JOURNAL OF CLINICAL MICROBIOLOGY, June 1989, p. 1377-1379
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0095-1137/89/061377-03$02.00/0
Copyright © 1989, American Society for Microbiology
Virulence of Campylobacterjejuni for Chicken Embryos
SANGEETA MAHAJAN AND FRANK G. RODGERS*
Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824
Received 28 December 1988/Accepted 16 February 1989
The pathogenicity of Campylobacterjejuni was examined in chicken embryos. In this system, mortality data
and histopathological findings induced by organisms and by bacterium-free filtered broth were identical. The
absence in chicken embryo tissues both of organisms and of an inflammatory infiltrate suggests a toxin etiology.
Campylobacterjejuni has been recognized etiologic
as an
agent of human gastroenteritis with a worldwide incidence
only within the last 10 years (1). Along with rotavirus and
enterotoxigenic Escherichia coli, this gram-negative, mi-
croaerophilic, vibriolike organism appears to be one of the
most common causes of diarrhea in developing countries (2,
3, 11) and is responsible for as many cases of gastroenteritis
as are the Salmonella species.
The pathogenesis of C. jejuni-induced disease is unre-
solved. Some reports consider adherence or selective adhe-
sion along with invasiveness of the organism to be important
virulence factors (4, 5, 12, 17), while others suggest that
mucus colonization is a prelude to intestinal infections (8).
The contribution of microbial adhesins, outer membrane
proteins, siderophores, and toxins to virulence remains
poorly understood. Systemic invasion of the blood and
extraintestinal organs has been documented in humans (7),
though the occurrence is low. However, strains are uni-
formly negative in the Sereny test (9), suggesting a lack of
invasiveness. C. jejuni enteritis varies from intestinal inva-
sion, characterized by blood and mucus in the stool, to
watery diarrhea with no sign of invasion. The objective of
the present study was to investigate the pathology induced
by C. jejuni in the fertile hen's egg and evaluate the chicken
embryo as a tool to study the pathogenesis of this disease.
A C. jejuni biotype 1 strain was isolated from a fecal
sample from a patient with acute gastroenteritis. It was
passaged once on buffered charcoal-yeast extract agar sup-
plemented with L-cystine (BCYE) and subsequently main-
tained at -70°C in 10% sorbitol with 1% calf serum. As
needed, portions were thawed and grown on BCYE at 37°C
in 10% C02 and 5% 02. To harvest the organisms, bacteria
were washed from the surface of the agar and suspended in
5 ml of phosphate-buffered saline (PBS), pH 7.3, to give a
density equivalent to 109 CFU/ml, as determined by den-
sitometry. Bacterial density data were confirmed by colony
counts on BCYE. Antibiotic-free, fertile White Leghorn
hens' eggs (University of New Hampshire Poultry Farm)
were inoculated with 10-fold dilutions of C. jejuni in PBS. By
using 10 eggs per dilution, the 50% lethal dose (LD50) data
for C. jejuni were calculated (13) for the allantoic cavity and
the chorioallantoic membrane after 10 days of incubation
and for the amniotic and yolk sacs after 6 days. Ali eggs were
candled twice daily to record embryo viability. Portions (0.1
ml each) containing 10, 100, or 1,000 times the yolk sac LD50
were inoculated into the yolk sac. Embryo organs were
harvested daily and fixed in 10% buffered Formalin. The
liver, heart, kidney, spleen, and gastrointestinal tract were
*
Corresponding author.
1377
Vol. 27, No.
embedded in paraffin in a Lab-Tek tissue processor (Miles
Downloaded from http://jcm.asm.org/ on August 19, 2020 by guest
Laboratories, Inc., Naperville, 111.), sectioned, and exam-
ined after hematoxylin and eosin staining. Organs from
similarly treated eggs were removed for bacterial colony
counts. Samples were rinsed in PBS, weighed, homoge-
nized, and serially diluted, and 0.1-ml portions were plated
on BCYE. Deparaffinized tissue sections and Formalin-fixed
homogenates were incubated with rabbit anti-C. jejuni serum
and fluorescein isothiocyanate-conjugated goat anti-rabbit
serum (Organon Teknika, Malvern, Pa.).
C. jejuni cells were grown at 37°C in thioglycolate broth
for 48 h under microaerobic conditions. A bacterium-free
supernatant was obtained by centrifugation at 15,300 x g for
10 min at 4°C and filtration through a 0.22-,um-pore-size
membrane filter. The toxic activity of the filtrate was assayed
by inoculation into the yolk sac of eggs. Similar assays were
performed after the filtrate was subjected to a 15-min expo-
sure to 60°C or 100°C, changes in pH, or treatment with 0.5
mg each of trypsin, protease, and lipase per ml for 30 min.
The LD50 of C. jejuni are shown in Table 1. The inoculum
required to kill the embryo ranged from 2.1 x 102 to 8.1 x
104 CFU/ml for amniotic and allantoic sacs, respectively.
Examination of harvested embryos and chorioallantoic
membranes showed no visible lesions. LD50 results demon-
strated that inoculation of relatively small doses of organ-
isms into the immediate environment or into the nutrition
source of the embryo killed the developing chick. Examina-
tion of organs inoculated with multiple doses of the yolk sac
LDO showed massive congestion of the blood vessels, slight
tissue damage due to edema, and small amounts of hemor-
rhage (Fig. 1). The lack of cell breakdown, necrosis, consol-
idation, and infiltrative inflammatory cells was striking. The
gastrointestinal tracts of these embryos, as well as all organs
from embryos previously inoculated with PBS or bacterio-
logical media (Fig. 2), appeared normal throughout the study.
Viable count and immunofluorescence studies showed no
microorganisms within embryo organs. Inoculation of yolk
sacs with bacterium-free broth induced a pathology identical
to that induced by organisms. Samples of filtrate originally
containing 5 x 107 CFU/ml retained toxicity when diluted to
10-4 but not to 10-5. Toxicity of the filtrate for embryos was
destroyed by exposure to 60°C, 100°C, pH 3.0, pH 8.0,
trypsin, or protease but not to lipase or long-term storage at
40C.
There is no suitable animal model for the study of the
pathogenic mechanisms of C. jejuni. Studies using chickens,
mice, calves, and pigs have failed to demonstrate a repro-
ducible infection (9, 14). Our preliminary studies using one
strain of C. jejuni suggest that the chicken embryo is a useful
tool for studying the pathogenesis of this organism at the
tissue and cellular levels. Fertile hens' eggs are relatively
1378 NOTES

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FIG. 1. Histopathological tissue sections of chicken embryo organs after yolk sac inoculation. Congestion, hemorrhage, edema, and the
lack of an inflammatory infiltrate are evident (hematoxylin and eosin; magnification, x70). (a) Liver at 1 day postinoculation with
bacterium-free filtrate; (b) heart at 6 days postinoculation with 100 times the yolk sac LD50 of C. jejuni; (c) kidney at 1 day postinoculation
with bacterium-free filtrate; (d) spleen at 8 days postinoculation with the organism at 100 times the yolk sac LD50.

easy to handle and inexpensive and can be used in statisti- chicken embryos were similar for C. jejuni and broth filtrate.
cally significant numbers. The lethal activity of the filtrate for fertile eggs was abolished
LD5. studies revealed a high degree of virulence of C. by preincubation with antibody to C. jejuni. These findings,
jejuni for the chicken embryo. In contrast to the findings of together with a lack of organisms in viable counts or immu-
Field et al. (5), our studies indicate that C. jejuni causes a nofluorescence studies, suggest a toxin etiology. Our results
noninvasive fatal infection in chicken embryos, as indicated on the heat lability of the cell-free filtered broth agree with
by a lack of dissemination beyond the inoculation site. The those of Ruiz-Palacios et al. (15) and Klipstein and Engert (6)
relative paucity of C. jejuni isolates from outside the gastro- but differ from those of McCardell et al. (10), who demon-
intestinal tract (16) suggests that the organism is rarely strated toxin stability at 100°C. It is possible that different
invasive. Mortality data had histopathological findings in strains of C. jejuni possess different types of toxin. Mucus
colonization of the gut by C. jejuni has been indicated as a
major factor in pathogenicity. Lee et al. (8) showed that in a
TABLE 1. LD50 endpoints for C. jejuni inoculated mouse cecal model organisms moved freely in the mucus and
into fertile hens' eggs did not adhere to or invade the epithelial tissues. Variability
Route of inoculation LD50 endpoint' in symptoms of Campylobacter infections may be due to a
number of pathogenic mechanisms, each of which may
Allantoic .................. 9.5 x1o4 predominate in different strains, as with E. coli. The wide
Amniotic ................... 2.1 x 102
Yolk sac .................. 3.3 x 102 range in the LD., of C. jejuni strains in fertile hens' eggs (5)
Yolk sacc ................... 1.0 x 103 indicates that invasiveness alone cannot account for the
Chorioallantoic membrane ....... ............ 8.0 x 103 entire spectrum of virulence shown by the organism in this
system. Our preliminary studies suggest that the production
a Control eggs inoculated with either PBS or bacteriological media showed of toxic factors by the organism is an important virulence
no mortality. LD50 data were derived by using 60 eggs for each route.
b Endpoints were calculated from three separate experiments. (An organ- factor and that, although pathogenicity is multifactorial,
ism dose of up to 100-fold lower was able to kill some embryos, while other invasiveness may not play the most important role in the
embryos survived inoculation with a dose 100-fold higher than the LD-5. disease process.
Death of embryos occurred from 3 to 8 days postinoculation and was
inoculum concentration dependent.) We thank Arthur Tzianabos for his help and valuable suggestions.
Organisms were washed in PBS prior to inoculation into the yolk sac. This work was supported in part by the Central University
VOL. 27, 1989
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S07-RR07108-14 from the National Institutes of Health.
LITERATURE CITED
1. Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B.
Reller, and W. I. Wang. 1979. Campylobacter enteritis: clinical
and epidemiologic features. Ann. Intern. Med. 91:179-185.
2. Blaser, M. J., R. I. Glass, M. Imdadul Huq, B. Stoll, G. M.
Kibriya, and A. R. M. A. Alim. 1980. Isolation of Campylobac-
ter fetus subsp. jejuni from Bangladeshi children. J. Clin.
Microbiol. 12:744-747.
3. Calva, J. J., G. M. Ruiz-Palacios, A. B. Lopez-Vidal, A. Ramos,
and R. Bojalil. 1988. Cohort study of intestinal infection with
Campylobacter in Mexican children. Lancet i:503-506.
4. Fauchere, J. L., M. Veron, A. Lellouch-Tubiana, and A. Pfister.
1985. Experimental infection of gnotobiotic mice with Campylo-
bacter jejuni: colonization of intestine and spread to lymphoid
and reticuloendothelial organs. J. Med. Microbiol. 20:215-224.
5. Field, L. H., V. L. Headley, J. L. Underwood, S. M. Payne, and
L. J. Berry. 1986. The chicken embryo model for Campylobac-
ter invasion: comparative virulence of human isolates of C.
jejuni and C. coli. Infect. Immun. 54:118-125.
6. Klipstein, F. A., and R. F. Engert. 1984. Properties of crude
Campylobacter jejuni heat-labile enterotoxin. Infect. Immun.
45:314-319.
7. Lastovica, A. J., and J. L. Penner. 1983. Serotypes of Campylo-
bacterjejuni and C. coli in bacteremic, hospitalized children. J.
Infect. Dis. 147:592.
8. Lee, A., J. L. O'Rourke, P. J. Barrington, and T. J. Trust. 1986.
Mucus colonization as a determinant of pathogenicity in intes-
NOTES 1379
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tinal infection by Campylobacter jejuni: a mouse cecal model.
Infect. Immun. 38:536-546.
9. Manninen, K. I., J. F. Prescott, and I. R. Dohoo. 1982. Patho-
genicity of Campylobacter jejuni isolates from animals and
humans. Infect. Immun. 38:46-52.
10. McCardell, B. A., J. M. Madden, and E. C. Lee. 1984. Cam-
pylobacter jejuni and C. col-production of a cytotonic toxin
immunologically similar to cholera toxin. J. Food Prot. 47:
943-949.
11. Neogi, P. K. B., and N. S. Shahid. 1987. Serotypes of Campylo-
bacterjejuni isolated from patients attending a diarrheal disease
hospital in urban Bangladesh. J. Med. Microbiol. 24:303-307.
12. Newell, D. G., H. McBride, F. Saunders, Y. Dehele, and A. D.
Pearson. 1985. The virulence of clinical and environmental
isolates of Campylobacterjejuni. J. Hyg. 94:45-54.
13. Reed, L. J., and H. Muench. 1938. A simple method of deter-
mining 50 percent end points. Am. J. Hyg. 27:493-497.
14. Ruiz-Palacios, G. M., E. Escamilla, and N. I. Torres. 1981.
Experimental Campylobacter diarrhea in chickens. Infect. Im-
mun. 34:250-255.
15. Ruiz-Palacios, G. M., J. Torres, N. I. Torres, E. Escamilla, B.
Ruiz-Palacios, and J. Tamayo. 1983. Cholera-like enterotoxin
produced by Campylobacterjejuni: characterization and clinical
significance. Lancet ii:250-251.
16. Tauxe, R. V., N. Hargrett-Bean, C. M. Patton, and I. K.
Wachsmuth. 1988. Campylobacter isolates in the United States,
1982-1986. CDC Surveillance Summaries 37:1-13.
17. Walker, R. I., E. A. Schmauder-Chock, and J. L. Parker. 1988.
Selective association and transport of Campylobacter jejuni
through M cells of rabbit Peyer's patches. Can. J. Microbiol.
34:1142-1147.