egg inoculation by c.ngullie

7/24/2019 Egg Inoculation by c.ngullie

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EMBRYONATEDEGG

INOCULATIONBy:Chinithung ngullie(M.sc 2nd year)

MODERATOR:Dr.Varsha A singh



irus cultiati!n

– is!late and identi"y iruses

– iral structure# re$licati!n# genetics %$ath!genesis

– accine $r!ducti!n

2



TEC&'*E+ O, VR*+C*-TVATO'

• EMBRO'ATED E// 'OC*-ATO'

•

T++*E C*-T*RE• A'MA- 'OC*-ATO'

0



ntr!ducti!n

• Cultiati!n !" iruses in chice3ry!

• di4erent ty$e !" a$$r!ach.

• ,!r all $ractical $ur$!ses they3ehae as tissue cultures

5



&ist!ry

Burnet

,irst used "!r cultiati!n !"iruses 3y Ernest GoodPasteurand Burnet (1601)

,.M. Burnet in thela3!rat!ry in the

early 16789s# ase;$erienting!n in<uen=airus genetics#using the

deel!$ing hen9segg.

goodpasture



EMBRO'ATED E//

>

state !" a"ertili=ed egg c!ntaining ane3ry! "!etus in its earlystages !"deel!$ents

es$ecially 3e"!reit has reached a

distinctiely



+electi!n !" egg

• ust 3e sterile

• shell sh!uld 3e intact and healthy.

•

sh!uld 3e !3tained "r! n!n?accinated# disease?"ree <!cs

@



• incu3ati!n ? 0 06C and >8 @8huidity.

• need t! 3e turned at least tice aday

!r

• r!lled c!ntinually in a s$eciallydesigned egg incu3at!r.



E// 'C*BATOR

6



Egg incu3at!r•

articially hatched ? c!ntr!lled and "a!ura3le c!nditi!ns• aintain "a!ura3le incu3ati!nF enir!nent ? c!nstant

te$erature !er a s$ecied $eri!d.

• electrically heated ther!stat

• intelligent c!ntr!l syste ? c!rrect easureent !" heat

Guantity #• ? adHusting hatching c!ntr!l

te$erature c!nstantly

• ariati!n !" te$erature ? a3ient t! @8 C

• c!ntr!lled 3y IJ*MOKF E/OK

/eran Ca$illary ther!stat haing accuracy !" L 8.7 c.• Capacity

• 78 t! 2888 eggs

•

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AD!ANTAGE" O# EMBRYONATEDEGG

deal "!r iruses t! gr!# !4ers seeral sites "!r iruscultiati!n

s!lati!n and cultiati!n !" any aian iruses and "eaalian iruses

+terile and ide range !" tissues and <uids

Ec!n!ical and Readily aaila3le

Maintenance easier

-ess la3!ur (n!t need "eeding and caging)

They d! n!t hae iune echanis lie anials t!

c!unteract irus in"ecti!n.

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D+ADVA'TA/E+

+!e iruses d! n!t sh! gr!th !n$riary in!culati!n int! the egg.

+light a!unt !" 3acterialc!ntainati!n in the in!culu ayill the e3ry!.

Eggs ay 3e c!ntainated ith

yc!$lasa and latent "!l iruseshich ay inter"ere ith the gr!th!" !ther irus.

12



E//+ *+ED ' VRO-O/

• &E' E//

• D*C E//

•

T*REN+ E//

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ADVA'TA/E+ A'D D+ADVA'TA/E+O, &E' E//

• ,!r aing teaching aterial

• ,!r characterisati!n !r accine$r!ducti!n !" in<uen=a irus

• An e;tra eth!d "!r is!lating dicultild strains !" in<uen=a A and B.

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D+ADVA'TA/E+

• *sed uch less n! a days.

• Tighter regulati!ns !er the use !"e3ry! tissuesPPPP

• Eradicati!n !" sall$!;PPPPP

• Deel!$ent !" si$ler and !re relia3leeth!ds "!r ty$ing her$es si$le; anddeel!$ent !" !lecular techniGues

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r!utes !" in!culati!n

1. Allant!ic caity

2. !l sac

0. Ch!ri!? allant!ic e3rane (CAM)

5. Ani!tic caity

1>



1@

1. CA'D-'/ O,

E//:t is a $r!cess !"h!lding a str!ng

3ea !" lighta3!e !r 3el! theegg t! !3sere thee3ry!.

Candling is d!ne ina darened r!! !r

in an area shielded



CA'D-'/

BOQ

1

A candling la$c!nsists !" a str!ng

electric 3ul3c!ered 3y a$lastic !r

aluiniuc!ntainer that hasa handle anda erture.



16

The egg is $laced

against thea$erture andilluinated 3ylight.



28

n!culatedeggs are

candleddaily t! seethe chicen

e3ry!sinside



21

• *nder the candling la$# the e3ry!a$$ears as a dar shad! ith the headas a dar s$!t (eye).

• &ealthy e3ry!s ill res$!nd t! thelight 3y !ing.



Dead e3ry!?Candling ill reeal a sall dararea and disru$ted 3l!!d essels.

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MAR'/ O, AR+AC

1. &!ld the 3lunt end !" the eggagainst the a$erture !" thecandling la$ and n!te the$!siti!n !" the head !" the

e3ry!.

2.Dra a line !n the shell aringthe edge !" the air sac.

0.Dra an ; a$$r!;iately 2a3!e this line.

5.The ; ars the in!culati!n site.

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E%&ryonated

Egg

25



27

ALLANTOIC

INOCULATION'( )*'+ days o,d egg re-uired

VR*+ &C&CA' BE

&ARVE+TED

',-E*'SA

M*M+

AVA'ADE'OVR*

+

'ECA+T-ED+EA+E

VR*+



+TE+• Candle the eggs and ar the in!culati!n site.

• uncture the shell !er the centre !" the airsac. A c!nenient $unch can 3e ade 3ycutting !4 a hy$!deric needle and tting it t!

a syringe 3arrel.

• nsert a 20? gauge needle# 1?1F2 inches inlength !n a 1 l syringe# int! the egg thr!ugh

the $uncture in the shell at a 57 angle t! thel!ng a;is !" the egg and aay "r! thee3ry!.

2>



ALLANTOIC ROUTE . INOCULATION"ITE DETERMINATION

2@



• nHect 2F18th (O.2l)!" <uid int! theegg.

• +eal the $uncture ith nail $!lish !rcell!$hane ta$e.

• !siti!n the eggs and incu3ate at0@! C.

2



&aresting !" Allant!ic ,luid

• Eggs ust 3e chilled t! !3tainallant!ic <uid "ree !" RBCs.

• Clean the u$$er hal" !" the shell ith@8 alc!h!l.

• Cut aay the shell a3!e the airs$ace.

• eel aay the hite !$aGue shelle3rane lining the air s$ace#e;$!sing the trans$arent allant!ic

e3rane directly 3eneath. 26



• Tear the allant!ic e3rane ith sterile

"!rce$s.

• Attach a 3all$!int needle t! a syringe

and insert the needle int! the caity.

• Re!e the <uid 3y sucti!n.

• Culture the harested <uid in a suita3leediu "!r a sterility chec.

08



O- +AC 'OC*-ATO'

• >? days !ld eggs reGuired.

•Ricettsiae# Chlaydiatrach!atis# C. $sittaci#3as!$hilic iruses( Beds!nia)

can 3e harested.

01



+TE+

• Candle the egg and ar !n the shellthe $!siti!n !" the air s$ace.

• uncture the shell !er the centre !"

the air cell.• nsert a 22 gauge needle#2 inches in

length !n a syringe# int! the egg

thr!ugh the $uncture in the shell.• !int the needle straight d!n "!r

de$th !" a3!ut 1?1F2 inches.

02



YOL/ "AC ROUTE

00



• E;$ress 8.7 l !" in!culu int! they!l sac.

• +eal the $uncture ith nail $!lish !rcell!$hane ta$e.

• ncu3ate the eggs.

05



&ARVE+T'/ O, O- +AC

• Disin"ect the u$$er hal" !" the shell.

• Re!e the shell# shell e3raneand underlying ch!ri!?allant!ice3rane.

• -i"t the e3ry! u$ ith sterile"!rce$s t! e;$!se the attached y!l

sac.

• ull the y!l sac "ree ith an!ther$air !" "!rce$s and $lace it in a

sterile $etridish. 07



Ch!ri!? Allant!ic Me3rane (CAM)

n!culati!n

• 11? 15 days !ld eggs reGuired.

• Viruses in!culated? &er$es si$le;irus# !; irus.

0>



+TE+•

Candle the egg and l!cate an area !nthe side !" the egg that is "ree !" large3l!!d essels. Mar this area ith a$encil.

• ith a sall grinding heel# grind ah!le thr!ugh the shell# 3ut n!t the shelle3rane # at the site ared.

• uncture a sec!nd h!le at the air s$aceend# this tie $enetrating the !utershell e3rane.

0@



CAM ROUTE O#INOCULATION

0



• lace a dr!$ !" sterile $hysi!l!gicalsaline !n the side h!le and gentlytease a$art the 3ers !" the shell

e3rane ith a 2@? gauge needle.• hen the shell e3rane has 3een

$enetrated# the dr!$ !" saline ill 3e

dran int! the egg as a result !"se$arati!n !" the CAM and shelle3rane.

06



• A$$ly negatie $ressure t! the air s$ace!$ening 3y eans !" !uth sucti!n

ith a ru33er tu3e. As the air isre!ed the CAM ill dr!$ "r! theshell ar!und the side h!le# creating an

articial airs$ace # !utline the liits !"articial airs$ace.

• E;$ress u$t! 8.2 l !" in!culu thr!ughthe side !$ening !nt! the CAM.

58



51



• +eal !$enings ith cell!$hane ta$e.

• /ently r!tate the egg t! s$read thein!culu !er the entire CAM under the"alse air s$ace.

• ncu3ate the eggs !n side ith "alse airs$ace u$ard.

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& ti th CAM



&aresting the CAMMe3rane

• Disin"ect the shell.

• ith sterile sciss!rs# cut thr!ugh theshell al!ng the l!ngitudinal a;is#

a3!ut 1F0 d!n "r! the u$$ersur"ace.

• /ently re!e the shell t! a discard

$an.• ith sterile "!rce$s#li"t the CAM# cut

"ree.

• lace the CAM in sterile saline and 50



AM'OTC CAVT 'OC*-ATO'

55

'( )*'0 days o,degg re-uired(

+(T1e a%nioticsac is %ain,yinocu,ated 2or

pri%aryiso,ation o2In3uen4a $irus

and Mu%ps$irus



+TE+

• Candle the egg and ar the$!siti!n !" the e3ry! and the!utline !" the airs$ace !n the shell.

• unch a h!le thr!ugh the shell at theedge !" the airs$ace directly a3!ethe e3ry!.

• *sing a 20? gauge# 1 inch needle !na syringe ae a sh!rt Ha3 thr!ughthe $unched h!le# t!ards the

e3ry!. 57



5>



• hen the ani!tic e3rane is$enetrated# the e3ry! ill 3e seen t!

"!ll! the !eents !" the needle.

• E;$ress u$t! 8.2 l !" in!culu.

• +eal the $uncture ith nail $!lish !rcell!$hane ta$e and incu3ate at 0@! C.

5@

&ARVE+T'/ O, AM'OTC



&ARVE+T'/ O, AM'OTC,-*D

• Re!e the shell and shelle3rane 3el! the air s$ace.

• Re!e the <uid "r! the allant!ic

caity # the ani!n sh!uld then 3eclearly isi3le.

• Re!e the ani!tic <uid ith a 28?

gauge needle and syringe.

5



CTOAT&O/E'CE,,ECT+

56



death !" the e3ry!

e3ry! cell daage

• "!rati!n !" ty$ical

$!cs !r lesi!ns

• !n the egg e3ranes

!edea !" the

deel!$ing e3ranes

inclusi!n 3!dies

resence !" iralantigen

in egg <uids 78

Viral gr!thand

ulti$licati!n in the egge3ry! is

indicated 3y

1

2

0

5

7

>



• The $r!cess !" cultiati!n !" iruses in e3ry!natedeggs de$ends !n the ty$e !" egg hich is used.

• A h!le is drilled in the shell !" the e3ry!nated egg#

and a iral sus$ensi!n !r sus$ected irus?c!ntaining tissue is inHected int! the <uid !" theegg.

• Viral gr!th and ulti$licati!n in the egg e3ry! is

indicated 3y the death !" the e3ry!# 3y e3ry!cell daage# !r 3y the "!rati!n !" ty$ical $!cs !rlesi!ns !n the egg e3ranes# !edea !" thedeel!$ing e3ranes# inclusi!n 3!dies in secti!ns

$re$ared "r! e3ry! tissues !r the $resence !"iral antigens in the egg <uids.

71

/ROT& O, VR*+ O' T&E



/ROT& O, VR*+ O' T&ECAM

• ,!rati!n !" characteristic $!cs.

• Vari!la $r!duces sall circular $!cs# d!esha$ed# n! surr!unding necr!sis !rhae!rrhage hereas Vaccinia irus larger

lesi!ns # <attened ith necr!sis andhae!rrhage.

72



• &er$es si$le; irus? sall # !alsha$ed ith n! eidence !" necr!sis.

70

/ROT& O, A VR*+ ' T&E O-



/ROT& O, A VR*+ ' T&E O-

+AC

PRE"ENCE O#BA"OP5ILICINCLU"ION

BODIE"

75

Vi hi h 3 h t d 3



Viruses hich can 3e harested 3yari!us r!utes:An Oerie

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*RO+E O, 'OC*-ATO'

• Diagn!stic? !;irus

&er$es si$le; irus

Mu$s irus

Vaccine r!ducti!n? n<uen=a irus

7>



&! Vaccines are r!duced inEggs

•

n egg culture# <u iruses are inHected int!chicen egg e3ry!s# here they ulti$ly. A"terseeral days !" incu3ati!n a achine !$ens theegg and harests the irus# hich is then $uriedand cheically illed.

• On aerage it taes !ne !r t! eggs t! $r!duce asingle d!se !" annual <u accine.

7@



RE,ERE'CE+

• rinci$les !" ir!l!gy: M!lecular3i!l!gy# $ath!genesis# and c!ntr!lU+.J ,lint# -.. EnGuist# R.M. rug# V.R.

Racaniell!# A.M. +ala.

7



T5AN/

YOU

egg inoculation by c.ngullie

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