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ANTIBODY RESPONSE OF CHECKENS
EXPOSED TC ENFECHQLES BROMCHETIS
‘flRUfi
Thesis fee: fhe Dagrea 3’5 M. S.

i‘ii-CZEEGAN S?AT'E CGflEGE

O-l69

This is to certifg that the

thesis entitled

"Antibody Response of Chickens
Exposed to Infectious Bronchitis Virus"

presented ln]
Calvin Ames Page

has been accepted towards fulfillment

oi the requirements for

M. S. dcgnx,h.Bacteriology

 

/ ‘l . j . f/j .J,L/t‘/" _,
Mnjnl‘ prulesmr

llate h? 6/7 ( V I L7\‘>-U
(

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“—v— --

AWTIBCDY RESPONSE OF CHICKEYS
EXPOSE TO ITFECTIOUS BRONCHITIS VIRUS

By

Calvin Ames Page

A Thesis

Submitted to the School of Graduate Studies
of Michigan State College of Agriculture
and Applied Science in partial
fulfillment of the requirements
for the degree of

Master of Science

Department of Bacteriology and
Public Health

1950

{THESIS

Acknowledgements

The author is grateful for the generous
help, guidance and instruction given to him
by Dr. C.H. Cunningham, Associate Professor
of Bacteriology and Public Health, and Dr. H.
J. Stafseth, Professor and Head of the Depart-
ment of Bacteriology and Public Health, Mich-
igan State College.

r367 I; fi‘f‘q
F,_':‘ "‘ h f p .

Table of Contents

Historic 1 Review ....................

Infectisus bronchitis of chickens

Cultivation of virus ........
Di gnosis ...................
EXperimental Procedures..........
Serum neutralization test....
Exposure of chickens.........

Tables and Figures of results....

1

jesults and discussion...........

Effect of pooling inmuqe serui

Effect of dilution...........
Sill-filigar‘yoooooooooooooooooooooooooo

Literitllr‘e CitedCOIOOOOCOOOOOOOOO

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171;: O Tnn PT‘IT‘TT-f

-1») butch-HI

Infect101s Bronchitis of Chicliens

 

Infectious bronchitis was first studied by Scha
3-7

and Pawn in 1931 in North Dakota as a respiratory dis-

9

ease of chicks. In 1925 Bushnell and Brandly reported
. _ fl. , 22 . t
the disease in Ka sas and $1bos reported 1t in mass-

achusetfs. 31nce that time the disease has been found
to be widely: ist riouted throu bout the country? The

t. . I 1
disease nas been recently reported in ingland and in

Infectious bronchitis teas ori inally thou;ht to

be confined to chicks, but it is now reCOQnized as an

. Yv‘ , I“ — " I) ‘ "O 1H . .l ’L . n (\ - f‘ "‘- 7fi~ .-
1nportant disease 01 CthKonb of all a_es -he moro-

idity rate in chicks is usually hiyh and the mortality

'2
rate mar be as hiyh as 90 per cent of those infected?7

In adult chickens the nor Ha ity rate is he 11 inle, but

in a laying flock there is a temporary cessation of egg
- i 1.. . ,16
proauccion which may zaers is t for set eral wee<s.’

The etiolo; cal a;=nt of the dis ease is a virus ca p-

—ble of passing thrCUQh all grades of :erkefeld ’ ’ 6 and
,1 16 ,
Seitz filters.

gm

Electron microscopy has shown the virus to be round

with a mean diameter of 90 mu. Filamentous projections

. 3
may be present on sons v1rus oa ticles. 5

A.

Affe ted chicks exhibit svnotoms of sneezin? gassing
J a .. , L .

c Q '2'
tracheal rales, depression and coarse chirping?’“"7 In

adult chickens the symptoms are less severe than those in

10,13 , . 5
chicks. The incubation period is from 3 to 7 days

and the duration of the disease is usually from 14 to 21
days? According to Hofsted ,5 the outstandin5 his patho-
lo5ic alterations of infectious bronchitis are a thicken-
ing of the tracheal mucous :embrane and submucosa due
primarily to e a1a and diifuse, leucocytic infiltration.
Inclusion bodies vere not observed. 31 nificant changes
were not seen in the liver, spleen and kidney. lross
lesions included mucus accumulations in the lower trachea
and bronchi, con5estion and edema of the lun_s and cloud-
iness of the air sac membranes. Facial swellin5s gay be
observed in youn5 chick32’4’16’25’37
The virus is found most abundantly in tracheal exud-
' o
ates and'in the lun 5 although Bus} ell and Srandly’ re-
ported succes ul tr ansm ssion of the disease with speci-
mens of blood, spleen, liver, and kidney from infected
chickens. Beach and Schalm2 demonstrated that the dis-
ease could oe re5uler1y transmitted to health y chiclcs by
intranasal or intratracheal injection of tracheal exud-
ate containing the virus. Delaplane and Stuart16 reported
successful transmission of he disease by subcutaneous
and intra peritoneal injections of the virus as well as
injections into the thymus 5land and the air Spaces of

the bones. Komarov and f-Eeaudette5’l stated that some

chickens recovered from the disease may be carriers a .1d
5 28

{U

serve as potential reservoirs of infection. Hofstad

demonstrated that recovered chickens could transmit the

virus as long as 35 days after recovery. Fabricant21

was able to isolate the virus in chickens up to 21 days
following exposure. Levine and Hofstad32 demonstrated
that the virus can be air-borne for a distance of at
least 5 feet and that ultra-violet irradiation was
ineffective in control of the spread of the disease.
Chickens recovered from infectious bronchitis are
immune to subsequent natural or artificial exposure to
the virus}6 Exposure of chickens 6 to 8 weeks of age
with chicken-propagated virus has been employed in an
immunization program in the New England area. Protect-
ion a5ainst the disease was attained in chickens during
the e55-layin5 period by employing this procedure.5’18’19
This pr05ram_has been extensively used with considerableu
success in hi5hly congested poultry areas but has not
been recommended in areas in which the disease is not
widespread}9
Neutralizing intibodies can be detected in serum of

5,26,30
recovered chickens with in vitro serological tests.

Jungherr and Terrell30

demonstrated by serum neutralization
tests a naturally acquired passive immunity in chicks
hatched from e55s laid by hens recovered from the disease.
The e55 yolk was the principal medium for the transference
of neutralizin5 antibodies. In yolk pools collected on

the 11th, 12th, and 16th days of incubation there was an

average of 9,513, 8,000 and 990 neutralizing doses respect-

ively. In pooled serums of chicks 1,2,3 and 4 weeks after

hatching there were 1,000, 675, 10 and 3 neutralizing
doses respectively. Random samples collected between
the 5th and the 17th week failed to show demonstrable
antibodies.

Hofstad and Kenzy29

confirmed the naturally acquired
passive immunity as reported by Jungherr and ”errell?O
but they were also able to produce infeCLious bronchitis
by injection of the virus into 4,6,7 and 10 day-old
chicks hatched from eggs laid by immune parent stock.
These findings would indicate that naturally acquired
passive immunity might not be effective in completely

protecting chickens against infectious bronchitis dur-

ing the first 10 days after hatching.

v-w

Cultivation 9: Virus in Embryonatin; Chicken 1348

1L1

 

Cultivation of the virus in embryonating chicken
eggs was first reported by Beaudette and Hudson? The
first passage of the virus via the chorioallantoic mem-
brane produced little observable effect on the embryo.
After 6 to 8 passaées in 8558 the virus became lethal
to the extent that a few of the embryos were killed.
Similar observations were made by Delaplane and

10’17 who reported that with succeeding trans-

Stuart
fers via the chorioallantoic membrane the virus became
proyressively more virulent to the embryo and less vir-
ulent to the chicken. At about the 65th passare the

virus was completely adapted to the embryo as evidenced

by mortality of all inoculated embryos. The virus at

lthis stage of adaptation was slightly virulent for
chickens. After the 90th passage the virgs was com-
pletely non-infective to chickens and was incapable
of stimulatiny the production of antioodies.
Delaplane20 reported that the virus could be more
rapidly adapted to the embryo by inoculation via the
allantoic cavity than via the chorioallantoic membrane.
Evidence of this adaptation was the noticeable dwarfing
of the embryo on the lst passage of the virus. Similar
results were reported by Fabricant21 who considered that
dwarfing and curling of embryos was pathognomonic of

7.2

infection with the virus. Loomis” substantiated the
20 nt21

-,

findings of Delaplane and Facrica that dwarfing

and curling of embryos during the first 8 passages of
virus isolated from chickens was pathognononic of inf-

ection with the virus. Licrosc0pic alterations of the

'27
//

embryo as reported by Loomis consisted of pneumonia,
hepatic necrosis, interstitial nephritis and congestion
in the spleen. The chorioallantoic membrane and the

asnionic membrane were edematous.

In vitro serum-neutralization tests have been

used as a diagnostic aid. The potentialities of the

test were first discussed by Van Roekel.’39

In studies of the distribution of eg5-adapted
strain Vll4D of the virus in embryos inoculated via
the allantoic cavity, Cunningham and El Dardiry13

found the greatest concentration of the virus in the

chorioallantoic membrane, followed in order by the
allantoic fluid, amnionic fluid and liver. The yolk
material was inocuous. The concentration of the virus
was greater in materials harvested from living embryos
than from dead embryos at the same postinoculation per-
iod. There was no advantage in the use of a 10-3 dil-
ution of virus as inoculum as compared to undiluted
virus—infected allantoic fluid. Cunningham and Stuart11
reported that an inoculum of 0.05cc. per e33 via the
allantoic cavity resulted in a higher concentration of
the virus in the allantoic fluid than when O.lcc. and
0.2cc. inoculums were employed. Groupe25 demonstrated
an interference phenomenon associated with the virus
harvested from infected embryos maintained at normal
incubation temperatures for 24 hours after death. Heat-
inactivated virus-infected allantoic fluid containing
the interfering material, if injected 30 minutes before
injection of the active infectious bronchitis virus,
delayed the rate of death of the embryos. This phe-
nomenon was not demonstrated in the allantoic fluid of
normal embryos, living infected embryos and infected
embryos dead less than 2 hours when treated in a sim-
ilar manner.

10

Cunningham and Stuart found that the following

chemical aéents usually employed for disinfection were

V

capable of inactivating the virus in 3 minutes or less;

/

phenol 33 and %; liquor cresolis saponatus, 3% and lfi;

sodium hydroxide l-20; potassium permgngznate, l-l,OOO
and l—lO,COO and mercuric chloride, l-l,OOO.

”The p? stability of the virus at 40C w:s studied
by Cunningham and Stu rt?2 The virus wys more stable
in an acid medium for the first 60 d ys, but from the
60th to the 170th d-y there was a shift to a greater
stability in an alkaline medium. The virus remained
active in allantoic fluid at pH 7.50 for 100 days and
for 100 d;ys and for 170 dsys in a phosphate buffer

at pl 7.79.

Diaynosis

 

The criteria usually employed for diagnosis of

t .J

r nchitis are the clinical history, symptoms

infectious b o
r '_~ 3r-
9

2nd lesions isolation of the virus with the

production of characteristic p tholoyic alterations of
. . . . 20,21,23 _ 3 5/
emcryonatln; chlczen eyys, and seroloylc tests.’ '

The virus does not a “lutin te chicken red cells. This

is of value for differentiation of infectious bronchitis
virus and fewcastle disease virus cs the “ewcestle virus

2
will afi'lutinate red blood cells.

C.) ".J

EXperimental Procedures

The object of thece experiments was to study the
antibody response of chickens at certain time intervals
following eXposure to infectious bronchitis virus as a
possible aid in the diaynosis of the disease by serum
neutralization tests.

Two different strains of infectious bronchitis virus
7114D and VR Lot 277 were used. Strain Tll4D was adapted
to cultivation in embryonating chicken ejys and was cap-
able of killing all embryos in 43 hours after inoculation
via the allantoic cavity. This strain was used as the
antiden in the serum neutralization tests. VR Lot 277
was a chicken-prOpa;ated strain of the virus and had not
been cultivctcd in enoryonutin; chicken eggs. This strain

hrouyh the courtesy of Dr. Henry Von Roekel,

C}-

was supplied
Department of Veterinary Science, University of a ssachu-
setts, Ahherst, and was used for exposure of the chick-
ens. All chichens were supplied throu;h the courtesy

of the United States Department of a

L

;riculture Regional

.a
'—_z

\

Poultry Laboratory, East Lansing, Lichiyan and had been

)

raised in complete iswlation under an unusually riyid

YR Lot 277 was received as a saline suspension of
meal washin;s from in;ucted chickens. Four 3—week-
old chicks were inoculated with 0.4cc. of the susaension

via the intranasal and intratracheal routes. Forty-eight

hours following inoculation the chicks displayed typical

 

symptoms of infectious bronchitis. The trachea and lungs
were collected, pooled, and Jourld with san in a mortar
and pestle and suSpended in 10 ml. of nutrient broth.
(Difco). The tissue suspension was centrifuged at 2,500
r.p.m. for ten minutes and the supernatant fluid was
transferred with a pipette into a sterile vial. The fluid

W 3

(I)

then treated with penicillin and st repto Liycin, 10,000
units each per ml. of suspension, and six 10-day embryon-
atin; chicken eyys were inoculated with O.lcc. of the
fluid via the allantoic cavity. The inoculum produced

characteristic dwarfin; and curling in the embryos by

the 3rd post-inoculation day.

14

In vitro scrum neutralization test

H

en- day 0-11“).er on atin; chic ten e w re used in the
neutralization tests. These e;ys had been incubated in
an electric, forced-draft incubator at 99—99.5 : eo-

C‘ T or“!
L'L) 2

wet bulb thermometer). The site for injection via
the all azltoic cavity was determined by trans-illumination
of the egg and selection of an area free of large blood
vessels about 3 an. below the base of the air cell. A

snall hole was drilled through the shell without piercing

rill attached to

Q:

the shell membrane, by means of a slall

Pb

the chuck 0 an ele tric motor. Another hole was drilled

(3

above the air cell to serve as an air vent to allow equal-
ization of the pressure produced by injection of the

inoculum into the eyg. The holes were painted with tinct-

ure of metaphen and the shell mehbrane above the air cell

\0

10.

was pierced with a sterile teasing needle.

Serial ten-fold dilutions of Vll4D virus-infected
allantoic fluid were prepared in sterile nutrient broth
(Difco) in the proportions of 0.7 ml. of virus to 6.3 ml.
of diluent. Serum-virus mixtures were prepared in sep-
arate tubes by mixing equal parts of each virus dilution
and undiluted test serum. It was necessary to dilute
some serums l in 10 with sterile 0.85 per cent TaCl to
pive enou;h volume to conduct the test. These mixtures
were incubated at room temperature (about 22-2500) for
45 minutes. Quantitative deterninations of the virus
dilution with sterile nutrient broth. In all mixtures,
0.5 ml. of the virus dilutions were mixed with 0.5 ml.
of the undiluted serum, diluted serum or nutrient broth.

Five eggs were used per dilution and each e5-

.3:
‘2

rec-
eived an inoculum of O.lcc. Inoculations were made with
a 1.0 cc. 3-D Yale tuberculin syringe fitted with a 27
gau;e, 5 inch needle. The 8558 for the virus titration
were inoculated last to make allowances for any possible
deleterious effect of incubation on the virus. After
inoculation the holes in the shells were sealed with
melted paraffin and the eggs were reincubated and candled
daily for 5 days. Death of the embryos during the first
24 hours was attributed to non-Specific causes and these
embryos were not included in the final results.

The results of the serum and virus titrations were

evaluated according to the 50 per cent end-point formula

ll.

of Reed and iguench36 expressed as LD50.

The difference between the reciprocal of the virus
titer and the reciprocal of the serum titer was considered
to be the neutralization index (WI). The antilog of the
NI represented the numbers of neutralizing doses (IL).
lhe II of the seruns diluted 1 in 10 was considered to
be 10 tines the difference between the virus titer and

the serum titer.

gxppsure _f Chickens

Eight lQb-day old Siane-Conb White Leyhorn cockerels
were used in the experiments. Six chickens were exposed
to strain VR Lot 277 and two chickens were maintained as
controls at the Regional Poultry Laboratory.

On January 25, 1950, blood was collected by cardiac
puncture from all chickens for determination of pre-exp-
osure neutralizing indices. At intervals of l,2,3,4,6,8,
10 and 12 weeks after exposure, 20 ml. of blood were
collected froa each chick n. The blood samples were
slanted, the serums collected after about 24 hours and
bacterial sterility tests were made. In a few instances
the Serums were contaminated with bacteria. These samples
were passed through a Swinney filter. All serums used
in the neutralization tests were bacteriologically sterile.

On January 25, 1950 the six experimental chickens
were exposed to strain JR Lot 277, as follows: Chicken
K131832, K1462Y3 and K1507VE were inoculated with 0.1 cc.

of the virus suspension via the intranasal route and with

0.1 cc. via the intratracheal route. Chickens K146023,
K1457S4 and K1G16F2 were inoculated with 0.2 cc. of the
virus suspension via the intranasal route and with 0.2 cc.
via the intratracheal route. On the 3rd day after eXp-
osure symptoms of infectious bronchitis were first ob-
served. These symptoms persisted for 5 days and were

accompanied by a loss of appetite.

Table 1 -- Chicken K1402F2

Control

13.

 

 

 

 

 

 

 

Virus dilutions Serum
Virus

Serum Titers 105% 10'2 10'3 10‘4 10-5 Titer* NI+ s++
Pre-exp. 5.16 5 4% 5 3 2 4.F8 .46 3
1 week 5.63 5 5 3 5.16 .47 3
2 weeks 5.63 5 5 2 4.83 .60 6
3 weeks 4.66 5 5 l 4.63 .05 l
4 weeks 5.83 5 5 5 35.00 ~83 37
6 weeks 6.00 5 5 3 g5.00 .40 33
8 weeks 4.63 5 4 l 4.50 .13 2
10 weeks 6.00 5 5 1 4.63 1.37 23
12 weeks 4.75 5 2 1 4.50 .25 2
These footnotes ;pp1y to Tables 1 through 11.

*Reciprocel of ne; tive exponent of logirithm base 10.
**Enbryos dead out of 5 inoculated per dilution.
+Neutralizution index.

++Heutrwlizing doses.

#One embryo of 5 inoculated died due to trauma.

fiSerum diluted 1 in 10.

Table 2 -— Chicken K1463T3
Control
Virus dilutions Serum
Virus

Serum Titer* 10;; 10"2 10’3 10'4 10"5 Titer* WI+ ND++
Pre-exp. 5.16 5 5 5 l, 0' 3.63 1.53 34
1 week 5.63 5 4% 2% 5.00 .63 4
2 weeks 5.63 5 5 2 4.83 .60 6
3 weeks 4.68 5 2 0 3.83 .b5 7
4 weeks 5.83 5 5 3 25.00 .83 §7'
6 weeks 6.00 5 5 l 4.63 1.37 23
8 weeks 4.63 5 2 0 3.83 .80 6
10 weeks 6.00 5 4 1 4.50 1.50 32
12 weeks 4.75 5 3 1 4.31 .44 3

 

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Table 3 --

Chicken K1460Z3

ixperinentjl

 

ewe

 

 

Virus dilutions Serum
Virus '0 *1 2 7 4 r
Serum Titerfi- 10-:3-21- 10 10- 10-) 10- 10-3 Titer-9% NI!- TTD++
Ere-exp. 5.16 4% 5 5 2 2 4.22 .94 9
1 week 5.63 5 5 5 4 4 o 4.25 1.38 24
2 weeks 5.17 5 4 3 0 0 3.00 2.17 143
3 weeks 4.68 5 4 1 0 1 0 1.63 3.05 1,122
4 weeks 5.63 5 2 1 1.00 4.83 67,610
6 weeks 6.00 4 1 o 0.50 5.50 316,250
5 weeks 6.00 5 2 0 0 0 0 0.83 5.17 147,900
0 weeks 6.00 5 0 0 0.50 5.50 316,250
2 weeks 4.75 5 l 0 0 0.63 4.12 13,190

 

Table 4 -- Chicken K1318B2

Experimental

1?.

 

 

 

HFJ

Virus dilutions Serum
Virus

Serum Titer* 1059 10'1 10"2 10-3 10-4 10"5 Titer* NI+ VD++

Pre-exp. 5.16 5 4; 5 3 1 4.32 .64 7
1 week 5.63 5 5 5 5 4 1 4.50 1.13 13
2 weeksfi 5.17 5 5 5 5 3 0 4.17 2.00 100
3 weeks 4.66 5 3 O 1.17 3.51 3,236
4 weeks 5.83 4 2 2 1.00 4.83 67,610
6 weeks 6.00 5 O 0 0-50 5.50 316,250
8 weeks 4.75 5 3 O 1.17 3.58 3,820
0 weeksfi 4.75 5 1 0 O 0.63 5.12 131,900
2 weeks 4.75 2 1 0 0 0.50 4.25 17,780

 

Table 5 -- Chicken K1457S4

Experinental

 

Virus dilutions Serum

 

Virus

.0 __
Serum Titer* 10+% 10 l

10‘2 10"3 10'4 10‘5 Titer* HI. vD++

Pre-exp. 5.15 5 5 5 1 2 3.88 1.28 19
1 week 5.53 5 5 4? 4 4 0 4.25 1.38 24
2 weeksfi 5.17 5 5 5 2 0 0 2.50 3.67 4,600
5 weeks 4.68 5 2 o 0.50 4.18 15,135

Chicken died Feb. 21, 1950. Cause of de th peritonitis.

 

Table 6 -- Chicken K1462Y3

Experimental

 

 

 

Virus dilutions ~ Serum
Virus
Serum Titer* 10;? 10"1 10"2 10"3 10"4 10‘5 Titer* El: ___§Q::
Pre-exp. 5.16 5 5” 5 4 1% 4.53 .63 4
1 week 5.63 5 5 4; 4 4 1 4.38 1.25 lo
4% 5 5 2 0 3.83 2.34 220

2 weeks# 5.17 5

Chicken died Feb. 8, 1950. Cause of desth ruptured artery.

 

20.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

      

 

 

 

 

 

        

 

    

 

 

 

 

 

 

 

 

 
 

 

 

 

 
 

 

 

 

 

 

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. . 1V . . V w . . 1 w w _ 1. 1 u . . . V
1 It . > . V . k 1 1 V . . . h 1 1 1 . 1 V
. m m) 1M1. _ 1 q 1 u . v m V. _ 1 . . u . “Mg 1 1. . H . . F .1.
V . ( V . a 1 . A . . _ . . a . 1 V2 1 .1 V . . . n
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1 1. 4.... 1 1 . 1 . 1 . . . 1 . 1 . 1 1 1 . 1 .. .11. . . 1 . .. . . . . 1
h . . . . V . 1 V 1 a . .. V .. .. 1 1 V . . . 1 1
. . 1 F .. . . 1 . 1 1 . . 1 . . 1 1 . . F . 1 1 _ . . _ 1
. 1 11 11 . . 1. . . 1 h . .Nr .1.. . - .. .. . 1 1 :1» . H .
. 11 . l.“ 11 1.. . 1 1 1.1fl\ . u . 1V .1 AfiN . .. 1
1 _ . m
.. 1 . . .1 V .. . . V . V 1 c 1 V . _ 1 _ « a . . . .. V
V .. .. . . . 1. 1 V 1 . H V . V V V 1 . 11 1 .1 . 1 V
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1 1 1 1 . . . . . . V V 1 V V . V . . . 11V 1 . 1 . 1 . . . a V
. . . 1 W V . +. 1 v . . V a. 1 1 _ 1 1 . V 1. . . .
1 1 . _ 1 1 . . 1 . 1. 1. . .1 . 1 . 1 S 1 . 1 1 . 1 1
. . . . . . . 1 1 a v . . p . 1 . a 1 . 1 . fl . .
1.1.1....11HI-..I..1IH-1 ['41.].1 1. 1. --- t.-. 1. 1.“1,. - 161 - I- 17-1 1 . U . .1 171. 011.11. .1. - 11-1.1-1M11-19I1. . .11 ”11.1-1-1... .- . .-..1.-1-11.-W.. .1- - 1 . 1 1. - .11.. . ... 1. . .1.-1.11.- . . aK ,. 1-141.111.41 . .1.. -.- --. TL . - . .1 1 . .V + 1 -111 - .1411- . 1 . - . -111 1.01.1- I V ...-31.1- .Iul‘I-ui
. W 1 ,1 1 1 n . . 1 1 .1 e 1 . 1 k .
V . . 1 . .. . 1 1. r V. V 1 1 1 a e . 1 fl V _ . e V
. .V . . . 1 V . e . . 1 V V . V . . . 1 _ . . .
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1 1. . _ . _ . 1 . . . .. 1 1 _ _ w - V _ 1 . . 1. .
V . . 1. . . . V . 1V . . . .V . . v 1 .
. . 1 1 . .. fl 1 _ 1 M . u H H . 1 _ . . 1 . w ..1
1 1, .1 . . . . . . . 1 _ .
1 1 . 1L. .. 11 . 1 1 1 :21 n1 L 1 . . 1 »> . . 1
. V . 1. 1 1. 1 H h . 1 . . d1 . FL I _. 1 .h . 1 . AIM . . 1
1 1V V . V m 1 w 1 e 1. . V N 1 1 1” i _ 1 _ W M. 1 . .. n V .
a .. V a . . 1 V V «- . . . 1 . . 1 1 1 . . , V
1 _ .. ,u . 1 . . 1 1 . . 1 V V . _ V 1 M . 1 bl 1
f1 . 1.1111 1.11%.- . 1 . Q1.... 9 v11 . H . 1 a . r . 1 11 V -- x 4 .1101 . r111. . . . .1011 . -14. .v .1”- . V V 0 1|- .1 . .... .- ... 1.71-11.11.10 o 91 .¢6|.1- . . ... LT! :l 11+.o-11011I. I . . 1+ . . . .0 . 1 . . ..... ... 1.41.. F 1 a 1. b . . 1 If..- 1 ...-1.1 4 0.11911 . .1111;
. . . V 1 . 1 . . 1 V . 1 . . +1 . 1 .. . V . . . ,
. V w .. _. 1 1 1 1 . V . . V . V 1 . . . OW 1 M _ . A . . 1H.
1 1 1 1 . 1 1 . 1 . 1 1 . 1 .1. TM. 1 .. 1 1 1 V e . . . .
1. V 1 1 . . 1 . a . . V 1 .1 O . . . . . V 1 . . 1 v
4 » 1191.111. I19 1.1.1.- Lr 411111.11 till..- +1.1. r J 111v! [L 11 101111-II1 11.1.11 9-91.11- - Oil-61111.- 1T 11- - . 1-11-1111. I 11111 -ll14111 1111011111 11.1 1. m .
.. . 1 _ V .1 V 1 a . V . 1 1 . V 1 V 1 ..
... . .. V M _ _ 1 1. 1 . . . . . . T . . . 1 1 . _ 1 . bl .1 .
V. V 1 . V . . . . . 1 . . 1 . . . .1 . . 1 1 1. 11 1 _ . A V . _ V.

r 1 . 1 ... . . 1 1 1V. 1 1 . 1 1 1 1 1. 1. . . 1 . 1 . _ . 1 . mu. . .
-.1..-.... .1.-. 11.. .....V-.1 ......... 1 .1 .11.. . ... 1-11. ... l 11 .1.... . . 1. 011- - 1 . .... . 1.11.? n .- -. . V 11. ..1 . . ...... V v. . . . 7.1.1....1. .. 11.. 1
. a . . V . 1. . W. 1 . . h . V m 1 1 A 14 V . . .A . . . . a 1 . 1 .

1 V V V . 1 V . 1. 1. V 1 1 V V. _ 1 . . 1 _ . o
. H .1 . 1 1 1 . 1 . .1 _ 1 . . 1 1 D h . 1. q p . 1 . 4 1 ..M 1
. . . V 1 . . 1 . . . V _ P V . . V
. H . .. 1. - 1A.. 1. 1r. 1 1. - - .+ 1 1. 1.
. 1 . 1 _ . V 1 - 1 . 1 . 1 h V .. . fl 1 . 1 1 u ”1 .
. . . 1. H. 1 1 V V V 1 1 u . . _ . d1 V _.1... .V1 1 1 1 . * . . V . 1
V V . o 1” 1 V 1 m . 1 . ~ N .V 4. V V 4 1 V * 1 V k y. .
fi. 1 V H w . . . . . . V N . R v _ 11 V V . . .V V 4 _ . .V1 .
. v .1-4- . 11.1. o . v 4 . 0.- 1.111. I . ¢ . 1 1 - . 1-11 - 9 - . . a. - .. 11V .41 t . 1111 u 11 . e 1111. o . 1 . o .111. 1+. . .11. - . 011-1. ...5 L o . . 1 . r1 . ? .1! 1; id 614...... .0. 1 ..1 V . - . p .1111 . - - . 1 1+ 1 . .11 r - 911117 - . . V 1 1 u - +1111 1 f. o 1.41 .111 0.. o ..1 v o 9-! 9111.14 -. v .1.14
. 1 . V 4 1V. . . . . . w . . . . . V . V . M n . 1. . . H _ . 1 ‘1.
. V 1 . . . 1. V . 1 1 1 .1 . . . . . 1 _ . . _ .. V . . . . .
1 1 . . H 1 1 . 1 1 1 q .1 . a H. . . . w 1 _ 1 ._ 11 1 D .1 . .1 . . . .H 1
1 — . . u . . . 1 . V . . — n 1 1 1 1 . 1 . . 1
111! o .19 o f. -«111 I?! -A - 01.1.4. , .1... .1161- ...ID?.-.101 - . - . .1 .1 1111.. 1 . - - 1&1. -Iv H1 1 . v .111 . . ... ..- V. . V.- r-Oll. 1 1 v . . ...“1? 1. -.5.V1.1.)1.1-r - - 11.11 141 - v. 61. I I 1 1 1.. V .. v1- . 11'- o . . . I ... 11. -..-1 bl. . H} A ...‘1 Pia-.5411 7.1.1111? 11111.1
. . a . V H V . _ 1 . “V . . 1 u 1 . . 1 g 1 . . 1 . V 1 .
1 V V V q _ . . . . m . 1 1 V _ V V V V. E 1 .1 1 V
. V V. 1 V V 1 1 1 . M V 1 . V” . 1 1 V _ 1 . 1 . . 1. V. . V
. . . . .v V 1 1 . . . a _ . 1 . 1 . . _ . . 1 . . 1D , . 1.. . - .1
v; . . 1o ... 1 4111 - t - 91. 11111.. 1 - .v H I .11. . a -1. v .1 . ..V. .1 1&1 I V . .11.. s w 1 I . . w . 111“ o . . «1..-- 6..111v ' 1 11L T9117 1» -1. . a . 1 a 1. 1 1 .1 ¢ . - . 11o; . - v . . . .. - 1. . 0.- I! . 1 -10 . - 111b- . W115 .0 $1111.11 41-6 1111-....1-1 41-1. ... 1
. V .m 1 + 1 1. 1 . F .. 1 fl 1 V _ V V . . .1 V . . . .
.1. . V V V V . 1 V . a . v 1. . . . . 1 . . V 1 . . 1 V . . . 1 . 1
.1 .. 1 . .1 _V 1. . . . . 1 a . _ . 1 A 1 . 1 1 H N. 1 1
. . . . 1 _ . . _ . . . n ..
L 1 1* 11 1 w . Dp1 1. 1 b1 1 1- 2 1 L1 .
.1 4 1 H41 . 1A1 .
.1 1 V . H a . u V n / ” . 1 . .1 4. V
. V 1 . . . . . 1 V . . . V . V . . V .. -. .
. 1. 1 ,1 1 . .1 . 1 u 1 1. 1 1 1 . .1. .... 1:11.. .
. . p _ W V V 1 . . _ . 1 1 V . . w 1 1V. . 41 ”
1..» .-41.-.... . 1... . . 1 ........¢. V .-.4_. . ..1- -.r. .11-...v....1..1 ... 1.4.111...“ 1*...1. . .o ....1 - . 11. . ...+-1.-111..-+-111-+.1..1.¢.o-.11
1 V V. . . V .V . » w . . . _ . V p 1 1 . . u 1 1V. .w a
. . V V . _ . 1 . . . h _ V. _ v 1V V. ,. V M w . 1 1. w. .
. _ b . . .1 1 . _ V 1 u . . . 1 M 1 . . 1 .V . . v
. . . . . _ . . 1 1 . . . . V . . . 1 1. . . _ V .1 V . . V
r11 W k t Y... 101.11-11.11111‘ .llnlvjl'llllulltl'tl‘ FIII.I- 1. 1" §L11I911§ 1.1.1.1! i. Mil-l- . 111.1111 r11- 11 4 H .
. . V 1 . 1 . . . V 1 V. . 1 V . . . . . . 1 4 1 1 V . V. o. .
. . .. . 1 . 1 . . m . 1 . 1 1 1 1 m 1 . ._ . 1. H . _ . 1
1 . V 1 1 . . . . a *1 . V . . V V - * 1 1 a .. . .1 1 . .V. .1 . h 1
. V . 1 V 1 .1. . 1 ~ ...1, ..V . . .. 1.. 1...:1 “ -1 .
I. - - . .1. v ..- v.11 1 1 - v .111 u v . - 1a - c 1.. . -+1 u 1.11. F . . .1.. . . w 1 1 p (1111* . TI...- 1‘ 111‘... . 9 . 11.. _ . cl. 1+..VOL .1" . # 1... 711w . e - . . .1+ . . . e . + V 4111114111 1‘ 1 it 7.131.019.1110 | v, . 1.110191? 1 v1.11;
. 91. w V . o 1 h .. . . . V . . V V m V . . m h .1. V 1 . V 1 . . 1 . . . V. J 1.
. 1. - . 1 . 1 . . . a. . * V . .~ 1 a. .1 . w . 1V 4 .. . 1 V V . . . . . .V . .. -
V V 1 w. . . 1 W V M . . . 1* h . a r V M u .. . _ . .1 . 1 . + H. H
w 1 1 1 1 V 1 .. 1 . 7 . .1. .1. 1 . .11. 1 . .. . D 1 1 . V.
_ . . a .1. 1 .1 ...1111 .... ......... .11 .1 1. 0 h . . . . . . 1- 1.1 .11 V... C. .1. 1 .
-1 .... .1 .....1 _ 1 1. . 1 .. . . V V. . .1..” . V .... 1 . . 11 . 1 1 V . .11 1..1 .. 17.1 .k 1. ..»..
1 * .1 .. ... . 1 . . 1 1 .1 1.... .«1 . .. 1. .- 1; .. 1 1 1 n . 1.... .-.1 .1 .V ....
. . 1 * . . . . . .. w V 1 O . . .. . O . . ... 1 H . 1 V p . V V 3' . V 1 . V .. .nv . . 1 .1 . . . V , . . L .
11111....1611 ..--.. -...11-.O. .1-.1.-...-1-.-.1.. .-,1.11.o.1- .-.11 «- ....1L-.-1..|f110.1.. 11....1-1.~. -1...-.-11.- -. -1.”.q1.....-+.-111V. .-. .. 1V... .111.-. .19-- 11ft-.1..1 11l11l»--111T-01o11+.-.111f|1.|1 .1.-1.4
.w . .. I . ' . 1 . fl . 1V. V w . . _. 1. V . . n ”1 . ' .. 1 h .1 + ~ I. . fl . 1 .' 1 V V A.. . . ”- _ . . . . M . . .—
. . . . .. . . . .. . . , . . u . . .. 1 . V . 1 - . o . 1 . . _ 1 . .. . . . . . . . . .
1 . ... 1 _ 1 . . .. .1 ..._1. . 1 V. V... _ . . 1 . 1 . .1 . , . .1. ....
. gN .5 1 4 r 1. 3 . . 1 2 . 1 ...-.1 ; .1 . . . 1N. 1 2 . 1 . 1 . .. .
111111 T -.6 0.11.1110. ..1‘10.+||9 1.16.1.- 1} 111 11.011 ...-1.4-011 0.1. .1. u -1. ... . . - W .1 1¢ . L .1014 091111411 ¢.. >1 _ .v .1 I. 1 11.7 .1 1 1T
. . .. . . W . 1 V w . . V . . V H . _ V 1 . V V w .1 . _ 1 ~ . V V V . . +. . V
v V 1 1 . . . . + . . V V .V . * . + . V 1 V . V ~ . . 1 o 1 V V. V V V .1 . 1. 1... . c 1. . . 1 . o . 1
. 1. .. . V a. . H p a , M . . h . . .1 1 _ u 1 1 1 . . V . . V . .. 1 1 1 1 . 1 1 v .. . .
..1.....V . ... .. w .1 . . 1 . V. . V 1 ... . 1 . ._. . M 1, 1 V 1 1 1 . . ..1. .V ...+..
1111-.....111011...-.. .111-.-~. ..1..11 ..... -111w .. ...41- .1111-V11....--.1T....V...-.?I.1...v. 10-.. .1.. L1....... 1.1 ... ..1..-.1. ..-..111 .-. ,v.-1.. 1. -11.».1111011114; -.- 1.141.114.11'o11Vo1111
. 1. . + - V V 1 1 . . ~ . .# . .n . V 4 . . 1 a V 1 A 1 1 . V, . H . . 1 . . V . a . - .1 a .11 .~ . . .. 1 1 .
_ . ¢ . . u _ . . .1 . V . 1- . a V V . V V - . . V . V 1 . v . . _ . . . . 1 . . V . . 1. . . . . . . V. .. . 1.
M1 1. . 1 .1. .1.“ .1 1. 1 1 1 1 . . . 1 H 1. .1. 1. .... .H ....1 . 1.-.. . H .1 11 .11“.
, . .1. .1. 1 1. 1. 1 1 1 .. 1 . 1 1 .1. .. 1 .1. 1r. 11 .. 1. . h . 1F .1 1 1. 1.

 

 

Table 7 -- Chicken K1507V3

Experimental

 

 

H14

 

Virus dilutions Serum
Virus '

Serum Titer-X- 10.19. 10"1 10'2 10"3 10"4 10'5 Titer)? 111+ 1717+.
Pre-exp. 5.16 5 5 5 4 0 4.55 .78 6
1 week 5.63 5 5 5 5 4 O 4.36 1.25 18
2 weeks 5.22 3? 2% l l 3.20 2.02 105
3 weeks 4.68 5 4 0 l 1.46 3.20 1,565
4 weeks Insufficient smount of serum

6 weeks 6.00 5 3 0 O 1.17 4.63 67,620
8 weeks 4.61 5 4 0 0 1.38 3.23 1,770
0 weeks 6.00 5 5 0 0 0 1.50 4.50 31,625
2 weeks 4.75 5 2 O l O 1.00 3.75 5,62;

 

 

 

 

 

 

 

 

A
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A 4V ,_ I I I1 N H J _ _ a
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V V . _ V _ P V . m V V
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V V . I _ 3 V V V m V V V V V V V V I
I . , . V . . . . V, V . . 4 I
_ w I _ f p 4 4 A I V V . .. 4 . H .
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V V. _ . V V V 4 V _ V * .
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V V V VV .V w . . I V V V . , , V V V .
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V V T V . _ , V
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v .V e C . W . V V V . H . V
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V V V . . V I A: . u a V V I . _ V p
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V V . H V a . V . V _ V ‘1). V V
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V a . . . V . V — . V n V . —
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V . V V _ V , _ V _ V _ . . . .
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V .. V I , V . V . . V V V V V. _ V _ . V
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V . . V . 4 . _ I V . V _ V . I V V .
w 4 . . . h . n _ . V . . .
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V H I V V V _ H V V ~ . V I V V
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szle 8 -- Chicken K1516F2

Experimental

23.

 

 

 

 

Virus dilutions Serum
‘.’irus

Serum Titer* 10;? 10"1 1010—3 10'4 10'5 Tiger* NI+ ND++
Pre-exp. 5.16 5 5 5 2 1 4. oo 1.16 14
1 week 5. 63 4% 5 5 5 2 0 3.33 1.80 ‘63
2 weeks 6. OO 5 5 3 O 0 l 2.31 3.69 4,296
3 weexs 5.17 4 2 O 0 0 0 0. 3 4.49 30,906
4 weeks 4.63 3 1 0 0 0 0.32 4.31 20 ,415
6 weeks 6.00 4 0 0 0 0.36 5.62 416,670
8 weeks 4.75 5 0 O 0.50 4.25 17,780
Chicken died Kircn 29, 1950. Cwuse of death unknown.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

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-.. ..1 . 1 1 1 1 .. .. 1 . 1 .1... 1.. . .1 1. .1 . 1 . 1 . . .. . ..1. .....1.
.1 . . v 1 . .1 . 1. 1 . 1 .. 1 H. 1 .. . 1 . 1 * . . m . 1 u . 1 1 . 1 . 1 1 1. ... 1. . . *. .
_1 ..1. _. 1. . fi. 1 .. . fl. 1 ... 1. .1 . .fl . 1 . . . . . . H. 1 . .1 .. .. . .. . . ._ .. ... 1 . . . . 1 . .
.. 1 . . 1. . 1 .. . . . 1 . 1 1 . .1 . . 1 1 . .. . . .
p. . . H 1 H w 1 Fl. L r W H . w * . H 1 1 .— .r L 1 r » 4 H . L. H

 

 

 

 

 

 

Tgble 9 -- Summstion of neutr:lization
indices and averiges for the
erperimentgl chickens

25.

 

H14

Neutr1lization indices

 

 

Serum K1450T3 K131552 K1457S4 K1462Y3 K150773 ¥1513F2 ”Ver';e
:re-e p. 0.94 0.84 1.28 0.33 0.78 1.16 0.94
l weed 1.3: 1.13 1.35 1.25 1.25 1.80 1.37
2 weeks 2.17 2.00 3.67 2.34 2.02 3.69 2.65
3 week 3.05 3.51 4.18 —- 3.20 4.49 3.69
4 weeks 4.33 4.63 -- -— -- 4.31 4.66
6 weeks 5.50 5.50 -' " 4'33 5'62 5'56
8 weeks 5.17 3.50 -- -- 3.23 4.25 4.05
0 weeks 5.5 5.12 -- -- 4.50 -- 5.04
2 weeks 4.12 4.25 -- -- 3.75 —— 4.04

 

.uv i.

T ble 10 -- jeutralizin; iniices of
pooled serums

 

Pooled Virus dilutlons oeruu
Serum Virus
3; pie Titer% 10;? 10-1 10-2 10‘5 10-4 10-5 Titer* NI+__

 

 

F.)
O LEON-bu: [U l-’

K1432F2 5.32 4; 5 3 5.17 .15
hl463T5 5.32 5 5 4 5.32 .00
week 5.32 5 0 0 3.50 1.82
weeks 5.32 0 1 0 1 0.70 4.62
weeks 5.52 2 1 0 0 0 0.50 4.82
weeks 5.52 5 2 2 1 0 1 1.63 5.69
weeks 5.52 5 3 0 0 1.17 4.15
weeks 5.32 5 1 0 0 0.5? 4.69
week 5.32 5 2 O O 0.05 4.49
weeks 5.32 5 1 0 0 0 0.32 5.00

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T ble 11 -- Effect of dilution on
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erum Titer* 19;? 10"1 10"2 10-3 10"4 10-3 Titer* NI+

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12 weeks

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30.

Results and Discussion

Chickens K1402F2, K1463T3, K1460Z3, {131552 and K1507V3
survived to the end of the experiment, but Kl462’3 died
from peritonitis, Kl45734 died from a ruptured artery,
and Kl5lSF2 died from unknown causes 2, 4 and 9 weeks,
reSpectively, after the bedinning of the experiment. As
a result, only 3 serum samples were collected from K1462Y3,
4 samples from Kl45734, and 7 samples from K1516F2.

Qhe neutralizing indices of the ore-exposure serum
samples and the serums from control chickens, Tables 1

15

to 8, closely a ree with the work of Cunningham who

b9 . 3'7
O. OO O 70

reported 7 10 j 1 1 0'37° or 101'917 z

. a’ 10 .

000776 — i
10 “ (X 3 30’ ) as the infectious broncnitis
neutralization index for normal chicken serum with Vll4D
antigen. According to these data, 99.7 per cent of the
serums from normal chickens should contain not more than

1+/
101.55 0

of 55 neutralizing doses as detected by the
serum neutralization test.

One week after exposure to the virus there was a
slight increase of the neutralization indices of all
serums. From the 2nd to the 6th weeks there was a marked
increase of the neutralization indices. The maximum
neutralization index occurred at the 5th week followed
by a decline at the 6th week, an increase at the 10th
week, and a decline at the ternination of the experiment
at the l2th week. Tables 5 to 8, Figures 3 t0 8.

The neutralization indices of the serums showed close

agreement at all intervals. During the initial period of
ascending indices a logarithmic pattern was followed. Exp-
eri n1ental evidence is not available to explain the sun-
seguent variations in the ind ices at the 3th, 10th and

l‘th weeks. Tables 3,4,7,6 and Bi5ures :,4,7,8. There

1.

are several possibie explanations for these variances.

The declines at the 6th and 12th weeks could go mssic ily be
explained on the basis of the interference phenomenon

25
reported by Groups since this decline was associated
with a virus of low titer which occurred when a 24-hour
har est fron dead enhryos inoculated with V1143 was
used as the anti en in the neutralization tests. Tables
l,2,7,c and F15,ures l,2,7,6. However, such a possibility
can be partially discounted on the basis of the data in
Tables 3 and 4. A sample of the virus which had a high
titer was used for titrations of the 6th, 8th, and 10th
week serums from 3:146 33. The decline and rise of the
neutralization indices were ooserved, but the decline was
not as pronounced as that observed for h1316o2, table
4 and Ligure 4, where the ct“, 10th and 12th week seru a
sa¢ples were titrated with a other sample of virus of low
titer. therefore, the decline in neutWLa ization indices
could possibly be due to the presence of interferin5 nat-
erials to the extent of the difference between the 0th

week neutralization index of $143023 and the nth week

201 ralization 11dex f KlilCLE. E15ures 3 81d 4.
Another possible 8X31 iution could 03 pcs ' o1 51n-

uent exposure at the 5th wee

virus fro1 another source. “0

were observed during thi

is also

0. the neutr iza ion indlces are a
0 ‘0 a fl 0 __ ~r «f -— 1 ’\ o _‘ A,,-.J- o
111. 01-1.; Sleep ,5. 1‘ till/1:71" l:-Vc:ol .

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J. \I‘ 1

33'1an the

.zal clsxrze:1 serun

in

titer of 1,030 r eutrahlizi

O n :
LR L‘»

file to the sossiple revs

of the virus,

in a Multiulication phase,

ht soon 0e ineffective and lJfEC

by Juixne1

symptoms of

C.‘
1..)

the possioilit; that flu

reported that

antioodies since

J

«p.

neutraliza
reported by

this experinent.

111

natural

1 ‘ I

526115 were

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-' ‘ l‘ rfi - a -
ha." O. b .3412}

U1
b
,—.

. x. x

ronchitis
infectious
period.
ctuations
occurrence

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ed frog eg‘s laid by

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“'40)

ti n index

1
.....1 Pulp
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An anticody

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5
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(7+
0

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01

7.

Burnet et a1 stated that inactivation of
certain viruses with iunune serum resulted primarily
from the union of antibody to the virus surface. This

was a reversible union and the time required to reach

)

an equilibrium was agproxiuat~ly proportional to the
concentration of the antibody. A permanent antibody-

virus union was possible after prolonged incubation.

'34.

The Effect f Pooling Immune Serums

 

 

Figure 9 shows the average neutralization indices

the serums at the several periods following exposure

1"!)

0
to infectious bronchitis virus as calculated from the
data in Table 9.

It was thou ht that by pooling aliquots of the ser—
ums, results would be obtained thpt would closely agree

with the averages :s Calculated above. However, the res-

I

ults obtained indicated a considerable deviation and
can not be explained at the present time. Further studies
of this phenomenon should be undertaken. Table 10, Figure

10.

The Effect f Dilution of Serum on
Teutralizntion

 

 

’\

Hirstd4 demonstrated th-t : dilution of l in 5 of
influenza immune serum resulted in a lO-fold decrease in
neutrilizing c;pucity when tested in mice.

Brandly gt §l6 showed that dilution of l in 10 of
Newcestle disease immune serum resulted in u lOC-fold

‘
a

decrease in neutralization cspscity, but a dilution of

1)

l in 5 resulted in u lO-to lOO-fold decresse in neutral-
izing capacity.

Ruched}4 found that Newcastle disease immune serum
diluted 1 in 10 in nutrient broth resulted in a 20-to
lOO-fold decrease in the neutralizing quaCity.

Table 11 shows the results of serum neutrulization
tests using serum diluted 1 in 5, l in 10, l in 15, ind
l in 20 in 0.85 per cent HaCl. Dilution of the immune
serum 1 in 5 resulted in a decre se in neutralizing cep-
scity lO-to 15-fold, Lnd further dilutions of the serum
did not appreciubly alter the neutrulizing capacity of

the immune serum.

S umm i; r‘

   

(a) Normal chickens were exposed to in uctive field

str in of infectious bronchitis virus. The serologicsl
response of the chickens st cert in time intervals were
evaluated by serum neutrulizution tests with the results
expressed as the neutr;liz:tion index.

(b) Ere-exposure neutralizstion indices were found to
be 101'53 or less.

(c) There wus found to be ; one week period in which no
signific nt production of neutr:lizin5 untibodies could
be demonstrzted by serologiCul tests.

(d) The logarithmic phuse of untibody production was
found to begin two weeks following exposure :nd the nox-
imum neutralizution index wis reached at six weexs fol-
lowing exposure.

(e) Dilution of immune Serum l in 5 in 0.85 per cent
NaCl resulted in a lO-to 15-fold decreuse in the neutral-
izins especity. Further dilution up to l in 20 did not
apprecisbly decreise the neutralizing capacity of the
immune serum.

(f) The results of serum neutralization tests on pooled

serums indiCute the need of further study of antibody-

snti5en reactions.

37.

Liter.ture jited

 

l38plin, F.D.: Identification of infectious bronchitis
in En5land. Vet. Rec. go, (1948): 485-486.

gnesch, J.3. and 0.x. Schalm: - filterable virus, dist-
inct from that of laryngotracheitis, the cause of a
respiratory disease in chicks. Poultry Sci., 15,
(1936): 199-206. ‘—

2 -

“Be:ch, J.R.: Chapter 19. Diseases pf Poultrv, edited by
P.E. Biester and L.H. Schw-rte. The Iowi State College
Press, hues, Iowa. 194:.

4Beudette, F.R. and C.B. Hudson: Cultivation of the virus
of infectious bronchitis. J. Am. Vet. hed. Assn.,
29. (1937): 51-56.

5Beaudette, F.R.: Twenty Years of Progress in Immunization
against virus diseases of birds. J. 1m. Vet. led.
Assn., 112, (1949): 367-38 .

Usir-.ndly, C.g., 9.3. hoses, and E.L. Jungherr: Transmission
of intiviral activity vi; the egg and the role of con-
genital passive immunity to Yewcastle dise se in chick-
€118. 5.!on Jo ‘feto Res. Z, (1946): 333-3420

7Burnet, F.i., E.V. Keogh and D. Lush: General Considerat-
ions on the reactions between animal-pathogenic viruses
and homologous antibodies. kus. J. Exp. Biol. and Red.
Sci., 12, (1937): 284-295.

C: ' "1 N
UBurnet, F.K., E.Y. Keogh and D. Lush: bummiry of tne im-
munological reactions of animal pathogenic viruses.
.us. J. EXp. Biol. and led. Sci., lg, (1997): 551-354.
C .1 , .i ,. . ,
/busnne11, L.U. and 0.3. Br ndly: Laryn;otr;cheitis in
chicks. Poultry Science, l3: (193j): 55.

loCunningham, C.H. and H.O. Stuart: The effect of certain
chemical agents on the virus of infectious bronchitis
of chickens. Am. J. Vet. Res., 1, (1946): 466—469.
llCunningham, C.H. and P.O. Stuart: Cultivation of the
virus of infectious bronchitis of chickens in embryo-
nated chicken eggs. Am. J. Vet. Res., 8, (1947):
209-212. "

l2Cunningham, C.H. and H.C. Stuart: The pH stability of
the virus of infectious bronchitis of chickens.
Cornell Vet., '21, (1947): 99-103.

leunningham, 0.3. and El Dardiry, A.H.: Distribution of
the virus of infectious bronchitis of chickens in fl
embryonated chicken eggs. Cornell Vet., :9, (194:):
Sal-ijaO

7r:
‘1'" .

1

4

Cunnin;nam, Cnirles H.: a Lab r tory Guide in
Virolo v. Burgess Publishing Co., Linneipolis,
Linnesot . 194G.

 

l 0 v -. - o o
5Cunningham, C. .: Personsl Co; unic-tion. (1950

\‘l

,
loDelapline, J.P. und H.O. Stuart: Studies of infectious

bronchitis. R.I. flér. Expt. Sti. aul., €273, (1939),

17Delaplane, J.P. ind 3.0. Stuart: The modification of
infectious bronchitis virus of chickens as the result
of props5ation in embryonated chicken eggs. R.I.
A;r. Exp. Sta. Bu1., $284, (1941).
13
Delaplsne, J.P.: The d
disesses of chicken
f258, (1943).

ifferentiation of the respiratory
So ROI. 3.”,1". EXpto Stu-Io ‘Ulo,

19Delhplune, J.P.: Panel discussion on poultry diseases.
J. in. Vet. Jed. “ssn., 10o, (1945): 91-103.

20Delaplane, J.P.: Technique for the isol tion of infect-
ious bronchitis or Heucastles virus includinb obser-
vations on the use of Streptomycin in overcoming
bacterial contaminants. Limeo Report. Nineteenth
Annual Pullorum Disease Conference. Raleigh, W.C.
June 11-12-15, 1947.

21Fabric;nt, J.: Studies on the diagnosis of Newczstle
disease and infectious bronchitis of fowls. II.
The diagnosis of infectious bronchitis by virus
isolation in chick embryos. Cornell Vet., 22,
(1949): 414-431.
22Gibbs, 0.3.: Bronchitis of baby chicks. Poultry Sci.,
13. (1933): 46.
23Groupe, V.: Demonstrction of an interference phenomenon
associated with infectious bronchitis virus (13?
of chickens. J. 3act., §§, (1949): 23-32.
24Hirst, &.K.: The quantitative determination of influenza
virus and antibodies by means of red cell agglutin-
ation. J. Exp. hed., 12, (1942): 49-64.

23Hofstad, M.S.: a study of infectious bronchitis in
chickens. I. The pathology of infectious bronchitis.
Cornell Vet., 2Q, (1945): 22—31.

26Hofstud, N.S.: II. Observations on the carrier sthtus
of chickens recovered from infectious bronchitis.
Cornell Vet., 2Q, (1945): 32-35.

27Hofstid, u.s.: III. attempts to utilize the chicken
red cell agglutin tion test as diuynostic aid in
infectious bronchitis. Cornell Vet., 35, (1945):
60-61. ‘7

[\J

CHofst-sd, 1.5.: IV. Further observations on the carrier
st-tus of chickens recovered from infectious bron-
chitis. Cornell Vet., 31, (1047): 26-34.

29Hofstad, k.S. and 8.3. Kenzy: Susceptibility of chicks
hitched from recovered hens to infectious bronchitis.
Cornell Vet., 32, (1950): b7—b9.

EOJunéherr, E.L. and y.L. Terrell: N turally acquired
passive imnunity to infectious bronchitis in chicks.
Am. J. Vet. Res., 9, (194o): 201-205.

filKomarov, A. and F.R. Begudette: Carriers of infect-
ious bronchitis. Poultry Sci., 11, (1932): 335-338.

52Levine, P.P. and H.S. Hofstad: Attempts to control
air-borne infectious bronchitis and Newcastle
disease of fowls with Sterilamps. Cornell Vet.,
21. (1947): 204- 210.

DjLoomis, L.H., C.H. Cunningham, h.L. Grey, F. Thorp Jr.:
Patholo y of the chick embryo infected with infect-
ious bronchitis virus. nm. J. Vet. Res., (In Print).

j[IR-ached, S.S.: Antibody response of turkeys vaccinated

with form lin-inactivated hewcastle disease virus.
Iich. Stite College Agr. Exp. Sta. Bul., #215, (1949).

j5Reagan, R.L., J.E. Fauser, X.F. Lillie and A.H. Craige:
Electron hicrogruph of the virus of infectious
bronchitis of chickens. Cornell Vet., 3Q, (194b):
190-191.

36 - _

’ Reed, L.J. and h. Luench: A simple method of fifty
per cent endpoints. in. J. hy;., 27, (1938): 493-497.

77 . . n . ,

"SChSlK, s.F. and h.C. hawn: kn ap,arently new respiratory
-. . . . - . n.
0138388 of baby cnicks. J. cm. Vet. sea. “ssn., 7c,
(1931): 413.

36

Swierstra, D.: Bronchitis infectioss bij Kippen in Ueder—
land. Tijdschr. Diergeneesk,'zg, (1947): 745—746.

)9Van Roekel, H.: Infectious bronchitis control. Proc.
of the 15th *nn. Confer. L-b. Workers in Pullorum
Dis. Control, F rmingdal L.I., Z, 1942. (Cited
by Jungherr end Terrell? ).

NV 29 '5h
80 3 '51

Apr 1? Ti:

W
MAY 2 31967!

,me USE ONLY