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Key words: Campylobacter jejuni, Phagocytosis, Inflammatory response. Campylobacter ...... Campylobacter jejuni and Campylobacter fetus subsp. fetus. Infect.
Microbiol.

Inflammatory

and

Phagocytic

Experimental

Campylobacter

Pedro L. Pancorbol, Aurelia M. Gallego2, and Gerardo Alvarez de Cienfuegos*,1 1

Response

Immunol.,

1994

to

jejuni Infection

Manuel

38(2), 89-95,

de Pablo1, Carmen

in Mice

Alvarez1, Elena Ortega1,

Department of Microbiology, Faculty of Experimental Sciences, University of Jaen, 23071, Jaên, Spain, and of Clinic Laboratory, Hospital "Ciudad de Jaen", 23007, Jaen, Spain

2Service

Received

June

21, 1993;

in revised

form,

November

1, 1993.

Accepted

November

10, 1993

Abstract: After intraperitoneal inoculation with Campylobacter jejuni BALB/c, Swiss and DBA mice show a peritoneal inflammatory response of different intensity. Only BALB/c mice have a strong peritoneal response. Simultaneous intraperitoneal inoculation of C. jejuni plus FeCl3 increase both inflammatory response and phagocytic activity in Swiss mice, without production of diarrhea. Some thermostable compounds of C. jejuni have a very strong chemotactic activity against peritoneal cells of mice, whereas a diffusible, thermolabile and glutaraldehyde-resistant factor has an inhibitory effect over murine peritoneal cell phagocytosis. Bactericidal activity of peritoneal cells increased after in vitro re-challenge with C. jejuni. Bacteremia is present in all the mice strains tested, but the clearance is quick in DBA and slow in BALB/c and Swiss mice. These experiments confirm that in mice, peritoneal non-specific mechanisms of defense, such as macrophages, play an important role in order to control C. jejuni infection. Key words: Campylobacter jejuni, Phagocytosis, Inflammatory

response

contribute to killing C. jejuni (22), although isolates from extraintestinal places show greater serum resistance than gastrointestinal isolates (9). Kiehlbauch et al (16) performed in vitro studies showing that C. jejuni is readily internalized by mononuclear phagocytes but it survives better inside monocytes or macrophages than in control

Campylobacter jejuni is a major cause of human diarrheal disease throughout the world. Man is usually infected from unpasteurized milk, raw or partially cooked foods, contaminated water (28), and by contact with animal carriers (8). The clinical presentation of infection is variable, from watery diarrhea to bloody diarrhea. Fever, abdominal pain and vomiting may appear. However, extraintestinal infection including meningitis (15), cholecystitis (13), urinary tract infections (11) and bacteremia (26) have been reported. Invasiveness and production of toxins are two potential virulence factors (5, 18). Although there is not universally accepted small animal model, mice have been used to produce an experimental infection by several authors, both via oral (6, 7, 14, 29) and via intraperitoneal (3, 19, 24). It has been established that mice are a suitable model to study the immune mechanism to prevent the illness after inoculation. C. jejuni isolates were generally susceptible to the bactericidal activity in normal human serum by both antibody and complement. Moreover, classical and alternative pathways may

preparations without phagocytes. Banfi et al (2) in their experimental model with guinea pigs reported high rate of survival of C. jejuni phagocytosed by macrophages. Also a recent study (27) shows high survival of C. jejuni inside human neutrophils. Other authors, on the contrary, described that normal levels of serum complement are not essential for host defense against C. jejuni infection in mice, and that macrophages are an important factor in the defense against Campylobacter infection (3). The purpose of this study is to examine the cellular defense mechanism that prevents intraperitoneal C. jejuni infection in mice, such as peritoneal inflammatory response and local phagocytes activation. Abbreviations:

* Address Cienfuegos, imental

correspondence Department

Sciences,

to

Dr . Gerardo

of Microbiology,

University

of Jaen,

Alvarez

Faculty 23071,

Jaen,

HBSS,

de

of Exper-

ly; LPS,

Spain.

saline;

89

Hanks

FCS, balanced

fetal

calf

salt

solution;

lipopolysaccharide; TSA,

Trypticase-soy

serum; PBS,

agar.

GC,

Gonococcus;

i. p., intraperitonealphosphate-buffered

90

P.L.

PANCORBO

ET AL

C.

Materials

and

Methods

jejuni

experimental

peritoneal

leukocytic

jejuni

Bacterial strain. C jejuni strain 38309-87 was isolated from a Mexican patient and kindly provided by Dr. G.M. Ruiz Palacios (Instituto Nacional de Nutricion, Mexico D.F.). Mice. For experimental infection Swiss, DBA and BALB/c male mice weighing 20-25 g were used. Mice received standard food and water ad libitum . Preparation jejuni

of

was

(TSA)

grown

(Difco)

under

the

inocula.

at

42

C

in

with

System).

The

bacteria

finally

PBS

by

adjusted

units

(CFU)

The

bacterial

checked

per

10%

were

obtained

ml

at

using

108

viable

on

blood. by

and

supplemented

of

serial

dilution

Miles

and

Misra

c mice ml

groups.

were

(107

mice

in

ml

CFU)

i.p.

and

bacterial

The

PBS.

BALB/c

last

bacterial

washed

3 times pellet

The LB/c

for with was

mice

were

Ten

to

into

CFU

the

Gonococcus

was hr

and

natants

under

were

filtered

membrane,

and

inoculated

with

VI:

Control

i.p.

inoculated

in

group. with

2

800 •~

through

ml

0.1

with

the

of

viable

age.

The

g

for

of

20

washed 2

pension

at in

0.22 ƒÊm

BALB/c

that

of

total

cells

following

functional

of

infected

i.p.

mice.

infected 10%

cell

assay

status

One

tube

at

with

C

at

were

dilutions

on and

Measure of the peritoneal leukocytic response to

200 •~

of

400

percent-

calculated

of

starting

was HBSS

and

distilled to

above.

CFU hr.

of After and

C. jejuni

in

10-fold with

aid

a

the

(non-associated

hr

at

The

shaken

4

collected

plating

48

double

number

107 for

viable

for

in

supplemented

tubes by

C.

from

incubated

CO2

conditions.

viable

cells

containing

supplemented

Intraphagocytic

the of

supplemented

was

g,

analyze

peritoneal

of

5%

enumerated

in

to

HBSS

FCS

centrifuge

TSA

vigorously

scribed

more

number

made

in

1 ml

incubating

resuspended

jejuni

ml

under

microaerophilic

C.

as

The

supernatant

of

bacteria)

cytolysis.

or least

phagocytes

FCS

37 the

supernatant

were

3

The

activity of peritoneal

mice

heat-inactivated

was

at washed

at

expressed

was

heat-inactivated

jejuni

blood

sus-

stain.

in

peritoneal

BALB/c

culture

centrifugated

PBS.

with

was

dish

percentage.

The

V:

i.p.

those

Assay of the bactericidal

jejuni

each

were Giemsa

mouse

the

monolayers

incubation

with

and

was

albicans

After

cells.

per

percent-

and

counted

adherent

cells

this

is,

number

as

For

monolayers

were

The

mouse

cell

ml.

non-

subtract-

percentage.

HBSS,

stained

hr,

the

per

adherent

the

adherent

incubation,

super-

and

cells

of

supernatant. and

fixed

cells

per

1 hr,

to

Candida

yeast

2

by

cells

heat-dead

included

cells.

C.

for

of

for

phagocytic

with

were

PBS,

expressed

fresh

5 •~108

CO2

yeasts

PBS.

Millipore mice

with ml

and

the

cells After

counted.

obtained

this

viable

for

was

from

phagocytosis

received

800 •~

culture

min,

starting

and

dishes.

and

adherent

cells

test,

106

CO2

non-adherent

incubated

10%

Viable

2 •~

5%

were

with

exclusion

removed

of

cells

petri

under

cells

number

were

C

C

of

cells

blue

salt

withdrawn

(FCS).

plastic

were

number

of

of

ml

37

was

containing

adherent

assay

at

inoculated The

at

of

with

was

condition

a

DBA ml

of

suspension.

bacteria-free

Swiss,

ml

0.1

were

BALB/c of

ml

ml

(Difco).

immediately 0.1

0.1

Two

with

jejuni

broth

at

of

suspension

microaerophilic

centrifuged

ml

centrifugation

C.

(GC)

incubated

2

trypan

into

cells

ing

min g 3

the ml)

serum

then

balanced

fluid

a

mice

and

Hanks

supplemented

calf

(2

to

Briefly,

peritoneal

HBSS

distributed

adherent

of

the

fetal by

suspension

from

800 •~

fresh

counted

age

0.1

treated

The

inoculated

of

mg/

60

with

IV:

bacterial

twenty

for

in i.p.

hr.

by

glutaraldehyde-treated

24

were

consecutive

adjusted

at

resuspended i.p.

of C

were

12

PBS

(10

resuspended

suspension

glutaraldehyde

g.

100

suspension.

Swiss

with ml

inoculated

bacterial

adjusted

FeCl3

Two to

0.1

II:

centrifugation

was were

of

in

phagocytic

with

inoculated

heated

by

pellet

BALB/

infected

ml

III:

PBS

mice

heat-dead

2.5%

0.1

was

with

times.

with

immediately

suspension

and

suspension.

suspension.

washed

DBA

(i.p.)

bacterial

treated

bacterial and

of

water

of

Swiss,

intraperitoneally

were

ml)

I:

and

C.

according

Peritoneal

heat-inactivated

with

Experimental

BA

suspended

ml

of

experimental

dislocation

3

centrifuged,

measure

(23).

cervical with

(HBSS).

and

to

performed elsewhere

by i.p.

solution

37

(20).

of

killed

inoculated

calculated

retrospectively

for

method

g

methods.

TSA

Counts

the

800 •~

colony-forming-

were

count

horse

washed

turbidimetric

suspensions

by

with

PBS

at

were

number

into

was

described

incubation a

Microbi-

7.2,

technique

The

response

infection

was

blood in

pH

centrifugation

with

horse

harvested

(PBS)

C.

agar

(BBL

were

saline

with

of

condition system

phosphate-buffered times

culture

10%

atmospheric

microaerophilic

ology

pure

Trypticase-soy

supplemented

reduced

CampyPak

3

A

infection.

water phagocyte

serial

10%

horse

C

under

42 pellet and

was the

tube

hypotonic

and

phagocyte-adhered

was

determined

as

de-

C. JEJUNI Assay mice

of

bacteremia.

inoculated

suspension (30,

60,

120

150

The

samples

blood

plemented

with

conditions

for

presence

of

horse

48

hr C.

were

At

0.1

C.

jejuni

of

times

blood

were

retroorbital

sinus.

in in

42

BALB/c

of different

ml

the

blood C

TSA

sup-

microaerophilic

to

determine

the

jejuni. Differences

C. jejuni-treated analyzed

significance

and ml

cultivated

at

analysis.

in

controls

min)

were

viable

Statistical

0.1

CFU.

from

10%

DBA

with 107

and collected

tical

i.p.

containing

aseptically

results

Swiss,

via

INFECTION

and

the

Student's

by

was

between

mice

assigned

at

those

mean untreated

test.

Statis-

P •ƒ0.05.

IN MICE

91

The response of DBA mice to C. jejuni experimental infection presents a slow increase of all the cellular populations tested that was significant with regard to the peritoneal and adherent cell number in day 3 post-infection (Fig. 2). Little or no change was observed in the percentage of adherent cells (Fig. 4). The low rate of phagocytic cells found both in treated and in control DBA mice was remarkable (Fig. 5). None of these mice showed diarrhea after C. jejuni experimental infection. BALB/c mice showed an intense peritoneal inflammatory response with a great and quick increase in the number of peritoneal cells, but very

Results Peritoneal Inflammatory Response to C. jejuni Infection The i.p. experimental infection of C. jejuni in mice led to a different inflammatory response in the mouse strains tested. Swiss mice have an early and significant decrease in the total peritoneal cell number and a later but not significant increase that reaches the maximun in day 6 post-infection. In day 9 all the values return to control values (Fig. 1). No significant differences were found in the percentage of adherent and phagocytic peritoneal cells between infected and control Swiss mice (Figs. 4 and 5). In spite of the poor phagocytic response, none of the infected mice showed gastroenteritis signs, although this mouse race is used as an experimental model in the study of diarrhea caused by this bacterium (19).

Fig.

Peritoneal

inflammatory

after

i.p.

C.

inoculation.

cells

per

mouse

values.

1.

* P•ƒ

jejuni (mean 0.05.

of

five

response Values experiments).

in are

the Day

Swiss

mice

number 0:

Control

Fig. i.p.

Peritoneal jejuni

mouse

after cells values.

inflammatory

response

inoculation.

(mean

*P •ƒ0

Fig. of

2. C.

of

five

Values experiments).

are

the Day

in

DBA

number 0:

mice of

after

cells

Control

per

values.

.05.

3. i.p. per

Peritoneal C.

inflammatory

jejuni

mouse * * P•ƒ0.01.

inoculation. (mean

of

five

response Values experiments).

in are

BA the Day

LB/c

mice

number 0:

Control

of

92

P.L.

Fig.

4.

Percentage

strains

after

mice

treated

ments.

of i.p.

C.

with

Day

0:

transitory,

(Fig.

(Figs.

4

may

be

LB/c

mice

jejuni

an

percentages

of

results

the

show

in

this

delayed

very

days

of

BALB/c jejuni

the

treated

of

diarrheic

ing

quickly

with

high

day

4

Also

FeCl3

3). first

the

in

day

mice in

tion

and

the

have

even (Fig.

the

These

activates

the

i.p.

mice

with

cells

by

fourth

heat-dead

per

leukocyte

cells

7).

live are

day

C

mouse

This

bacteria

(Fig.

8);

after

and

is

inoculation between in

spite

of

hardly live

*P •ƒ

until

increase

predominant

BALB/c

jejuni of

led peritoneal

with

mice

bacteria

to

a

with progressive cells,

inflammatory

the

number Day

0

FeCI

3 of

0:

Control

after

response

cells

i.p.

C.

per

mouse

values.

jejuni

in

Swiss

**

inoculation.

mice

(mean * P •ƒ

of 0.001.

Values five * *

experiP•ƒ0.01.

.05.

reach-

counts

proportion than

Peritoneal

treated

showed

response,

peritoneal

caused

6.

infection.

ments).

(Fig.

of C.

P •ƒ0.05.

and

5).

against

infected

peritoneal

smaller

number

Swiss experi-

2

this,

arises, inocula-

9).

hyde-treated

five

a strong

(Fig.

treatment

via

phagocyte

Inoculation

in

107

that

of

have

infected

they

inflammatory

Adherent

active

being

great

resident

than

(Fig.

*

mean

to

illness.

treated

4•~

are

inflammatory

defense

and

post-inoculation

greater

the

a

values.

mouse Fe:

i.p.-infected

specially

activity

of

the

show

Values

different Swiss+

to

response

and

cells

4).

phagocytic

mice

FeCl3.

Control

in

factors.

are

symptoms

0:

cells

inoculation.

5 post-

response

significant

adherent

mechanisms

None

with

phagocytic jejuni

and

host

effective

C.

adherent

4 and

FeCl3-treated

(Fig.

that

treated Day

of i.p.

2

Fig.

peritoneal

after

mice

day

strong

some

counts

6).

of mice

increase

experi-

in

of

FeC13

cell

(Fig.

non-infected

a

cells

with

high

post-infection

Percentage

infection.

and

with

5.

strains

ments.

is

most

ET AL

Fig.

Swiss

five

values

by

the

treated

intense

of

percentage

5);

mouse Fe:

P•ƒ0.01.

There

caused

showed

mice

response

high

and

different

mean

control

the

intraperitoneal

Swiss show

3).

peritoneal

infection

C.

to

in

infection

BA

* *

back

phagocytic

are

values.

increase

in

Swiss+

Values

Control

coming

significant

cells inoculation.

FeC13.

post-infection

and

adherents

jejuni

PANCORBO

glutaraldeincrease reaching

a

maximum in day 4 (Fig. 7). This increase occurs essentially in adherent cells. There is a significant decrease in phagocytic cell rate maintained between the first and the fifth day (Fig. 9). In BALB/c mice inoculated i.p. with C. jejuni culture supernatant the inflammatory response shows a kinetics similar to that caused by live C. jejuni inoculation (Fig. 7), but with less adherent cell rate ( Fig. 8). Moreover, there is a decrease in the phagocytic cell rate, though more brief, only two days, than the one produced by glutaraldehydetreated bacteria ( Fig. 9).

C. JEJUNI

Fig.

7.

after

Peritoneal

i.p.

Values

inflammatory

inoculation are

of

the

experiments).

number Day

response

different of

0:

BALB/c

preparations

cells

Control

in

per

of

mouse

values.

INFECTION

mice C.

(mean

* * P•ƒ0.01.

jejuni. of *

five

IN MICE

Fig.

9.

i.p.

inoculation

Values **

P•ƒ

93

Percentage

are P•ƒ0

of of

mean

.01.*

of

phagocytic

cells

different five

in

BALB/c

preparations

experiments.

mice of

Day

0:

C.

after

Control

jejuni. values.

P•ƒ0.05.

0.05.

Table 1. Bactericidal activity of BALB/c mice peritoneal cells against C. jejuni

Fig. p. are **

8.

Percentage

inoculation

of

mean P•ƒ0

of

of .01.

different

five *

adherent

cells

in

preparations

experiments.

BALB/c of

Day

0:

mice

C.

jejuni.

Control

after

i.

Values values.

P•ƒ0.05.

Survival of C. jejuni in the Peritoneal Cavity None of the mice inoculated i.p. with 107CFU of C. jejuni showed viable bacteria in the peritoneal cavity 24 hr after inoculation. This fact shows the efficacy of control mechanisms against i.p. C. jejuni infection. Bactericidal Activity of Peritoneal Cells The bactericidal activity of murine peritoneal cells after a second challenge with C. jejuni is given in Table 1. Peritoneal leukocytes after a contact with the bacteria were activated towards it and were capable to kill it on a second challenge. This effect

remains

at least

infection

but

8 days has

after

disappeared

the

first in

and day

in vivo 13

post-

infection. Bacteremia

The i.p. inoculation of C. jejuni make an early bacteremic status appear in BALB/c and Swiss mice, whereas DBA mice show a relative resistance to bacteremia. The disappearance of C. jejuni in the blood occurs slowly in BALB/c mice, whereas Swiss mice give an earlier bacteremic clearance (Table 2). The simultaneous treatment with FeC13 and the i. p. C. jejuni infection decreases the bacteremic Swiss mice number. No differences occur in bacteremia between FeC13-treated and non-treated BALB/c mice.

94

P.L.

PANCORBO

Table 2. Bacteremia after i.p. C. jejuni inoculation (10 CFU) in different strains of mice

Discussion

The use of mice as an experimental model for the study of the C. jejuni infection has been proposed by several authors (6, 19, 21, 24). In the present study we have employed 3 mouse strains; two of them have been previously used in this type of investigations (BALB/c and Swiss) but the third (DBA) has not been described as an experimental model to C. jejuni infection. The results obtained show differences between mouse strains in the peritoneal response to infection. BA LB/c mice exhibit a more intense inflammatory peritoneal response, together with the greatest stimulation of the phagocytic capacity of the peritoneal cells. BALB/c mice may be a suitable model to study the role of macrophages in the control of C. jejuni infection. Moreover, BALB/c mice have the advantage of being genetically well known. None of the mice tested showed either symptoms of illness or diarrhea after i.p. inoculation of C. jejuni. This proves the resistance of adult mice to this infection (6, 19, 29). Although Fe3+ is considered as a stimulating factor of bacterial infective capacity (1, 10), Swiss mice treated with FeC13 and simultaneously inoculated with C. jejuni showed no diarrhea, contrary to the reports of McCardell et al (19) in Swiss mice and Stanfield et al (24) in BA LB/c mice, with infective doses of 105 to 1010 CFU of C. jejuni using the same via of inoculation. The smaller infective dose and the absence of virulence factors in our bacterial strain may be the cause of these differences. In our model treatment with FeCl3 produced an increase in the peritoneal inflammatory response and in the activity of peritoneal phagocytes that may explain the control of the infection in the peritoneal cavity. The high rate of peritoneal adherent cells, macrophages and polymorphonuclears found in BA LB/c mice inoculated with heat-dead C. jejuni shows a chemotactic effect on the phagocytic cells

ET AL

by some thermostable bacterial compound. This compound may be the lipopolysaccharide (LPS) used in the thermostable antigen typing scheme (12). This chemotactic effect was described by Ullmann and Krause (25) both in a suspension of living bacteria and culture supernatants. These authors proposed enterotoxin as the chemotactic factor of C. jejuni, but enterotoxin is thermolabile (17) and our data do not support the possibility that enterotoxin is the chemotactic factor. Moreover, the smaller peritoneal inflammatory response induced by living bacteria shows the existence of compounds in the bacterial surface with capacity to inhibit this response which would otherwise become inactivated by heat treatment, thus allowing for thermostable compounds (LPS) to exert their potent chemotactic effect. The inoculation of glutaraldehyde-treated bacteria gave a more delayed and less intense peritoneal inflammatory response, suggesting the lack of chemotactic factors. Moreover, the early decrease in the rate of phagocytic cells can be due to the existence of a bacterial phagocytosis inhibitory factor just as has been reported by some authors (2). Supernatants of bacterial culture produced also the decrease of the rate of phagocytes but more transitory, perhaps due to dilution of the factor in the medium. According to our results C. jejuni may present a diffusible, thermolabile and glutaraldehyde-resistant factor inhibitory of the phagocytic activity. The resistance of mice against C. jejuni infection reported by several authors (6, 19) may be due to the existence of non-specific mechanisms of defense. The role that polymorphonuclears and macrophages play in defense against C. jejuni infection has been reported in both in vitro (2, 16, 27) and in vivo (3) assays. Our results show the important role of phagocytes in the infection of C. jejuni in mice. Twenty-four hr after the inoculation there are no viable bacteria in the peritoneal cavity. Moreover, the phagocytic activity increased after a second challenge, but the phenomenon of in vitro intracellular bacterial survival reporter by Kiehlbauch et al (16) for non-activated murine macrophages was not detected. The presence of bacteremia in mice after the infection with C. jejuni has been reported both via i.p. (7, 24) and oral (6). Our results in the bacteremic assay were similar to those described by Stanfield et al (24), but in our assay there are no differences between FeC13-treated and non-treated BALB/c mice. The fact that treatment with FeC13

C. JEJUNI

INFECTION

favors bacteremia in DBA mice may be due to weakness of defense mechanisms of mice, similar to other animals (4). On the contrary, in Swiss mice, the treatment with FeCl3 leads to an increase of clearance of bacteria in blood that, together with the high peritoneal phagocyte activity, points out that the treatment with FeCl3 exerts an immunostimulating effect in Swiss mice. References 1) Autenrieth, I., Hantke, K., and Heesemann, J. 1991. Immunosuppression of the host and delivery of iron to the pathogen: a possible dual role of siderophores in the pathogenesis of microbial infections. Med. Microbiol. Immunol. 180: 135-141. 2) Banfi, E., Cinco, M., and Zabucchi, G. 1986. Phagocytosis of Campylobacter jejuni and Campylobacter coli by peritoneal macrophages. J. Gen. Microbiol. 132: 2409-2412. 3) Bth.,W. 1988. Role of murine macrophages and complement in experimental Campylobacter infection. J. Med. Microbiol. 26: 55-59. 4) Bell, J.A., and Manning, D.D. 1990. Pathogenesis of Campylobacter jejuni in intraperitoneally or intravenously inoculated ferrets. Curr. Microbiol. 21: 47-51. 5) Black, R.E., Levine, M.M., Clements, M.L., Hughes, T. P., and Blaser, M.J. 1988. Experimental Campylobacter jejuni infection in humans. J. Infect. Dis. 157: 472-479. 6) Blaser, M.J., Duncan, D.J., Warren, G.H., and Wang, W.L.L. 1983. Experimental Campylobacter jejuni infection of adult mice. Infect. Immun. 39: 908-916. 7) Blaser, M.J., Duncan, D.J., and Smith, P.F. 1984. Pathogenesis of Campylobacter infection: clearance of bacteremia in mice. Microecol. Ther. 14: 103-108. 8) Blaser, M.J., Taylor, D.N., and Feldman, R.A. 1984. Epidemiology of Campylobacter infections, p. 143. In Butzler, J.P. (ed), Campylobacter infections in man and animals, CRC Press, Boca Raton, Florida. 9) Blaser, M.J., Perez-Perez, G.I., Smith, P.F., Patton, C., Tenover, F.C., Lastovica, A.J., and Wang, W.-I.L. 1986. Extraintestinal Campylobacter jejuni and Campylobacter coli infections: host factors and strain characteristics. J. Infect. Dis. 153: 552-559. 10) Bullen, J.J., Ward, C.G., and Rogers, H.J. 1991. The critical role of iron in some clinical infections. Eur. J. Clin. Microbiol. Infect. Dis. 10: 613-617. 11) Davies, J.S., and Penfold, J.B. 1979. Campylobacter urinary infection. Lancet 1091. 12) Dawn Mills, S., Bradbury, W.C., and Penner, J.L. 1985. Basis for serological heterogeneity of thermostable antigen of Campylobacter jejuni. Infect. Immun. 50: 284-291. 13) Drion, S., Wahlen, C., and Taziaux, P. 1988. Isolation of Campylobacter jejuni from the bile of a cholecystic patient. J. Clin. Microbiol. 26: 2193-2194. 14) Fauchere, J.L., Veron, M., Lellouch-Tubiana, A., and Pfister, A. 1985. Experimental infection of gnotobiotic

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95

mice with Campylobacter jejuni: colonization of intestine and spread to lymphoid and reticulo-endothelial organs. J. Med. Microbiol. 20: 215-224. 15) Goosens, H., Kremp, L., Boury, R., Vlaes, L., van der Borre, C., Henocque, G., Rocque, J., Alanis, G., Hemelhof, W., Macart, M., and Butzler, J.P. 1986. Nosocomial outbreak of Campylobacter jejuni meningitis in newborn infants. Lancet is 146-149. 16) Kielbauch, J.A., Albach, R.A., Baum, L.L., and Chang, K.-P. 1985. Phagocytosis of Campylobacter jejuni and its intracellular survival in mononuclear phagocytes. Infect. Immun. 48: 446-451. 17) Klipstein, F.A., and Engert, R.F. 1984. Properties of crude Campylobacter jejuni heat-labile enterotoxin. Infect. Immun. 45: 314-319. 18) Konkel, M.E., Babakhani, F., and Joens, L.A. 1990. Invasion-related antigens of Campylobacter jejuni. J. Infect. Dis. 162: 888-895. 19) McCardell, B.A., Madden, J.M., and Stanfield, J.T. 1986. A mouse model for the measurement of virulence of species of Campylobacter. J. Infect. Dis. 153: 177. 20) Miles, A.A., and Misra, S. 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38: 732-749. 21) Newel, D.G. 1984. Experimental studies of Campylobacter enteritis, p. 113. In Butzler, J.P. (ed), Campylobacter infections in man and animals, CRC Press, Boca Raton, Florida. 22) Pennie, R.A., Pearson, R.D., Barrett, L.J., Lior H., and Guerrant, R.L. 1986. Susceptibility of Campylobacter jejuni to strain-specific bactericidal activity in sera of infected patients. Infect. Immun. 52: 702-706. 23) Ruiz-Bravo, A., Jimenez-Valera, M., Alvarez de Cien fuegos, G., Ruiz, C., Kouwatli, K., and RamosCormenzana, A. 1985. Immunomodulation in mice by experimental infection with Yersinia enterocolitica. Microbiol. Immunol. 29: 1089-1097. 24) Stanfield, J.T., McCardell, B.A., and Madden, J.M., 1987. Campylobacter diarrhoea in an adult mouse model. Microb. Pathog. 3: 155-165. 25) Ullmann, U., and Krausse, R. 1987. The reaction of Campylobacter species on the chemiluminescence, chemotaxis and haemagglutination. Zbl. Bakt. Hyg. A 226: 178-190. 26) Van der Meer, J.W.M., Mouton, R.P., Daha, M.R., and Schuurman, R.K.B. 1986. Campylobacter jejuni bacteremia as a cause of recurrent fever in a patient with hypogammaglobulinaemia. J. Infect. 12: 235-239. 27) Walan, A., Dahlgren, C., Kihlstrom, E., Stendahl, O., and Lock, R. 1992. Phagocyte killing of Campylobacter jejuni in relation to oxidative activation. APMIS 100: 424-430. 28) Walker, R.I., Caldwell, M.B., Lee, E.C., Guerry, P., Trust, Ti.,. and Ruiz-Palacios, G.-M. 1986. Pathophysiology of Campylobacter enteritis. Microbiol. Rev. 50: 81-94. 29) Yrios, J.W., and Balish, E. 1986. Colonization and infection of athymic and euthymic germfree mice by Campylobacter jejuni and Campylobacter fetus subsp. fetus. Infect. Immun. 53: 378-383.