Reduced intestinal colonisation with F18-positive enterotoxigenic

Escherichia coli in weaned pigs fed chicken egg antibody against

the ¢mbriae

Armando Zuènì igaa, Hideaki Yokoyama

b, Pia Albicker-Rippinger

a,

Ernst Eggenbergerc, Hans Ulrich Bertschinger

a;

*

aInstitute of Veterinary Bacteriology, University of Zuërich, CH-8057 Zuërich, Switzerland

bImmunology Research Institute in Gifu, Gifu-City 501-11, Japan

cBiostatistical Services, Faculty of Veterinary Medicine, University of Zuërich, CH-8057 Zuërich, Switzerland

Received 24 April 1997; accepted 24 April 1997

Abstract

Newly weaned pigs were fed a basal diet containing either egg antibody against fimbriae F18 at a high or low level, control

egg powder or no egg, and challenged with enterotoxigenic Escherichia coli with fimbriae F18. The challenge was repeated after

termination of the antibody treatment. Antibody-containing egg powder was produced by vaccination of hens with semi-

purified fimbriae of the two variants F18ab and F18ac. Pigs eating egg powder with antibody against the same fimbrial variant

were fully protected, even if the vaccine for the hens was produced with a different serotype devoid of enterotoxins. The effect

was dose-dependent. The high dose of antibody against the heterologous variant of fimbriae F18 reduced colonisation at a level

which was not significant. Ingestion of egg antibody partially suppressed the build-up of anti-colonisation immunity. Oral

application of egg antibodies offers a promising approach for the prevention of infectious diseases of the digestive tract.

Keywords: Enterotoxigenic Escherichia coli ; Fimbriae F18; Pig; Post-weaning diarrhoea; Prophylaxis; Egg yolk antibody

1. Introduction

Post-weaning Escherichia coli diarrhoea (PWECD)

and E. coli enterotoxaemia (ECET, oedema disease)

continue to cause considerable economic loss [1,2].

In both diseases the clinical signs and death of the

pigs are caused by one or several toxins released by

bacteria colonising the small intestine. No more than

two families of adhesive ¢mbriae, F4(K88) and F18,

are involved in this colonisation. The ¢mbriae F18

occur as two antigenic variants, F18ab (formerly

F107) and F18ac (formerly 2134P, Av24 and 8813)

[3,4].

Colonisation of the intestine with F18-positive E.

coli leads to strong anti-colonisation immunity asso-

ciated with the appearance in the serum of anti-¢m-

brial antibody of the IgA class [5]. However, so far

no e¡ective vaccination protocol has been developed

[6]. The situation is in many regards comparable to

human cholera and enterotoxigenic E. coli (ETEC)

0928-8244 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

PII S 0 9 2 8 - 8 2 4 4 ( 9 7 ) 0 0 0 3 5 - 7

FEMSIM 760 27-8-97

* Corresponding author. Tel. : +41 (1) 635 8602;

Fax: +41 (1) 635 8912.

FEMS Immunology and Medical Microbiology 18 (1997) 153^161

infections. Passive immunisation may therefore be an

attractive alternative. It may be achieved by the in-

gestion of antibody against essential virulence deter-

minants. In the case of porcine ETEC, colostral anti-

bodies against the adhesive ¢mbriae were shown to

protect against intestinal colonisation and disease

[7,8].

Egg yolk obtained from immunised hens is an in-

teresting source of large amounts of antibody (IgY).

Antibodies extracted from chicken eggs are thera-

peutically and prophylactically e¡ective in neonatal

pigs infected with ETEC when continuously applied

per os [9,10]. In the gastrointestinal tract of some-

what older pigs, the puri¢ed antibodies are enzymati-

cally inactivated [10]. However, egg yolk antibodies

pass the small intestine intact, if fed as whole egg or

mixed with egg white [11].

Fimbriated E. coli preincubated with egg antibod-

ies against the ¢mbriae do not adhere in vitro to

enterocytes [12,13]. Deprez et al. [14] reported that

egg powder containing antibodies against the ¢m-

briae fed to weaned pigs o¡ers excellent protection

against PWECD caused by F4-positive ETEC. Pro-

tection goes together with reduced faecal shedding of

the pathogen. In a double-blind ¢eld trial, Erhard

and Loësch [15] observed a signi¢cant reduction of

PWECD and of the need for treatment with a feed

enriched with 5% of a similar egg antibody prepara-

tion fed to the newly weaned pigs on a farm with a

high incidence of PWECD due to F4-positive ETEC.

Whole egg powder obtained from hens hyperimmu-

nised with puri¢ed ¢mbriae F18ab kept down the

faecal numbers of E. coli with either ¢mbriae

F18ab or F18ac in pigs experimentally infected

with such strains [16]. The inhibition was more

marked with the bacteria bearing the homologous

than with the bacteria producing the heterologous

¢mbrial variant.

In view of the economic feasibility, the present

investigation aimed at testing the preventive e¤cacy

of di¡erent concentrations of antibody-containing

whole egg powders in the feed against intestinal

colonisation by ETEC with the homologous or

the heterologous antigenic variant of ¢mbriae

F18. In addition interference of the passive im-

mune protection with the development of ac-

tively acquired anti-colonisation immunity was

studied.

2. Materials and methods

2.1. Production and puri¢cation of ¢mbriae for

vaccination of hens

The bacteria (Table 1) were grown for 20 h on Iso-

Sensitest agar (CM471, Oxoid, Basingstoke, UK)

with 0.0625% (w/v) alizarin-yellow (Art. No. 5570,

Fluka AG, Buchs, Switzerland) [17] in an atmo-

sphere with 5% CO2 at 37³C and growth was sus-

pended in saline. The ¢mbriae were detached by in-

cubating the suspension at 60³C for 20 min and the

bacteria removed by centrifugation at 26 000Ug for

20 min at 4³C. Fimbriae were precipitated by the

addition of 20% (v/v) saturated ammonium sulfate.

The mixture was held overnight at room temperature

and centrifuged at 41 000Ug for 45 min at 4³C. The

pellet was resuspended in Tris/EDTA bu¡er (10 mM

Tris, 150 mM NaCl, 1 mM EDTA, adjusted with

HCl to pH 7.3) and dialysed against Tris bu¡er (50

mM Tris, adjusted with HCl to pH 8.8). The precip-

itation of ¢mbriae was repeated with 10% (v/v) sat-

urated ammonium sulfate. Further steps were per-

formed as described above.

2.2. Vaccination of hens and production of egg powder

Five-month-old White Leghorn chickens (strain

Hyline W36; 100 chickens) were utilised for immu-

nisation. Each dose of ¢mbrial vaccine containing

0.5 mg (dry weight) of ¢mbrial antigen (F18ab,

strain 107/86 and F18ac, strain 8199) was mixed in

equal parts with emulsion oil containing 5% (v/v)

Arlacel 80 (Maine Biological Laboratories, Water-

ville, ME, USA). 1 ml of this emulsion was injected

intramuscularly (0.5 ml injected into each breast

muscle of the hens). Eight weeks after the initial in-

jection a booster injection was given in the same

manner, and eggs were harvested 2 weeks later for

about 1 month.

Whole egg contents were applied to a spray-dry

machine (Model L-12, Ohkawara Kakohki, Kanaga-

wa, Japan) operated at an air-inlet temperature of

140³C and an air-outlet temperature of 72³C. The

material was introduced into a feeder at the rate of

5 l h

31with a pump and then sprayed in rotary

fashion at a high speed (22 000 rpm) in the applica-

tion zone where it was mixed with temperature-con-

FEMSIM 760 27-8-97

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161154

trolled air. At the bottom of the dryer, the dried

material was transported by a £ow of lower-temper-

ature air to the collection vat. The dried powder

containing antibody was stored in a desiccator in a

cool room until use.

2.3. ELISA for antibody titration in the egg powder

Highly puri¢ed ¢mbriae for use as antigens were

produced with the same strains (Table 1) and proce-

dures as for vaccination. The centrifuge pellet after

precipitation by 20% saturated ammonium sulfate

was resuspended in Tris bu¡er pH 8.8 and dialysed

against distilled water for 24 h at 4³C. The suspen-

sion was applied onto a chromatographic column

packed with Sephadex QAE Sephadex A-50 (Phar-

macia, Uppsala, Sweden). The column was equili-

brated with Tris bu¡er pH 8.8. For fractionation,

Tris bu¡er pH 8.8 with increasing NaCl concentra-

tions (300, 500, 700 and 1000 mM) was used as elu-

ent. Fractions (1.5 ml) were collected and protein

was assayed spectrophotometrically at 280 nm. Pro-

tein-containing fractions were pooled. The purity of

each ¢mbrial preparation was analysed by SDS-

PAGE in 11% acrylamide gels. Gels were transferred

onto nitrocellulose by electroblotting. Rabbit anti-

sera against the whole organisms of the production

strains diluted 1:100 were used as primary antisera.

Bound antibody was visualised by peroxidase-conju-

gated goat anti-rabbit IgG developed with hydrogen

peroxide and 4-chloro-2-naphthol. No bands were

recognised except ¢mbriae F18. The suspension was

dialysed against distilled water before lyophilisation.

The protein content of the pool was determined with

the Lowry method, and volumes corresponding to

0.5 mg protein freeze-dried under vacuum.

Microdilution plates (Immuno 2; Dynatech Labo-

ratories Inc., Chantilly, VA, USA) were coated with

100 Wl of a 5 Wg ml

31suspension of highly puri¢ed

¢mbriae in 0.05 M carbonate bu¡er (pH 9.6) per well

at 4³C for 18 h. The plates were emptied and blocked

with 150 Wl of PBS containing 3% bovine serum

albumin per well at 37³C for 1 h and then washed

with 0.02% Tween 20-saline three times. Egg pow-

ders were reconstituted in 0.05% (v/v) Tween 20-PBS

1:100 (w/v). 100 Wl of twofold serial dilutions of egg

antibody in Tween-PBS were added per well. The

plates were held at 37³C for 1 h and washed as

described above. Rabbit anti-chicken IgG conjugated

with horseradish peroxidase (Cappel, Organon Tech-

nika, West Chester, PA, USA) diluted 1:8000 in

0.05% Tween-PBS was applied and plates were incu-

bated at 25³C for 30 min. The o-phenylenediamine

dihydrochloride substrate was added, and the colour

reaction stopped after 20 min by the addition of 3 N

H2SO4. OD at 490 nm was determined in a micro-

dilution plate reader (MR 5000; Dynatech). Titres

were calculated based on the highest sample dilution

yielding an OD490 nmv 0.5.

2.4. Design of pig inoculation experiment

Weaner pigs were fed the basal diet containing

dried egg antibody powder at a high or low level,

control egg or no egg powder and challenged with E.

coli with ¢mbriae F18 of the homologous or the

heterologous antigenic variant (Table 1). The chal-

lenge was repeated after termination of the antibody

treatment (Table 2).

2.5. Experimental pigs

Sixty-eight Swiss Landrace secondary SPF pigs

were obtained from the herd of the institute. They

were bred for susceptibility to F18-positive E. coli

[18] and found at slaughter to belong to the suscep-

tible phenotype by means of a previously described

enterocyte adhesion test [19]. The pigs were weaned

between 23 and 30 days and a mean body weight of

8.6 kg in experiment 1 and 6.9 kg in experiment 2.

Pigs in each treatment group (¢ve or six) were kept

in separate £atdeck holdings. The £atdecks were

equipped with nipple drinkers and a self-feeder,

and located in isolation rooms held at 22þ 1³C.

The pigs were fed a compound feed for weaner

pigs containing crude protein 19.5% (w/w), digestible

energy (pig) 13.2 MJ kg

31and crude ¢bre 3.4% (w/

w). Organic acids and anti-microbial additives were

omitted. Enro£oxacin 150 ppm (Baytril I.E.R. pre-

mix 2.5% (w/w), Bayer AG, Leverkusen, Germany)

was included for the ¢rst 6 days after weaning.

2.6. Experimental treatments

Six treatments were compared (Table 3). Alloca-

tion of pigs to treatment groups was based on litter

FEMSIM 760 27-8-97

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161 155

and weight. The feeds for treatments designated high

egg antibody were the basal feed plus 5% (w/w) un-

diluted egg powder from immunised hens, whereas

the feeds for treatments called low egg antibody were

the basal feed plus 1% undiluted egg powder from

immunised hens, plus 4% (w/w) egg powder from

non-immunised hens. Two control groups received

the compound feed with 5% (w/w) dried egg powder

from non-immunised hens, and the compound feed

without egg powder. The egg powders were mixed

with the feed by Ufamed AG, Sursee, Switzerland.

2.7. Challenge inoculations

The pigs were challenged and re-challenged in the

¢rst experiment with E. coli strain 3064/84

STM6.7 log

cfu ml

31and in the second experiment with strain

8199RIF

6.7 log cfu ml31

(Table 1). The E. coli was

grown aerobically in static trypticase soy broth

(BBL), at 37³C for 20 h. The culture was diluted 1

in 100 in sterile demineralised water. On the 3 days

of inoculation, the pigs received 50 g per pig of feed

without egg powder containing 5 ml of the bacterial

suspension in the morning. The rest of the morning

feed was given 2 h later.

2.8. Monitoring of the faecal haemolytic E. coli

To exclude spontaneous infections the haemolytic

E. coli £ora was monitored beginning 9 days before

weaning and throughout the experiment. Rectal

swabs were taken at 1^2-day intervals and plated

semi-quantitatively on selective and non-selective

media. The faeces adhering to the swab were sus-

pended in 1.5 ml trypticase soy broth and homoge-

nised with a Vortex (model G-560E Merck ABS,

Dietikon, Switzerland). The suspension was inocu-

lated onto double layer blood agar without antibiotic

supplement, double layer blood agar with 1000 ppm

streptomycin sulfate (S-6501, Sigma) and double

layer blood agar with 100 ppm rifampicin (R-3501,

Sigma). The double layer blood agar was made of

Trypticase0 Soy Agar (BBL) with additional granu-

lated agar (11849 BBL) to a total concentration of

2.5% (w/v) to prevent swarming of Proteus bacteria.

In the double layer blood agars, sheep blood (5% v/

FEMSIM 760 27-8-97

Table 2

Time schedule of pig inoculation experiments 1 and 2

Activity Experimental day(s)

Weaning from the sow 1

Medication of feed with enro£oxacin 1^6

Adaptation to feed with egg powder 7^9

Feed with egg powder according to treatments 10^29

Bacterial inoculation during treatment 11^13

Viable counts during treatment 12^18a

Bacterial inoculation after treatment 30^32

Viable counts after treatment 31^35

a

aCounting was stopped when numbers fell below 1U10

4cfu g

31of faeces.

Table 1

Characteristics of the E. coli strains used for production of antigens for immunisation of hens and for inoculation of pigs

E. coli strain Serotypea

Fimbrial

variant

b

Toxinsc

Resistance marker Reference(s) Strain used for

107/86 O139:K12(B):H1 F18ab SLT-IIv Streptomycin [3], [24] Immunisation of hens

3064/84

STMO157:K3 :H3 F18ab STIa,

STII

Streptomycin [5] Inoculation of pigs in experiment 1

8199RIF

O141ab:K3 :H4 F18ac STIa,

STII

Rifampicin [3], [5] Immunisation of hens and inoculation

of pigs in experiment 2

aWHO Escherichia and Klebsiella Center, Statens Seruminstitute, Copenhagen, Denmark.

bAgglutination, immuno£uorescence and PCR.

cPCR.

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161156

v) was contained in the upper layer only to improve

perception of haemolysis.

If present, three colonies with haemolysis from the

agar without supplement were subcultured on plain

blood agar, blood agar with streptomycin sulfate and

blood agar with rifampicin. Additional con¢rmation

was obtained from slide agglutinations of one colony

per sample in the sera O157 and O141ab. Haemolytic

isolates di¡erent from inoculated strains were further

examined for serotype including ¢mbriae.

Viable counts on faeces were made beginning on

day 12 and day 31, respectively, and continued until

the count of the inoculated bacteria was below

1U10

4cfu g

31(Table 2). Faeces were collected

once a day and counts done on the appropriate se-

lective medium with the drop count method [5].

2.9. Statistical analysis

One-way analysis of variance and pairwise mean

comparisons (Sche¡eè test [20]) were realised at the

0.05 signi¢cance level.

For these analyses, the viable counts on days 2^5

after the ¢rst challenge inoculation and on days 4

and 5 after the re-challenge inoculation were com-

pared with the counts in the treatments without egg

powder. The reason not to include the ¢rst 2 days

and the ¢rst 4 days, respectively, was that on these

days the viable counts could be a¡ected by the in-

oculated bacteria. Counts lower than 2.0 log cfu g

31

could not be exactly determined, and for statistical

analysis a count of 0.5 log cfu g

31was substituted.

3. Results

3.1. Antibody contents of egg powders

ELISA titres of F18ab (107/86) and F18ac (8199)

speci¢c antibody powders are shown in Table 4. Sig-

ni¢cant cross-reaction was observed. Control egg

powder from non-immunised hens yielded titres be-

low 1 in 100.

3.2. Intake of antibodies by experimental pigs

Two experiments following the experimental de-

sign were done: the ¢rst with the challenge strain

FEMSIM 760 27-8-97

Table 3

Spontaneous intake with feed of egg powder containing antibody against ¢mbrial variants F18ab or F18ac by experimental pigs

Treatment with egg powder containing Egg powder with antibody per pig/day (g)

Experiment 1 Experiment 2

Day 10

aDay 15

bDay 10

aDay 15

b

Homologous antibody high 10.0 27.5 15.0 20.0

Homologous antibody low 2.0 5.5 3.0 3.5

Homologous antibody high 10.0 25.0 15.0 15.0

Homologous antibody low 2.0 5.0 3.0 2.5

Egg powder from non-immunised hens None None None None

No egg powder None None None None

Means of the ¢ve or six pigs per treatment are shown.

aDay before start of inoculation with E. coli.

bDay of approximately maximal colonisation with inoculated E. coli.

Table 4

Means ( þ S.D.) reciprocal ELISA titres of seven batches each of egg antibody powder against highly puri¢ed ¢mbriae F18ab and F18ac

produced by the E. coli strains 107/86 and 8199

RIFand of control egg powders from non-immunised hens

Egg powder from hens vaccinated with ELISA titre to ¢mbrial antigen

F18ab F18ac

F18ab (107/86) 27 300þ 2 900 11 700þ 1 200

F18ac (8199

RIF) 7 200þ 1 200 26 500þ 3 500

Non-vaccinated control 6 100 6 100

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161 157

E. coli 3064/84

STMwith the ¢mbrial variant F18ab

and the second with the E. coli strain 8199

RIFwith

the ¢mbrial variant F18ac.

At the start of challenge inoculation, on day 10,

mean feed intake was lower in experiment 1 than in

experiment 2, despite the higher body weights at

weaning in experiment 1. Mean feed intake did not

increase in a regular way. There were periods of

stagnation and even reduction. These periods corre-

lated sometimes, but not always, with vomiting and/

or diarrhoea with individual pigs. Feed consumption

during the critical phase of intestinal colonisation

stagnated more in experiment 2. This led to a daily

gain between days 1 and 28 of 309 g in experiment 1

and of 236 g in experiment 2. In experiment 1, the

calculated daily intake of egg powder at the start of

inoculation was 10 and 2 g per pig in high and low

treatments, respectively. In experiment 2, the corre-

sponding ¢gures were 15 and 3 g, respectively (Table

3). The intake increased during the critical period of

intestinal colonisation in experiment 1 and stagnated

in experiment 2 where a lower intake of antibody

resulted.

3.3. Clinical observations

Mild to severe diarrhoea in some pigs was ob-

served at variable intervals after weaning and in

nearly every treatment. No di¡erence was observed

in the proportion of treated and control piglets that

became diarrhoeic. Diarrhoea was not related to fae-

cal viable counts of the inoculated E. coli. In the ¢rst

experiment a high frequency of diarrhoea was ob-

served between days 33 and 35 that was not seen

in the second experiment.

3.4. Faecal shedding of inoculated bacteria during

treatment

One day after challenge, all the pigs except four

shed the inoculated strain of E. coli, which could be

identi¢ed by means of growth on the appropriate

selective medium and serologically. The pigs contin-

ued shedding the organism until day 13 after the ¢rst

inoculation in experiment 1, and day 11 in experi-

ment 2. No relationship was found between duration

of E. coli shedding and treatment. No infection with

other pathogenic E. coli occurred before and during

the two experiments.

In the ¢rst experiment involving challenge with the

F18ab-positive E. coli, the homologous treatments

with high and low antibody concentrations reduced

mean bacterial counts in the faeces by 3.6 and 3.2 log

cfu g

31, respectively, in comparison to the treatment

without egg powder (Table 5). These di¡erences were

statistically signi¢cant (P9 0.05). On the other hand,

25 g of egg powder with heterologous antibody did

not confer signi¢cant protection on day 15, at max-

imal colonisation. The highest viable counts were

observed between days 2 and 4 after the start of

inoculation (Fig. 1). In treatment groups without

signi¢cant protection, most of the pigs were colon-

ised at a level with counts above 6.7 log cfu g

31of

faeces. This level had earlier been shown to be asso-

ciated with a risk of developing oedema disease,

when a toxigenic strain was used [18]. The counts

FEMSIM 760 27-8-97

Table 5

Viable counts (log cfu g

31) of inoculated E. coli in the faeces of the experimental pigs

Treatment with egg powder containing Experiment 1 Experiment 2

During treatment

aAfter treatment

bDuring treatment

aAfter treatment

b

Homologous antibody high 5.2 (4.4^6.2)

�4.3 (2.3^5.9)

�3.6 (2.4^4.0)

�3.7 (6 2.0^5.9)

�

Homologous antibody low 5.6 (4.2^7.0)

�5.8 (4.9^6.2)

�7.7 (6.2^8.2)

n:s:1.0 (6 2.0^2.6)

n:s:

Heterologous antibody high 6.8 (6.0^8.9)

n:s:3.2 (6 2.0^4.3)

n:s:6.0 (5.6^6.3)

n:s:1.6 (6 2.0^3.2)

n:s:

Heterologous antibody low 6.7 (5.1^8.5)

n:s:2.2 (6 2.0^3.9)

n:s:7.7 (6.7^8.5)

n:s:0.7 (6 2.0^2.0)

n:s:

Egg powder from non-immunised hens 7.2 (5.8^8.5)

n:s:2.4 (6 2.0^3.3)

n:s:7.5 (6.0^8.7)

n:s:1.9 (6 2.0^2.7)

n:s:

No egg powder 8.8 (8.0^10.0) 1.3 (6 2.0^3.4) 7.3 (5.5^8.0) 1.2 (6 2.0^3.0)

Means of the ¢ve or six pigs per treatment and extreme values are shown.

aMeans of counts on days 2^5 after the start of inoculation during treatment.

bMeans of counts on days 4 and 5 after the start of inoculation after treatment.

�Signi¢cant,

n:s:not signi¢cant at the P9 0.05 level in comparison with no egg powder.

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161158

started to decline on day 5. The egg powder from

non-immunised hens resulted in counts that were by

1.6 log cfu g

31lower in comparison with the `no egg

powder' treatment. This di¡erence was not statisti-

cally signi¢cant.

In the second experiment involving challenge with

the F18ac-positive E. coli, the treatment with a high

homologous antibody concentration was the only

treatment bringing about a reduction of bacterial

numbers that was statistically signi¢cant (P9 0.05).

The di¡erences for the rest of the treatments were

not signi¢cant (Table 5).

Pigs fed the high concentration of heterologous

antibody had a somewhat reduced excretion, and

in experiment 2 mean counts did not exceed the crit-

ical level of 6.7 log cfu g

31.

Viable counts from the `no egg powder' treatment

were 1^2 log cfu g

31lower than counts from the

corresponding treatment in experiment 1 and equal

to the `control egg powder' in experiment 2.

3.5. Faecal shedding of inoculated bacteria subsequent

to re-challenge after treatment

Pigs which were protected during treatment

against intestinal colonisation shed higher bacterial

FEMSIM 760 27-8-97

Fig. 1. Means ( þ S.E.M.) of the faecal viable counts of inoculated bacteria beginning with the ¢rst day of inoculation (day 0) with E. coli

F18ab (experiment 1: A,B) and E. coli F18ac (experiment 2: C,D). Two treatments each with ¢ve or six pigs per treatment are shown:

egg powder with homologous antibody high (closed squares) and control without egg (open squares). Challenge and viable counts were

done during egg powder treatment (A,C), and 14 days later after termination of the treatment (B,D).

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161 159

numbers following repeated challenge after termina-

tion of egg powder application. In experiment 1,

`homologous high' and `homologous low' yielded

the highest viable counts with a statistically signi¢-

cant di¡erence compared with the `control without

egg' (Table 5). In contrast, with control pigs without

egg powder bacterial numbers subsequent to termi-

nation of the egg powder treatment were the lowest.

In experiment 2 the pigs treated with the high level

of homologous antibodies showed the highest excre-

tion after termination of egg powder treatment,

being statistically signi¢cant in comparison with the

`control without egg' group.

In both experiments, subsequent to the re-chal-

lenge after egg powder treatment the level of faecal

shedding associated with a risk of disease was not

attained by any pig, and the viable counts declined

already from day 3 on.

4. Discussion

The present study con¢rms earlier reports on the

e¤cacy of chicken egg yolk antibodies for reduction

of intestinal colonisation by ETEC in weaned pigs

[14^16]. Full protection was achieved against strains

with the same antigenic variant of ¢mbriae F18, even

in experiment 1 where the ¢mbrial vaccine was pro-

duced with a non-enterotoxigenic strain belonging to

a di¡erent serogroup.

In agreement with Yokoyama and coworkers [9],

who studied puri¢ed antibodies in neonatal pigs, the

e¡ect was dose-dependent. In the present experiment,

a daily intake of 5.5 g of egg powder was protective,

whereas 3.5 g was insu¤cient for homologous pro-

tection. Feed intake immediately after weaning is

usually low, and the increase is irregular. Inclusion

in the feed of antibody-containing egg powder at a

constant ratio requires considerable overdosage. In

the experiments performed by Imberechts and cow-

orkers [16], a constant daily dose of 30 g of egg

powder was used (H. Imberechts, personal commu-

nication). This mode of application avoids overdos-

age at times when the pigs eat the full ration.

Actively acquired local anti-¢mbrial immunity has

been shown to be cross-protective against the other

antigenic variant of ¢mbriae F18 [5], and Imberechts

and coworkers [16] have observed such cross-protec-

tion with a high dose of anti-¢mbrial egg antibody.

In the present investigation, the intake of antibody

was most probably too low. Comparability of exper-

imental results as well as of prospective commercial

products su¡ers from a lack of standardisation of

antibody contents. The present data do not exclude

a colonisation-reducing e¡ect of egg powder from

non-immunised hens at the highest dose of 25 g (ex-

periment 1). However, no such e¡ect was reported

by Erhard and Loësch [15] or by Imberechts et al. [16]

despite a similar or higher dose of control egg. Com-

ponents of chicken egg such as lysozyme [21] and

ovotransferrin [22] display anti-bacterial e¡ects.

The latter may become more marked if higher doses

are used. Our data preclude conclusions regarding

the feed value of dried egg, because the numbers of

pigs per treatment were too small and the individual

weights were not well balanced.

In the porcine species, weaning precipitates enteric

infections due to the withdrawal of the sows' milk

which is rich in antibody produced locally in the

mammary gland [23]. The suppression of local

build-up of immunity was also demonstrated in the

present investigation. After termination of treatment

and re-challenge, mean faecal viable counts of the

inoculated bacteria were in inverse proportion to

the counts obtained during antibody treatment.

This relationship gives indirect evidence that bacte-

rial numbers in the faeces at least statistically re£ect

the level of small intestinal colonisation. Thanks to

incomplete colonisation inhibition during egg anti-

body treatment, bacterial numbers did not reach crit-

ical levels after withdrawal of the egg powder. How-

ever, ¢eld tests without experimental inoculation are

indispensable to see if in-feed egg antibodies interfere

with the natural spread of the pathogen through a

population at risk.

In conclusion, oral application of egg antibodies

against enteric pathogens o¡ers a promising ap-

proach for the prevention of infectious diseases of

the digestive tract.

Acknowledgments

This work was supported by Lohmann-LTE, Cux-

haven, Germany. The examination by PCR of the

bacterial strains by Hein Imberechts, Brussels, Bel-

FEMSIM 760 27-8-97

A. Zuènìiga et al. / FEMS Immunology and Medical Microbiology 18 (1997) 153^161160

gium, and blending of the egg powders with the feed

by Ufamed, Sursee, Switzerland, are acknowledged.

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