Pathogenesis of Salmonella enteritidis Infection in Laying Chickens. I. Studies on Egg Transmission, Clinical Signs, Fecal Shedding, and Serologic Responses

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Pathogenesis of Salmonella enteritidis Infection in Laying Chickens. I. Studies on EggTransmission, Clinical Signs, Fecal Shedding, and Serologic ResponsesAuthor(s): H. L. Shivaprasad, J. F. Timoney, S. Morales, B. Lucio and R. C. Baker

Source: Avian Diseases, Vol. 34, No. 3 (Jul. - Sep., 1990), pp. 548-557Published by: American Association of Avian PathologistsStable URL: http://www.jstor.org/stable/1591243Accessed: 14-09-2015 19:18 UTC

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AVIAN

DISEASES

4:548-557,

1990

Pathogenesis

of

Salmonella enteritidis Infection

in

Laying

Chickens.

I.

Studies on

Egg

Transmission,

Clinical

Signs,

Fecal

Shedding,

and

Serologic

Responses

H. L. Shivaprasad,AD. F. Timoney,B S. Morales,c

B.

Lucio,A

and

R.

C. Bakerc

ADepartment

f

Avianand

Aquatic

Animal

Medicine,

College

of

Veterinary

Medicine

BDepartment

f

Microbiology,

Immunology

and

Parasitology,

College

of

Veterinary

Medicine

CDepartment

f

Poultry

and Avian

Sciences,

College

of

Agriculture

and Life Sciences

Cornell

University,

Ithaca,

New York

14853

Received

14

September

1989

SUMMARY.

Laying

hens

were

inoculated

orally, intracloacally

IC),

or

intravenously

IV)

with

Salmonella

enteritidis

phage

type

8

isolates from a human

(E700-87),

eggs

(Y-8P2),

or

the

ovary

of a hen

(27A).

Oralor

IV inoculation of 2

x

108

o

4

x

108

colony-forming

units

(CFU)

of

E700-87

caused

depression,

anorexia,

reduced

egg

production,

diarrhea,

and some

mortality.

Lower doses

resulted

in milder clinical

signs.

S. enteritidis

was cultured from

the

shells of

a few

eggs

but

not from

egg

contents. Fecal

shedding

persisted

for

up

to 6

weeks

in

some birds.

Isolate Y-8P2

106

CFU)

also caused

anorexia,diarrhea,

nd a

drop

in

egg

production.

Hens

inoculated

orally

or

IC

were

less

severely

affected than those inoculated IV.

Fecal

shedding

was

intermittent

and lasted

up

to

18

days. Eggshells

from

the IC-inoculatedbirds had the

highest

rate of

contamination,

and

S.

enteritidis was isolated from the albumen of 11 and

yolk

of three of

726

eggs.

Oral inoculationof 106CFUof isolate 27Aresulted in a bacteremic nfection with seeding

of the

liver,

spleen,

peritoneum,

ovule,

and

oviduct.

However,

the

birds remained

clinically

normal with

normal

egg production.

S. enteritidiswas

cultured

from the

yolk

and

albumen

of

a small

number

of

eggs

until

11

days postinfection.

Antigen

prepared

rom

S.

enteritidisdetected

antibody

n

more sera than

did

commercially

available S.

pullorum

antigen

in

agglutination

tests.

RESUMEN.

Patogenesis

de

la

infeccion

por

Salmonellaenteritidisen

ponedoras.

I.

Estudios

de

transmisi6n

a

traves

del

huevo,

signos

clinicos,

diseminaci6nfecal

y respuestas

erologicas.

Gallinas

ponedoras

fueron inoculadas

por

las

vias

oral,

intracloacal o

intravenosa,

con

Salmonella

enteritidis

fago

tipo

8

aislada

de

humano

(cepa

E700-87),

de

huevos

(cepa

Y-8P2)

o del ovario de una

gallina

(cepa

27A).

La noculaci6n oral o intravenosa on una dosis de 2 x 108a 4 x 108unidadesformadoras

de

colonia

(UFC)

de la

cepa

E700-87

aus6

depresi6n,

anorexia,

disminuci6n

en la

producci6n

de

huevos,

diarrea

y alguna

mortalidad.Dosis

menores

produjeron

ignos

clinicos mas leves.

Se

cultiv6 S. enteritidis

a

partir

de

las

cascaras

de unos

pocos

huevos

pero

no del

interiorde

ellos. La

diseminaci6n

fecal

persistio

por

hasta 6

semanas en

algunas

aves.

La

cepa

Y-8P2

(106

UFC)

tambien caus6

anorexia,

diarrea

y bajas

en

producci6n

de

huevos.

Las

gallinas

inoculadas

por

las vias

oral o intracloacal ueron

afectadascon menor

severidad

que

las

inoculadas

por

la via

intravenosa.

Las

cascaras

de los

huevos de las

gallinas

inoculadas

por

la via

intracloacal uvieron el

mayorporcentaje

de

contaminaci6n

y

de

un total

de

726

huevos,

se aisl6

S.

enteritidis de la

albuimina e

11

huevos

y

de

la

yema

de

3

huevos.

La noculaci6n oral de 106

UFC de la

cepa

27A

result6 en

una infecci6n

bacteremicacon

diseminaci6n al

higado,

bazo,

peritoneo,

6vulos

y

oviducto. Sin

embargo,

las aves

perma-

necieron clinicamente normalescon unaproducci6nde huevos normal.Secultiv6 S.enteriti-

D

Presentaddress:

California

Veterinary

Diagnostic

Laboratory ystem,University

of

California,

Davis,

Fresno

Branch,

2789

South

Orange

Avenue, Fresno,

CA

93725.

548






S. enteritidis

n

layers

disde

la

yemay

de la alb6mina

e un

pequenio

6merode

huevos

hasta os 11 dias

despues

de

la infecci6n.

En as

pruebas

e

aglutinaci6n

ealizadas l finaldel

experimento,

l

antigeno

preparado

con

S. enteritidis

etect6

anticuerpos

n un

mayor

fimero e sueros

que

el

antigeno

de

S.

pullorum

disponible

omercialmente.

A

dramatic increase

in

the number of out-

breaks

of

food

poisoning

in

humans

due

to Sal-

monella enteritidis

has been

observed

in

the

northeastern and mid-Atlantic

regions

of the

United States

over

the

past

10

years

(22,30).

Epidemiologic

studies have determined

that

grade

A shell

eggs

is

probably

he

major

ource

of

infection

(30).

Because the exterior

of

grade

A shell

eggs

is

washed and

sanitized,

it

has been

suggestedthat he salmonellacontaminationwas

derived

from the

interior of the

eggs,

which

became infected

by

means of

transovarian

n-

fection before

eggs

were

formed

(30).

S.

pullorum

and

S.

gallinarum

commonly

lo-

calize in

the

ovary

and

produce

transovarian

infection

(20,25),

but there are few

reports

of

ovarian nfection of chickens

by

other non-host-

adapted

salmonellae.

Attempts

to

produce

experimental

transovarian nfection have been

inconclusive

(2,10,15,16,18).

A

few non-host-

adapted

salmonellae such as S. enteritidis, S.

heidelberg,

and S.

typhimurium

may

occasion-

ally

and

infrequently

be

transmitted ransovar-

ially

in

chickens,

turkeys,

and ducks

(13,14,24,

29,35).

Although

S.

enteritidisis a common

pathogen

in

rodents

(4,7,35),

it has been

isolated infre-

quently

from birds

(1,6,13).

It

was

occasionally

isolated from ovules of

infected chickens

(13).

S.

enteritidis

is

commonly

found on

duck

eggs

(3),

but it has not often been

associated with

disease

of

ducks

(35).

In

England,

S.

enteritidisphage

type

4,

which

was

rarely

found

before

1987,

has caused

clin-

ical

signs,

mortality,

and

pathological changes

in

broiler chickens

(19,33).

This

phage

type

has

also been isolated

from the

oviduct and ovules

from hens that

died

in a

commercial

layer

flock

implicated

as

a source of

contaminated

eggs

(17).

During

January

1988,

17

of

24

separate

incidents in

which S. enteritidis

was

isolated

were of this phage type. The proportion of S.

enteritidis

isolates

from

humans

in

the United

Kingdom

increased from

9%

n

1982

to

33%

n

1987

and

51%

in

1988

(9).

Two-thirds

of

the

isolates were

phage

type

4,

a

type

not found

in

commercial

poultry

n

the

United

States,

where

most isolates

are of

type

8 or

13

(B.

Rowe,

per-

sonal

communication,

Central

Public

Health

Laboratory,

London,

England).

Human infec-

tions due to S. enteritidis have become more

frequent

in

Norway,

Sweden,

Ireland,

Yugosla-

via,

Canada,France,

Spain, Italy,

and Zaire

(R.

V.

Tauxe.

Salmonella enteritidis

workshop,

Al-

bany,

N.Y.

Sept.

27-28, 1988);

in the

United

States,

United

Kingdom,

and

Spain,

they

have

been attributed o the consumptionof products

that contained

eggs

contaminated with

S. en-

teritidis.

Few

reports

have described

pathogenicity

of

experimental

S.

enteritidis

infection in

young

or adult

chickens,

and none have dealt with

egg

transmission

(21,26,29,32).

The

ability

of

S. en-

teritidisto

penetrate

the

intestinal

mucosa

was

found to fall

rapidly

with

increasing

age

of

birds,

and the caudal

regions

of

the intestine

(ileum

and

ceca)

were common sites of infection

(32).

One-day-oldor 2-week-oldbirdsdeveloped se-

vere

gross

and

microscopic

lesions

in liver

and

spleen

but

failed

to

develop

antibodies

by

7-

14

days postinoculation

(PI).

Lesions in

adult

birds were

minimal or

absent,

and

antibodies

were detected at

4

and

18

days

PI.

The

present study

describes

experimental

n-

fection of

laying

hens,

with

particular

mphasis

on

egg

transmission,

using

three different

U.S.

isolates of S. enteritidis

of

phage

type

8

from

humans,eggs,

and

poultry.

MATERIALS

AND METHODS

S.

enteritidisisolates.

E700-87,

solated

in

upstate

NewYork

roma human

with

egg-associated

almo-

nellosis,

and

27A,

recovered rom

pooled

ovaries f

hens,

were

provided

y

Dr.

Mehdi

Shayegani

f

the

New

YorkState

Department

f

Health.

solateY-8P2

was

recoveredrom

pooled

yolks

of

eggs

implicated

as

a

possible

ourceof

salmonellosis

n

humans

t a

NewYork

ity

hospital y

Dr.C.E.

Benson,

University

of

Pennsylvania.

All

isolates were of

phage

type

8 and

contained

plasmids

of

54

kb.

Chickens.

Commercial

hite

eghorn

ayers,

ac-

cinated

against

Marek's

isease,

Newcastle

disease,

and

infectious

bronchitis,

nd

free

of

any apparent

549






H. L.

Shivaprasad

et al.

diseases

throughout

the

growing

and

laying

periods,

were used at

age

18

months

(Expts.

1,

2),

2

years

(Expt.

3)

or

9

months

(Expt.

4).

Housing.

All

birds,

including

the

controls,

were

held

in

wire-bottom

batteries

in an

isolation room

with one bird

(Expts.

2, 3), two birds

(Expt.

1),

or

three

birds

(Expt.

4)

per

cage.

Birds were acclimated

for

3

days

before

inoculation.

Feed

and water were

provided

ad

libitum.

Wax-paper

sheets were used

un-

der the

wire floors

of

each

cage

to catch

droppings.

Sloped cage

floors allowed

eggs

to

roll

away,

reducing

fecal

contamination.

Inoculations. Birds were

inoculated

orally

by

in-

stilling

1.0 ml of

broth culture into the

upper

esoph-

agus using

a

syringe

and a

12.5-cm

blunt-ended

cath-

eter.

Intracloacal

(IC)

inoculations were

made

by

depositing

inoculum

directly

into

the cloaca. The

wing

vein was used for intravenous

(IV)

inoculation.

Clinical and

pathological

examinations.

Clin-

ical

signs,

egg

production,

and

mortality

were re-

corded

throughout

the

experimental periods.

Birds

that

died

were

necropsied,

usuallywithin

a few

hours,

and examined for

gross

and

microscopic

lesions.

Bacteriologic

examinations.

Feces.

Approxi-

mately

5-10

g

of fresh

feces were added to

15-25

ml

of selenite F broth and incubated

overnight

at 42

C.

Approximately

50

,l

of

broth was then

streaked

on

brilliant

green

agar plates

and incubated

overnight

at

37 C. Salmonella-like colonies were tested by plate

agglutination

using

an antiserum

against

the

9,12

O

antigens

of

S.

enteritidis.

Eggs.

Eggs

were stored

2-5

days

at 4

C

before

shells

and

yolks

were cultured for

S.

enteritidis.

Eggs

were

placed

for

5

minutes

in 10 ml

of selenite

F

broth

in

plastic bags

and then

removed,

and the broth was

incubated at

42

C

overnight

before

streaking

on

bril-

liant

green agar.

S.

enteritidiscolonies

were

identified

as above. Yolk was cultured

by swabbing

the

pointed

end

of

the

egg

with

70%

alcohol and then

puncturing

the shell

with

sterile

forceps.

Albumen was allowed

to

drain

out without

breaking

the vitelline membrane.

The vitelline membrane

was cut with sterile

scissors,

and

1 ml of

yolk

was collected with a

syringe

and

incubated

overnight

in 15 ml selenite before

being

streaked

on brilliant

green

agar.

Albumen was

cul-

tured

the same

as

yolk.

Organs.

Yolk,

collected

on

swabs

from the

interior

of

ovules

after the exterior was sterilized

by searing

with

a

hot

spatula,

was

placed

in 10 ml

of

selenite

F

broth.

The

exterior

of

the

oviduct was seared at the

junction

of

the

magnum

and

isthmus,

and

5-6

ml

of

selenite broth

was

injected

into the lumen.

The

pos-

terior end

of the

oviduct

was lifted

slightly

so that the

broth traversed almost

the entire

length

of

the

mag-

num. After

5-10

minutes,

the contents

(2-3 ml)

from

the

magnum

were

poured

into

tubes

containing

5-6

ml

of selenite

broth,

taking

care to avoid

contami-

nation

from

the

peritoneum. Similarly,

the exterior of

the

liver was

seared,

and

its

interior

was

sampled

on

a

sterile

swab that was then cultured.

The exterior of

the

spleen

was sterilized

by

dipping

in

70%

alcohol,

flaming

it

over a

burner,

and

cutting

it into

pieces

with

sterile

scissors. The

pieces

were cultured

in 10

ml

selenite

F broth.

In

some

cases,

1-2

g

of various

organs

were

cultured

in 20 ml

of selenite broth. Heart

blood

(0.5

ml)

was also cultured

in

selenite

broth,

as

were the contents of the jejunum, cecum, and colon.

Selenite cultures were

incubated

overnight

at

42

C

and streaked on brilliant

green

agar

as above.

In

Expt.

1,

tissues from birds that died were streaked

directly

on brilliant

green

agar.

Serology.

Serum

agglutinins

were

assayed

in

a

rapid

serum

plate agglutination

test

using

both com-

mercially

available

pullorum

antigen

and an O

an-

tigen

suspension

made from S. enteritidis

700-87

as

described

(8).

Table

1.

Design

of

experiments showing

the

various isolates

of S.

enteritidis,

source of

isolates, doses,

and inoculation

route of the

bacteria used

and

number

of

hens

per

treatment

in

each

experiment.

Isolate Inoculation

No.

of

Dose

Expt.

Isolate

source

route birds

(CFU)

1

E700-87

Human

Oral

32

4

x

108

Intravenous 16 2

x

108

Controls

12

None

2

E700-87 Human

Oral 14

104

Intravenous 20

106

Intracloacal 14

5

x

107

Controls

6

None

3 Y-8P2 Yolk Oral 14 104

Intravenous

20

106

Intracloacal 14

5

x

107

Controls

6

None

4

27A Chicken

Oral 66

106

(ovary)

Controls 6

None

550






S.

enteritidis in

layers

z

LJ

I

cr

c

O

-

C W

aw

.

-

z

n

bd

0

14

28

DAYS

AFTER INOCULATION

Table

2.

Expt.

1.

Isolation of

S.

enteritidis

E700-

87

from

organs

of

laying

hens 42

days

after inocula-

tion.

No.

Perito-

Groups birds neum Ceca Liver Spleen

Oral

10 0 2 0 0

Intravenous

11

3

2 2

1

Control

5

0 4 2

1

42

Fig.

1.

Expt.

1.

Effectof

S. enteritidis

E700-87

on

egg

production

following

oral

or IV

inoculation with

4

x

108 or 2

x

108

CFU,

respectively.

Phage

and

plasmid typing.

All

S.

enteritidis

so-

lates were

phage-typedby

Dr. B.

Rowe,

CentralPublic

Health

Laboratory,

London,

England,

as

described

(34).

Confirmation

f

plasmid

content was done

in

our

laboratoryusing

routine methods

(5).

Experimental design.

Table

1

shows

isolates

in-

oculated,

routes

of

inoculation,

dosages

of

organisms,

and number of hens

inoculated

for all

four

experi-

ments.

Expt.

1.

Control

birds

were

housed

in one

battery

in the same room as the inoculated birds.Orally n-

oculated birds were

held

in

two

batteries,

and

IV-

inoculated birdswere in a

separatebattery.

Preinocu-

lation

samples

of

eggs,

blood, feces,

and

feed were

collected

for

bacteriologic

examinations.

During

a 42-

day

experimental period

after

inoculation,

clinical

signs,

egg production,

and

mortality

were

recorded,

and feces and

eggs

were

collected

daily

from

each

cage

for

culturing.

Two ml of

blood

were collected

for

serology

from the

wing

vein of

several birds in

each

group

at 28

days

PI

and from the

heart of some

of the

surviving

birds at 42

days

PI.

Birds

that

died

were necropsied and examined for gross and micro-

scopic

lesions.

Samples

of

liver,

spleen,

cecal con-

tents,

peritoneum, ovary,

and

heart blood

were cul-

tured on

brilliant

green

agar.

At

the end of

the

experiment,

all

the

surviving

IV-inoculated

birds,

10

of the

orally

inoculated

birds,

and five

control

birds

were

euthanatized,

and 1-2

g

of each

body organ

was

cultured.

Growth

of S.

enteritidis

E700-87

in

egg yolk

was

studied

by

inoculating

1-ml

samples

of

yolk

with 0.01

ml

of a

suspension

of

S.

enteritidis

containing

103

colony-forming

units

(CFU)/ml

in LB

broth.

The

yolk

was mixed thoroughlyand incubatedovernightat 37

C before

plating

on

brilliant

green agar.

Expt.

2.

Feces and

eggs

were collected

and cul-

tured as described for

Expt.

1. In

addition to shell

and

yolk,

albumen

was also culturedfor

S.

enteritidis.

The

experiment

was terminated

19

days

PI,

when

all

birds

were

bled

for

serology.

Representative

irds rom

each

group

were

necropsied,

and

their

organs

were

cultured.

Expt.

3.

This

experiment

was

similar

to

Expt.

2

except

that he birdswere observed or29

days

nstead

of

19

days.

Expt.

4.

On

days

4, 7, 12, 19,

and

30

PI,

two

to four

experimental

birds

plus

one

control bird

were bled

for

serology,

then euthanatizedand

necropsied.

Sam-

ples

of

heart

blood, liver,

spleen, peritoneum,

jejun-

um,

cecum, colon, ovules,

and oviduct

were

cultured.

At 42

days

PI,

all

remaining

birds were bled for se-

rology

and then

necropsied.

The liver

and

cecum

of

all birds and the

ovules, oviduct,

peritoneum, jejun-

um, colon,

and heartblood of 11 birds

and

one

control

bird were cultured.

Eggs

were collected

daily,

and

the

yolk

and

albumen were cultured

separately

as

described

for

Expts.

2

and

3.

Feces was cultured on

three occasions

during

the

experiment.

RESULTS

Expt.

1. Most

of

the hens

inoculated

orally

or IV with

E700-87

were

depressed

and

anorec-

tic.

Many

had

watery

diarrhea

lasting

for

7-14

days.

IV-inoculated

hens

were most

severely

af-

fected,

and five died

during

the

first

14

days.

At

necropsy,

all these birds were

very

dehydrated,

with

fluid-filled

intestines and

a

fibrinous

exu-

date

in

the

peritoneum.

S. enteritidis

was

re-

covered

from

heart

blood of four and

from the

peritoneum

of

two

of

these five birds. Two

birds

in

the

orally

infected

group,

which

died

after

17

or

25

days

due

to severe visceral

gout

or

cannibalism,

were

negative

for

S.

enteritidis.

Egg

production

was

greatly

reduced for

about

14

days

PI in

the

IV-inoculated birds

(Fig.

1)

and had not returned to the levels of the control

birds

by

42

days

PI.

There was

also

a

slight

depression

of

egg

production

in

birds inocu-

lated

orally,

especially during

14-28

days

PI.

Most

orally

infected birds shed

S. enteritidis

in

the feces

for

5-16

days (Fig.

2),

and a few

551






H. L.

Shivaprasad

t al.

z

30-

'25-

C)

LL

0

20-

U)

15-7

o

/

0,/

,

9

1112 13 14 1 6 17 18 19202122232425 262728293031 32

*

Birds

died

CAGE

NUMBER

Fig.

2.

Expt.

1. Mean fecal

shedding

of S. enteritidis

E700-87

by cages

of hens

following

oral or IV

inoculation.

birds shed

for

up

to 21

days.

Shedding

was much

more

prolonged

in

the IV-inoculated

birds and

continued

for at

least

42

days

in

a few

birds.

At

necropsy,

four

birds

from

the IV-inoculat-

ed

group

had mild

peritonitis,

and

three

of these

had

yellow

caseous

granulomas

attached to the

peritoneum

near the

ovary.

S. enteritidis

was

isolated

from

these

granulomas

and

also

from

the

cecal

contents, liver,

or

spleen

of a few

oral-

ly

or IV-inoculated birds

(Table

2).

S. enteritidis

was also cultured from the

liver,

spleen,

or ce-

cum of four of

five

clinically

normal,

lesion-free

control

birds. Fecal

shedding

of

S.

enteritidis

was not monitored

in

the control

birds.

S. enteritidiswas

not isolated

from the

yolks

of

250

eggs produced

by

infected

hens but was

found

on the shells of

two of

80

eggs.

All

eggs

from control

birds were

negative.

Albumen was

not cultured

in

this

experiment.

Overnight

in-

cubation at

37

C

of

yolk

inoculated

with

1-3

CFU of

E700-87

resulted

in massive

overgrowth

of

the

organism

following

plating

on

brilliant

green

agar.

Serology

is

reported

in Table

3.

Positive birds

Table

3.

Expts.

1 and 2.

Agglutination

reactions of sera from hens

following

inoculation with

S.

enteritidis

E700-87.A

Data show the number of reactors

o S.

pullorum

and

S.

enteritidis.

Expt.

1

Expt.

2

28

days

42

days

19

days

Group

SPB

SEC

SP

SE

SP

SE

Oral

0/10D

6/10 4/30

10/30 1/14

2/14

Intravenous

5/5

5/5

5/11 11/11 10/19

12/19

IntracloacalE

0/14

3/14

Control 1/5 1/5 2/12 3/12 0/6 1/6

ANumbers of

organisms

inoculated are

given

in Table 1.

Commercial

S.

pullorum-stained

antigen.

'Suspension

of

O

antigen

of

S.

enteritidis

E700-87.

No.

hens

tested/no.

positive.

'No

intracloacal

group

in

Expt.

1.

552






S.

enteritidis

in

layers

Table 4.

Expt.

3.

Isolations of S. enteritidis

Y-8P2from

eggs

of hens

inoculated

by

the

oral, intravenous,

and

intracloacalroutes.

No.

of isolations from

No.

eggs

Group

examined Shell

Albumen

Yolk

Oral 221 2

(3)A

6

(5, 7, 11,

14)

0

Intravenous

274 5

(1,

5,

14)

8

(2,

6)

2

(2,

4)

Intracloacal

231

12

(1, 3, 5,

11,18)

4

(7,

14)

1

(27)

Control 124

1

(3)

0

0

ANumbers

n

parentheses

are

the

days

on

which

S.

enteritidiswas

cultured.

were detected

in

all

groups,

including

the con-

trols,

at

all

three

samplings

from

19

to

42

days

PI.

S.

enteritidisantigen

was

more sensitive than

S. pullorum antigen.

Expt.

2.

IV-inoculated birds were

moderate-

ly

anorectic and

depressed

and had

watery

diar-

rhea

lasting

6-8

days

in

most

birds and as

long

as 18

days

in a

few birds.

A

slight

decrease in

egg

production

occurred,

probably

due in

part

to

molting

in

some birds.

A

few

orally

and IC-

inoculated birds

were

mildly depressed

and

an-

orectic

and had mild

diarrhea

lasting

for 12-18

days. Egg

production

was not

noticeably

affect-

ed

in

these

two

groups.

One

IV-inoculated bird

died on day 17 PI with severe dehydration and

visceral

gout

but was

negative

when

cultured

for

S.

enteritidis.

Fecal

shedding

lasted for

17,

6,

and

17

days

in

the

oral, IV,

and

IC

groups,

respectively.

S.

enteritidis

was cultured from

one control

bird

6

days

after

infection. Two of

five

IV-inoculated birds

euthanatized

at

19

days

PI had mottled livers

and

atrophied

ovaries.

However,

five

orally

inoculated

birds,

two

IC-

inoculated

birds,

and two

control

birds were

free

of lesions.

S.

enteritidis

was not

recovered

from

liver,

spleen,

ovary, peritoneum,

or cecum

of

any

bird

in

any

group.

S.

enteritidiswas

cul-

tured from

8-10

eggshells

in

each

experimental

group

but not from

the

yolk

or

albumen.

Shells

and contents of

eggs

from

control

birds were

negative

for

S.

enteritidis.

Serology

done at

19

days

PI

(Table 3)

showed

that the

antigen

made

from S. enteritidis

was

more

sensitive in

detecting

antibody

than

the

antigen

from

S.

pullorum.

A

control bird

that

was serologically positive was also positive by

fecal

culture for

S.

enteritidis

on the sixth

day.

Expt.

3.

Most of the

birds

infected

with S.

enteritidis

isolate

Y-8P2 had

mild-to-moderate

watery

diarrhea,

anorexia,

and

decreased

egg

production

in

the

first

5-7

days.

IV-inoculated

birds were most

severely

affected;

six of

these

birds

had diarrhea

20-28

days.

Five

orally

in-

oculated birds had mild

diarrhea

lasting

10-20

days. Birds inoculated IC were least affected,

and all control birds remained

normal. One

IC-

inoculated bird died on

day

3

PI,

one

IV-inoc-

ulated bird died on

day

5

PI,

and one

orally

infected

bird died on

day

25

PI.

All

of

these

birds

were

dehydrated

and had

mild

exudate

in

the

peritoneum.

S. enteritidiswas

cultured

from

the

liver,

spleen,

cecum,

peritoneum,

and

ovary

of all

three.

Egg

production

was

appreciably

reduced

in

the three

experimental

groups compared

with

the control birds. The reduction was greatest in

the

IV

group

and least in

the

IC

group.

Fecal

shedding

of S.

enteritidiswas

intermit-

tent and

lasted

for

about

5,

9,

and 18

days

PI

in

the

IV, oral,

and

IC

groups,

respectively.

Con-

trol birds

remained

negative

throughout

the ob-

servation

period.

At

necropsy,

three of

five IV-

inoculated birds

had

atrophied

ovaries and

mild

fibrinous exudate in

the

peritoneum.

There

were

no

lesions in

two

birds each

from the

IC and

control

groups.

Cultures of

liver,

spleen, peri-

toneum,

cecum,

and

ovary

were

negative

for

S.

enteritidis.

Table

5.

Expt.

3.

Plate

agglutination

reactions of

sera of

hens

29

days

after

noculation

with S.

enteriti-

dis

(Y-8P2).

No.

No.

reactors

reactors

to S.

No. of

to S.

enter-

Group birds pullorum itidis

Oral

13

1

1

Intravenous

19

10

16

Intracloacal

13

2

1

Control

6

1 1

553






H. L.

Shivaprasad

t al.

Table 6.

Expt.

4. Isolations of S.

enteritidis

(27A)

from

hens

orally

inoculated with 106

CFU.A

No.

positive/no.

cultured

on

days

PI

Organ

4

7

12

19 30

42

Heart blood

1/2 1/2 1/3

0/4 0/3

0/11

Liver

2/2 2/2 1/3

0/4 0/3

1/52

Spleen

1/2 2/2

1/3 1/4

0/3

NCB

Peritoneum

1/2

1/2 2/3 1/4

0/3 0/8

Jejunum

2/2 2/2

3/3 1/4 0/3

0/11

Cecum

2/2 2/2

1/3 0/4

0/3 0/52

Colon

1/2 1/2 1/3

0/4 0/3

0/11

Ovule

0/2 2/2 0/3

0/4 0/3

0/11

Oviduct

2/2 2/2 1/3

1/4 0/3

0/11

AThe

rgans

of

six

uninoculated control

hens were

negative for

S.

enteritidis.

BNot ultured.

Table

4

shows isolations of S.

enteritidisfrom

eggshells

and contents.

Eggshells

from

the

IC-

inoculated birds showed the

highest

rate of con-

tamination;

one

eggshell

from

a

control

bird

was also

positive.

Albumens from a

few

eggs

from the

three infected

groups

were

positive,

as were two

yolks

from IV-inoculated birds and

one

yolk

from an

IC-inoculated bird.

Most of

the isolations from

albumen

were made

be-

tween

5

and

7

days

and on

day

14

PI.

Eggs

from

which S. enteritidis

was

isolated from

the

al-

bumen

generally

did not

yield

the

organism

from the

yolk.

Moreover,

eggs

that carried the

organism

on

the shell did

not

carry

the

organism

in

albumen or

yolk.

Table

5

shows

serological

test

results.

There

were more reactors

in

the

IV-inoculated group

to S.

enteritidis

antigen

than

to

S.

pullorum

an-

tigen.

Sera from the

other

groups

reacted

equal-

ly

well with

both

antigens,

although

only

a

few

sera

were

positive.

One

control bird

was also

positive.

Expt.

4.

Expt.

3

proved

that

S.

enteritidis

isolate Y-8P2

may

not

only

be

transmitted

in

yolk

but also in albumen.

Expt.

4 was

performed

to

confirm this result

with an isolate of

ovarian

organ and to establish whether the source of

the

organism

in the

egg

was the

ovary

or

the

oviduct.

Moreover,

because the natural route

of

entry

for

chickens is the oral

cavity, only

this

route was

used to

infect the hens.

Except

for mild

diarrhea

in

the first

few

days,

all

birds remained

clinically

normal

with

no

mortality

or

reduction

in

egg production.

One

bird

died at

day

18

PI of

cannibalism

and

pro-

lapse

of the rectum.

Some birds shed S.

enter-

itidis

in

feces for

18-25

days.

Table 6 shows isolations of S. enteritidisfrom

various

organs

in birds killed at

4,

7, 12,

19,

30,

and

42

days

PI. Most of the

organs, including

ovule and

oviduct,

were

positive

for

S.

enteriti-

dis at

7

days

PI. The

organs

of

many

birds

were

still

positive

at 12

days

PI. The

number

of

iso-

lations

decreased

substantially

by

19

days,

and

by

30

days

the

organs

of all birds

were

negative.

At the end

of

the

experiment

at

42

days,

the

liver of

one

of

52

birds was

positive.

All

other

tissues from

representative

birds

were

negative.

A total of

889

eggs,

including

25

eggs

from

control

birds,

were examined

during

the

ex-

periment.

Table

7

shows

the isolations of S.

enteritidis

from

eggs

for 11

days

after inocula-

tion,

when 11

yolk samples

and six

albumen

samples

were found

positive.

S.

enteritidis was

found

in

the

yolk

as

early

as

1

day

PI

and

as

late

as

11

days

PI but

was

not found in

the

albumen

until

7

days

PI. The

organism

was

never

cul-

tured

from

the

yolk

and

albumen

of

the

same

egg. After 11 days, all eggs were negative for S.

enteritidis.

Only

three of

57

sera

gave

positive

aggluti-

nation

reactions with S.

enteritidis

antigen

at

30

and

42

days

after

inoculation. No

serum

re-

acted with

S.

pullorum antigen.

Table

7.

Expt.

4.

Isolations of

S.

enteritidis

(27A)

from the

albumen and

yolk

of

eggs

of

hens

inoculated

with

isolate

27A.A

Egg

No.

positive/no.

cultured

on

days

PI

part

1

2

3

4

5

6

7

8

9

10

11

Albu-

men

0/32

0/37

0/24

0/22

0/30

0/28 2/24

1/29

0/34 0/34

3/20

Yolk

2/32

3/37

0/24

0/22

0/30

0/28

0/24

0/29

0/34

1/34

5/20

ATwenty-five

eggs

from six

control hens

were

negative

for

S.

enteritidis.

554






S. enteritidis in

layers

DISCUSSION

Our results indicate that

isolates

27A

and

Y-8P2

of S. enteritidis

can be

transmitted

in

the

albumen

or

yolk

of the

egg. However,

the num-

ber

of

eggs

that

harbored

organisms

was

few,

and

shedding

occurred

predominantly

during

the

first

2

weeks

PI.

This

finding

contrasts with

those

noted

for S.

pullorum-

or S.

gallinarum-

infected

birds,

in which

30-50%

of

eggs

were

culturally

positive

(20,25).

Albumen was

more

frequently

contaminated than

yolk,

which

sug-

gests

that

the

source

of

the

organism

was the

oviduct or

peritoneal

cavity.

The

conclusion is

supported

by

the

fact that

S.

enteritidiswas iso-

lated from these locations in some birds during

the

first weeks

following

infection.

Recovery

of

S.

enteritidisfrom

eggs

at this

stage

of

infection

suggests

bacteremic

seeding

of

organs

and tis-

sues

such

as the

peritoneum

and

oviduct.

Fecal

contamination

of

the shell

appeared

to be

a less

likely

source of the

organism

for

the

interior of

the

egg

because fecal

shedding

was

light

and

intermittent

in

Expt.

3,

in

which 21

isolations

of

Y-8P2 were made from

yolk

or

albumen.

Moreover,

shell

contamination in

that

experi-

ment did not correlate well with the

presence

of

the

organism

in

the

yolk

or

albumen.

Expt.

1,

with

isolate

E700-87,

provided

even

stronger

evidence

against

the shell as an

important

source

of the

organism

for the

interior of

the

egg.

Most

of the hens in

Expt.

1

developed

a

severe

watery

diarrhea and shed

S.

enteritidis

in

the

feces for

up

to

16

days.

The

organism

was

recovered from

the

shells of

about

3%

of the

eggs

but not

from

the

yolk

of

any egg.

In

another

study

in

this

laboratory

(31),

it

was shown

that an

isolate of

S.

enteritidis

phage

type

4

may

be shed in

the

yolk

or

albumen of about

10%

of

eggs

during

the

first

2

weeks

following

experimental

oral

inoculation. This

isolate

behaved

similarly

to

isolates

27A

and

Y-8P2 in

that

it

produced

in-

vasive infections

following

oral

inoculation

and

did not

seriously

affect

egg production,

al-

though

it

was

present

in

the

egg-producing

or-

gans

of

most

birds

examined

following

inocu-

lation.

Although the present study is the first direct

experimental

evidence of

transmission of non-

typhoid

salmonellae

such as S.

enteritidis

not

only

from the

ovary

but

also

from the

oviduct,

there

is

some

earlier

circumstantial

evidence

for

the

phenomenon.

Several

serotypes,

including

S.

enteritidis,

S.

typhimurium,

S.

thompson,

and

S.

heidelberg,

have been detected

in

chicken

egg

contents

(27,28,35).

In the case

of S. en-

teritidis

and S.

typhimurium

in duck

eggs,

di-

rect

infection

of the

ovary appears

to have

oc-

curred

(3).

More

recently,

S. enteritidis

phage

type

4

has been

isolated from

eggs,

ovules,

and

oviducts

of a commercial

layer

flock

implicated

as a source of

eggs

that caused food

poisoning

in humans

(17).

Egg production

in this

flock

was at

expected

levels,

although

some

birds

died

with lesions from which

S. enteritidis was

cul-

tured. This

observation,

together

with

reports

of infection

of

young

chicks

by

this

phage

type,

suggests

that S. enteritidis

phage

type

4 is

very

invasive, causing lesions in various body organs

(19).

In the

present study,

the isolations of

iso-

lates

27A

and

Y-8P2

from the

liver,

spleen,

ovi-

duct,

peritoneum,

heart

blood,

and ovules from

hens killed at

5,

7,

and 12

days

after oral

inoc-

ulation

suggest

that

some isolates

of

phage type

8

may

also be

unusually

invasive

for the

adult

hen.

Older

chickens have a

high

level of

resistance

to invasive

infection

by

salmonellae other

than

S.

pullorum

and

S.

gallinarum

(32);

it

was

not

possible

to demonstrate invasion of the intes-

tine or

significant

intestinal colonization

of

6-month-old

chickens

following

intraoral

in-

oculation of

7.5

x

108 CFU of an avian

isolate

of

S.

enteritidis.

A

similar oral dose of

E700-87

in the

present study appeared

to be more vir-

ulent,

causing

diarrhea,

depression,

extended

fecal

shedding,

and localization of the

organism

in the

liver of some birds killed at

42

days

PI.

However,

isolate

E700-87

differed

from

27A

or

Y-8P2 in

that it was never

detected

in

yolk

or

albumen

of

eggs

of

any

bird,

whether

inoculat-

ed

orally

or IV

(Expts.

1,

2).

Because

E700-87

was of

phage

type

8

and carried a

plasmid

of

54

kb,

identical

in

mass to that in

isolates

27A

and

Y-8P2,

the difference

in

its

behavior

in

the

hen

seems to be

unrelated

to

phage

type

or

plasmid

content.

However,

proof

of this

awaits

further

research,

as

does the elucidation of

the

genetic

basis

and

origin

of

the enhanced

virulence of

S. enteritidis

clones

that have

apparently

emerged independently in laying hens in Eu-

rope

and

North

America

in

recent

years.

Based

on

the

studies

referred to

in

the

present

paper,

and on

related

studies of

S.

enteritidis

phage

type

4

(31),

we

suggest

that the

recently

emerged

epidemic

clones of

S.

enteritidishave

enhanced

555






H. L.

Shivaprasad

t

al.

ability

to

invade and survive n the

bloodstream

and infect

organs

such as

the

oviduct,

perito-

neum,

and ovules involved

in

egg

production.

Of

practical importance

in

detection

of in-

fected flocks is the observation that even the

more invasive

isolates

of S. enteritidis

(27A

and

Y-8P2)

were cleared from the tissues of

most

birds 28

days

to

42

days

PI.

In

the

case of

Y-8P2,

the

organism

could not be detected in

birds

29

days

after inoculation. These

findings

suggest

that

retrospective

epidemiologic

trac-

ing

of infected flocks could

be

difficult and must

rely

in

part

on environmental

sampling

and

de-

tection

of serum

antibodies.

However,

some

birds

in

some

flocks

conceivably

could remain

indefinitely infected and shed the organism

when stressed at

a

later time.

Predictably,commercially

available

S.

pullo-

rum

antigen

reacted

with serum

of

some birds

infected

with S. enteritidis

isolates

E700-87

and

Y-8P2.

However,

more reactors

were detected

with

homologous

S.

enteritidisantigen

than with

S.

pullor2m antigen

after

infection

with

isolate

E700-87

in

Expts.

1

and

2

(Table

3).

A

similar

phenomenon

was noted for

birds inoculated

IV

with

Y-8P2

(Expt.

3).

However,

in

this

experi-

ment,

only

a

very

few birds

inoculated

orally

or

IC made detectable

agglutinin

responses.

Sim-

ilarly,

in

Expt.

4,

only

one bird of nine

tested

following

oral inoculation with isolate

27A

was

serum

antibody-positive

to

S.

enteritidis30

days

after infection.

At 42

days,

only

two of

48

birds

tested

in

Expt.

4

were

positive using

S.

enteriti-

dis

antigen,

and no sera reacted with

S.

pullo-

rum

antigen.

The variations

in

antibody

re-

sponses

could be

attributed to differences

in

the

quantity

and

quality

of

antigens

in

the cell

wall. This

is

well known

among

S.

pullorum

isolates,

some of which

possess

form

variations

in

antigens

XII2

and

XII3

(11,12),

which can

have

a

marked effect on the

agglutination

test

(11).

It

is

possible

that

similar

antigenic

varia-

tions

exist

among

isolates of

S. enteritidis.

It is

noteworthy

that the number of

reactors

among

birds

infected

orally

with

various iso-

lates of

S.

enteritidiswas low in all

experiments

and some birds

did not

become

positive

until

30 daysPI (Expt.4). This resultmayreflect the

low

sensitivity

of

the

rapid

plate

test,

which

is

known

to be less

sensitive

and

less

reliable than

the tube

agglutination

test

(20,23,25).

How-

ever,

it

is clear

from our

results that S.

enteritidis

antigen

is more

sensitive and

specific

than

com-

mercially

available

pullorum

antigen

for

the

de-

tection of

serum

antibody

to

S.

enteritidis in

laying

hens.

Few

control hens

became

infected,

as

evi-

denced by culture of S. enteritidis from the or-

gans

and

by

serology (Expts.

1, 2,

3).

The

most

probable

mode of

infection of

the control

hens

is

by

aerosolization of

feces from

the

experi-

mental hens that were in

contact with

the con-

trol hens.

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ACKNOWLEDGMENTS

We

thank

the

following

for their

assistance

in

var-

ious

ways:

Dr.

C.

E.

Benson of

the

University

of

Penn-

sylvania

and Dr.

M.

Shayegani

of

the

New York

State

Department of Health provided the salmonella iso-

lates. Alan

Keyes,

Judy

St.

Leger,

Amy

Crawford,

Joy

Agger,

and

Moira Resnick

provided

technical

assis-

tance,

and

Phyllis

Ibsen and

Gwen

Troise

typed

the

manuscript.

The

financial

assistance

of

the

South-

eastern

Poultry

and

Egg

Association is

gratefully

ac-

knowledged.

557

55 5

Documents

Pathogenesis of Salmonella enteritidis Infection in Laying Chickens. I. Studies on Egg Transmission, Clinical Signs, Fecal Shedding, and Serologic Responses