o f cyc l e 2 months �

D c v c l o?wen t o f .

-

E �

I 4!

:•

•

I

I

!

Il I

I

I

u

..

0

z

.,'

I

•

••

• !�

I

'

II : '; I

I

'

.,

�1 .1� .: l I

I

l:..

•

•

I •

;..

0

•

•

I •

3 � 1

,....

I

• 24 BHK ee l 1 ·

I

I

• .,

s

•

• •

10

½

1i

I I

'

j

' I l ' 8 / I • • 4 "' • • • • • 1

:

f

15

t u r e s er i es

' • • t

'

I

J

I

•

j

•

/

• • I

20

%

•

- 2..5. -

. � �a.blo �o . 5 a l l dat.a relative to such a cul ture cyc le are collecte d . They are s :�i l ar to ��ose o f other l engthy cycles and can be c onsidered typi cal tor euok cul � �r e a \.:.:'. �e: ��e cond iti ons of the Brescia laboratory.

TABLE

5

�ata co=ros�ondi n g to the BHX cel l culture cycle represented in F i gure :Xo . 2 .

II

r::'ota.l of days o f culture

54

Tota l of cultures (?fo . )

24

Avera 6e durati o n of each culture (hours )

54

7

number o f viable Avera cel ls ml

l

At the beginning of the cultures At the end of the cultures

-

Average mult i pl i cati on for each culture ':'otal number o f v iable c e lls

At the beg i nning of the cycle Produced i n 54 cultures

To t al mul t iplicati on in 54 cycles Average number of ro l ler bottles, 'Which can be prepared -with the cells harvested from one c u lture

4. 6 2.8 6

•-X

10 5

X

10 6

X

46 X 109 6870 x 109 163

X

18, 000

Int errupti ons o f tne cul ture cycle� are attributed mainl y to machinery breakdowns or contaminati on of the suspensi on.

On s everal occasi ons we had t o interrupt the culture f or reasons inherent to �ta qua lity of calla.

W e ha ve observed that after many s uccessive cultures, the ca l l a do no t c o n t i nu e to multiply satisfactori l y in monolayers, although regular gro� t h may conti nue in suspensi on .

Since t h i s phenomenon i s due to ?. �haracteriat � h ich t h e cell s acq�ire during ext ended suspensi on culture, it seems advisable �er iodi ca l ly t o ret ur n t o t he bas i c cell stock.

Tte medium us ed i s Eag le ' s medium with a double quan t i ty of amino-ac ids and v 1 �arnins , t o whict ar e added 10 p ercent tripticase Soy broth (BBL), 10 percent C J ttle serum and O.l percent Edifas (I CI ) (Te l l ing and El sworth, 1 965 ) .

S i nce , t h i s i s a n expe nsiv e m edium which can be difficult to pre pa re on an industri a l scale , w e o ubsti tuted t he amino-acids (wi th the exception of g lutami n ) �Y l actal buc.in hydr o l ysa te and the grow th o f B HJC celle was found t o b e almost the a . �o ao tha t on t he o r i gi na l m•d 1 U ffl e Eo� e v e r , hav i n g found s ources o f suitable ami no-acids and v i tami n s, whi c h � � J u ced t r. e pr i c u o f the o r i gi n a l med ium a p proxi ma t e ly 10 fold , � e d i d not adopt t :·. e r.-. od i f ::. ed rr,ed 1 um c:1.s i..e feared that cha n ge s in the c harac t e:ris t i c e of the cell ! 1 :-. e �• rr. 1 t� = � t o c cur .

- 26 -

(2)

C � l t � r e " f B HK c o l l s i n mono l a v e r

� t e c e l l s u3 �e n� l o n f r o� the c u l t ures i n 1 50-l i t re tarJcs , i s kept i n 5-li tre bo t t l � a lO tr.ti r 13 f.r i e;vrd t o r at •· 4 ° c , hav i ng ens ured tha t the pH i s no t l ower than 7 . 2-7 . 4 .

I t i s c ar e ful ly checked for bacter i a l s teri l i ty and the capabi l i ty of the c e l l s t o grow on m o no l ay e r s i n r o l l e r b o t t les .

Thus , 'the s us pens i o n i s not us e d :' o r t ha p r e ; ara t i o n o f mono l ay e rs less than three days after c o l lect io n and ma.Y , i n f a c t , b e a t o rod for a s l o ng as t w o veeks .

The m e thod o f p r e p a r i ng mcno l �yers i s the one a lready des c ribed by us , name lyr Di l u t i on o f t he c e l l s us pe ns i o n i n mono l ayer c u l t ure medium ( Po l�tni ck and Sac hr a c h , 1 96 4 ) t o p r o v ide a ce l l �o ncentrat i on vary i ng from 50 , 000 t o 15 0, 000 cel l s per m l .

Ed ch b o t t l e r e c e i v e s 200 m l . o f t hi s suspens i on , equal t o 10 - 30 x 106 cel l s . rhe d e v e l o p m e r. t o f t he monol aye r i s obtained i n t hree days wi th the greater i ni t i al �uant i ty o f ca l ls o r i n five d ays w i th t he smaller quant i ty, wi thout changi ng the �e d i um# A t the t i�e o f t nocu l a t i o n w i th v irus , t he t o ta l number of cel l s per bot t l e i s no t l e s s ttan 2 50 x 10 ; i f i t i s �os s i b l e t o extend the ineubg t i on peri od b y o ne to t v o day s , the numbe r o f cel l s p e r bot t le r i ses to 3 50-400 x 10 .

The pres e n t 1 ns t a l l a t i o n i s c apable of handl i ng 28 , 800 b o t t les per week wi t h t he equi pce nt and �e thodo d e s c� i bed for calf kidney ce l l s .

I t i s 1 nt e re s t i ng to n o te that , wi t h the BHX oel l s i n m onolayers , i t i s poss i b le � n a � emere--o n cy , t o i n c rease the p o ten t i 3 l of the p l a n t , with the same incubat ion capa c i ty. l n fa c t , by l i mi t i ng t he i ncuba t i o n of mono l ayers to three days and w o rk i ng e v e ry day produc t i on could be increased by 5 0 percent , with ident i c a l resul ts .

G r ow t h fai l ur e s are a t t r i butab l e t o : a) b) c) d)

the us e o f cel l s cul t i vated conti nuously t or several months i n s us pens i o n ; c ons e rv a t i o n o f the c e l l s i n the refri&erator beyond two w eeks or at a pH l ow e r t han 7 ; c a t t l e s erum o f i nfer i or qua l i ty ( The serum used i n Bre s c i a i s p r e p · r e d as a l r e ady d e s c r i bed f o r cal f k i dney cel l s ) . c e l l s o r i si n a t i ng from h i gh l y co n c e n trated s us pe ns i o ns a nd o b t a i ne d by a x t e nd i r g i nc u ba t i o n beyond 7 2 hours ( i . e � ce l ls n o t i n l o ga r i t hm i c p has e ) .

� 1 t h 3:fi c e l l s t r. � ? O r c e nta&e o r contami n a t i o n i s much l ower than tha t observed w i t h c a l f k i dney c u l t ur e s .

Th i a pe rmi t ted reduc t i o n o r b o th the number and the 1ua n t i ty o f a n t i b i o t i c s pre sent i n B HK cel l cul tures .

(3)

P rod u c : i o n o f Ff.:D v i rus o n m o no l ayers

':'h'3 i n f e c t i o n of Bnl< c e l l m o n o l ay e r s i n r o l l e r b o t t l e s i e c a r r i ed o u t a e d e s c r i b e d f o r C d l f k 1 dneJ c& l : a . ;n:.n v 1 r �s adap t e d t o DniC c e l l s i a us ed f o r i no cu l a t i on i n t h e p r o p o r t i on o r J . l - C , C 5 TCD jO �o r c e l l . :, o d i f f i c u l ty hd s bd e n e nc o u n t e red :r n ye t i n adap t i ng

f:.J)

e t r n i n a t o BHk c a l l a .,

- 27· F:.:.:i v i rus ad.1 :Yted t o c a l f kidney c� l l s i s not aui table as i noculwn tor BHJ< ce l l s ( l ov c,:r t o ;, ..1 t hi c .:i fto c t ) ; how e v e r , the vi rus adapted t o BHK cel l cul tures can be Th i a has a pra c tical aspect w hen produc­ u t i l i zud i o r i n:e c t i c 0 c � l f kidney ce l l s . ti o n in c a l f ki dney c e l l s and BHK ce l l s i s carried out e im u l t aneous l1 . ? h e v i r us i s tarv e a ted aft e r 20-24 hour s o f i ncubat i o n with total ce l l lys i e . �abl e N o . 6 a bov e t he a v e r a ge i nfe ct i ve t i tr e s o f the three types o r FMD virue , A. and C , produced over th e four-year per i od 1 96 5-196 8 , on BHK ce l l monol ayers i n rol l e r b o t t l e s .

o,

TABLE 6 Fli:D v i rus cul t u r e a o n BHK mono lay e r s i n ro l le r b o t t l e s . i nf e c t ive t i tres i n s uck l i ng m i c e .

I

Year

Number o f i noculat ed bo t t l es

Lo gl O LD 5 o/ml

A7

01 Sw i t z e r l and 1 9 6 5

Parma 1 96 2

Bre sc ia 1 964 7.7

1965

1 2 5 , 215

7.6

7.7

1966

2 4 5, 824

7.5

8.1

1 967

1 96 8

Average

7 .7

3 57 , 4 5 5

7 .7

7.9

e.o

8. 5

7.9

8 3 2 , 170

7.7 -

Tab l e 7 i ndi cates t he v ar i a t i ons i n infec t ive t i tres o f the bat ches o f vi rus produced o n BHlC oe l l mono l ay e r s in ro l le r b o t t l e s in 1 968 .

TABLE

7

Var i ab i l i ty of i nf e c t i ve t i tre� i n mi ce o f tte b a t che s o f FMD v i r�a pro d uced i n 1 968 o n BHK ce l l mono l ay e rs i n ro l le r bo t t l e s .

Typ e o f v i r us

N wn b e r o f batche s p roduced

Ave rage v o l u.me of e ach batch in i t r e s

21

900

0

3

7

15

330

0

C

0

51

6 90

0

w i th t i t re

Numb er

16

11

15

29

?�od u c � : o n o f F�� vi rus i n su spension As a lreaq i'.":,rnt i o ned , this technique is regarded as being still at an experi­ �ental s tasa at t�e 3rea cia Institute.

However, a pilot plant equipped v ith a 6 0t l i tre �an.k has beon brought into o peration.

100 litres of cell suspens i o n are trans ferred t o thia tank froc a 150 litre tank and, following the addition o f culti cediu.m to cake a total of 500 litres, a cell culture in suspension is prepared. I 1 4 8 h ours satisfactory developoent i s attained ( 2 - 2.5 x 10° cells/ml ) . At this p oint 0 st irring i s stopped, with refrigeration the suspension temperature is reduc, t o +4 C a nd the c ells are allowed t o sediment for 12-24 hours.

The supe rnatant fluid is discarded , w hile the sedimented cells remain in the tank, in a volume equ! t o 1 0 per cent of �he total initial volume.

During this operation about 20 percen1 of the / ce lls are lost.

Culture medium is added until a cell concentrati on of abot 3 x lo b cell/ml is o btain ed.

This cell suspension is inoculated with virus newly harves ted from B� monolayers ; the virus is c ollected after about 30 hours . To dai v i ru s multiplica t i on has been satisfactory with '\�irus type C ( LD c;o 7.0 - 7.5/ml ) bti for types O and A, the cultures carried out so far have shown a tather low infecti� titre ( LD 5 0 6 . 5 - 7 . 0/ml) .

The maj or sources o f difficulty encountered wares

a) I n securing supplies of F:MD antibody-free cattle serum ; a two percent con cent ration of whi ch is indispensable f or suspended call cultures that are to b used directly for virus cultivation.

Wi �h cell cultures of 500 litre, eedi­ �anta tion and aspiration, large quantities are obviously needed.

As already mentio ned , this problem does not arise with virus cultures in monolayers, wher the growth medium containing serum is almost compl�tely eliminated at the time o f inocula tion. b) Sec ondly, the loss of cells whioh takes place when the supernatant flui d i s dis carded is a drawback.

A 1 though this method of cultivating suspend.ad cells is not ourrently being use1 for the p reparation o f vaccine for bovines, experiments continue and a 1,200 litre tank ia a bout to be installed, with the aim of raising the laboratory ' s potential.

I I I . PREP A R ATION AND CONTROLS OF VAC CINE Vaccines are always prepared i n monovalent form.

After i na ctivation they are s to red under refri geration in 50 litre cans awaiting titration for potency in cat t l1 ( Pn 50 ) .

The trivalent vacoine is then prepared by mixing t he monovalent vaccines i n appropr iate pro p o r ti ons.

(1)

M o n o va l e nt va c ci ne

r ��e diately af ter harvesting , the F}U) virus is refrigerated . 1 . 2 percent c h l o r o forn ia added, the mixture is s ti � red for two hours at +4 ° C and centrifuged � t 8 , 000 x g. A fur t her a.a per cent chloroform ia added and the mixture stirred overnight at +4 ° c , then centrifuged in two centrifuges ( 8, 000 x g ) in series, with an i nterpo s e� cooling cy c l e . �he tr� a ted virus is added t o alumi n i um hyd r oxide ( l . 4 percent Al 2 0 3 ) , glyc o c o l buf fered a t p n 6 . 0-d. 2 , and inac t ivated by form ali n at a f inal concentration of 0 . 6 � parts per tho us � nd ).

7to 'iuan t i ty of aluminium hyd ro x i de 1a C dlculated on the ba a ia of 3 . 1 5 m l for each 1 � l o f v � ru a .

-------

•

° A f t e r 45 ho�ro o f i na�tivat i o n at +25 c the vacc i ne i a conce ntrated b1 re mov i n g �he gre ate r part o f the liquid which has aeparated trom the aluminium gel .

Fr om L0. 1 5 cl o f vacc i ne ( of whi ch 3 . 1 5 ml alumi nium hydroxide and 7 ml virus ) 7 .15 ml ot L iqui d can b e e xtracte d .

For t. h i s opera t i o n a s pe ci a l 2 50 11 t r e sta inless ste e l concentrator i a used. tn i t a i nner cavi ty hanes a cyl i ndri cal t i ss ue bag o r 150 litres capaci ty . The .acci ne is poure� i n t o t he bag w he re a he l i co i dal screw ■tira i t co ntinuoua ly. The l iquid i a e lici nated through the meshes or the bag whi l e the gel be comes more and � o re c o nce ntrated.

Adhes io n o f the gal to the w a l l s ot the bag i s prevented by the 1gi tat i on. 2 50 li tres o f l iquid can be elimi nated i n thi s w ay per hour and the l oss Jf F!::::) ant i ge n is ne gl i gi b l e .

T h i s techni�ue of conc e n t ration v i th a luminium hydroxid e was developed at Bres c i a ye ars a go t o con ce n trate FV.D antige n from cal f k idney cells cul t ivated i n Roux b o t t l e s as i t w a s found t o be too d i l ute:l for sffi c ient immuni zation. Thia iiffi. c u l ty has been o v e r come by the use of ro l le r bot t l e s and BHK cells w hi c h giv e Howeve r , conce ntration on aluminium a sati s f a c to ry ant i ge n i n tha cul ture liquid. gel i a s t i l l routi nely practised for two main purpo •e & S a)

the concentration o f FMD ant i gen makee i t pos s i bl e to prepare t rivalent v a c c i ne s whi ch can be adminis tered i n amall doses w i th s t imul a t io n of s tron g immunity;

b)

t he e limina t i o n o f a l l the s ubstances present along w i t h the v irus i n the cul ture media after ce l l lys i a , vhioh are not adsorbed by alumini um hydroxi de .

(2)

Triv a l e n t v a c c i ne

As a l ready s tated , the m,novalent vacc ines are mixed o n the baoi s of their Thia titration i • sys temati cal ly carr ied anti t;;e n i c i ty t itrations ( PD 50 ) in cat t l e . out in the i s o la t i o n s tables o f the Bre s cia I ns t i tute (Ubertini !! !l• , 1968 ) .

The t rivalent cat t l e dose i s so formulated that i t co ntains about 10-20 catt l e A t the mixi�g s tage 3 m g o f saponi n are added for each catt l e dos e . ?D �o p e r type .

Th& s a p o n i n comes from i ndust r ial ly produc ed batches w hi o.b. are tested tor non­ spe c if i c antibody production prior to use i n vac c ine .

(3)

I nno cui ty t e s ts

Each batch o f monovalent vaccine i s tested tor 1 nnoouity i n suckling mi ce by i nt raporitoneal i nj e ction. Not l e s s than 50 mi ce are used for each batch or v a c c i ne ..

Tri v a l e nt v a c c i r.e i e t e s t e d i n a us _c e pt ible catt le by i n tr adermo-lin gua l. i nj e c t i o n , i n add i t i o n t o t h e tests i n mi c e .

(4)

Ant i &en i ci ty t i t r a t i on o � mo nova lent vac c i n� s

Dur i ng the pre para t io n of the vari ous batches o t monovalent vaccine , titrati on o f i mmuni z i n g po� e r i s sys t e ma t i cal ly car r i ed out in guinea-pi gs as a routine con t r o l ove r prod u c t i o n a nd t o c he ck t h e v a c c i ne produced, � i th v i r a l s t rai ns or r ec ent 1oolation. �1 xturea o f v a r i ous ba t c h e s or monovale n t vaooin• ar• teste d ( PD 50 ) i n cat t l e . Au3 t r i a n ca t t le fre e o r F►rD ant i bodie s are used , and are kept i n i s o lat ion s ta b l e 3 d ur 1 ne t h e e x� e r i�e n t a .

The v a cc i ne i s a i l u t e d i n buffer s o l u t i o n , a nd no t l e s s t han three d i l u t i ons �re 1 nJ e c t � d e u L c u t a na o ue ly into c a t t le each in a 5 ml_ dos e .

For ea ch d i l ut i o n f 1 v o t o o e v e n c , t t l e a r e us e d .

- .39 Af t er three weeks the animal s are challenged by intradermo-lingua l injec tion

or 1 0, 000 I. U. of the homolo gous virus strain. '!'M o non-vaccinated ca t t ). e whi ch

serve as contro la are infected simult aneously.

Tha animals are observed f or e ight days. bas i s of their res istance t o generalization.

(5)

The PD50 is calcu lated o n the

Efficacy t e s t s o f tri valent vaccine

The trivalent vaccine is tested under the supervision ot the I talian Oovernmc It i a in jected in a 1 : 8 diluti on int o 18-20 two years old Aus trian ca t tle, the cattle d oae being 5 �l.

After 21 days the animals are divided in three groups of s ix t o seven cat tle and each group is i nfected with a different virua type usir Tw o non-vaccinated animals of the group s erve i the technique already des cribed. contr o l s for each of three virus types.

The trivalent vacci ne i s cons idered efficient it at the injected 1 : 8 dilutior. a t least half of the animal s are protected against generalization of all three types of' virus.

LITERATURE

Capatick , P. B., Telling, R.C . , Chapman, w. a. and S tewart, D. L. ( 196 2 ) Grow th of a cl oned s train of hamster kidney cells in suspended cul t ures and t heir s us ceptibility t o the v irus of Foo t-and-Mouth Disease.

Nat ure , ill, 116 3 .

Capet ick, P. B. , Garland, A.J . , Chapman, W. G. and Maa tere, R. C. ( 1965 ) Production of F o ot-and-Mouth disease virus antigen from BHK 21 cl one 13 cells grown and i nfected in deep suspensi o n cultures.

Nat ure, 2 05, 1135• )�owa t, G . N. and Chapman, W. G. ( 1962 ) - Growth ot Foot-and-Mouth disease virus in a fibroblastic cell line derived from hams ter kidneys.

Nature, ill, 253.

Nardelli , L. , Ubertini, B., Dal Pra t o, A . , Panina, a. and Santero, G. ( 196 0 ) Wei tera Erfahrungen Uber die Massenheratellung dea MXS Virus i n Kalbernierenzellkulturen.

Arch. Exp. Vet. Med., ll, 276.

Panina, G. ( 1 96 5) - Coltura i n vi tro del v irus attoso : utilizzazione della cor t icale o della midoll are del rene di V i tello. Atti S oc. I tal. S o.

Vet. , .12, 7 57 Panina , G. and Dal Pra t o, A . { 1965) - Congel ament o in vapori di a z o t o l iquido del l a l i nea cel lu lare BHK 2 1 . Atti Soc. I ta l . S o . Vet . ,!2, 7 51 Polatnick, J . , and Bachrach , H. L. ( 1964 ) - Product i on and purifica t i on of cil l ig ram a�ounts o f Foot-and-Mouth disease vir us from baby hams ter kidney call cultu re s . Appl. Mi crobiol . , 12 , 368 Telling, R . C. and Els� orth, R. ( 1965 ) - Submerged cul ture or hamster k i dney cel l s in a s tainless s teel ves sel. Bi otechnol. Bi oeng. , 1, 4 1 7 Uber t i ni, 3., Nardelli, L., Barei, s. and Santer�, G. ( 1956 ) - Etudes sur la cul ture in vi tro du v irus aphteux s alon Frenkel. Zbl. Vet. Med • .J., 4 1 9 and 767 uber tini, B . , Nardell i , L . , Santero , G. and Panina , o. ( 1 96 0 ) - P r o ces s Rep o rt : Large scale p roduction ot F)!D virus . J . Bi och. Micro bia l .

Te c hn . Eng . , 2 , 3 2 7 Ube rtini, B . NardeTli , L . , Dal Prat o , A . , Panina , a. and Santero, O. ( 196 3 ) Lar ge aca l e cultivat i on o f FKD virus on cal f kidney cel l monolayers i n r o l l i n � bo ttl e s . Zbl. Vet . J.�ed . , R e ihe B . , 1.£, 9 3 Ubertini , B. , �ard6lli , L . , Pe nina, a. and Lode tti , E. ( 1 967 a ) - S ome N otes on T e chn1q u a s o f Foot-and-�outh Diseas e Vi rus production us ed in B re s c i a .

Re po r t �d d t1ng �esearcb Group FAO , Long I sland , N . Y . , USA, 2 9 .

- 31 tibert ini , B . , Nar de l l i , L. , Dal Prato , A. , Panina, G. and Barei , s. ( 1 96 7 b ) 3H:.( 21 ce 1 1 cultures for the large-s c;:ile productio n or FJ.iD virus .

Zb l . Vat . !,! e d . , Rei he B . , ll, 432 Ube rtin i , B . , Narde l li , L. , Bare i , s. , Panina , a. and Lode tti , E. ( 1968 ) Con tro l l i di efficacia su bovini di vaccini antiattos i monovalenti e t riva l e nti preparati con virus da ce l lule BHX. Atti Soo. I ta l . Sc .

Ve t . , ll,, 8 9 5 .