INFECTION AND IMMUNITY, Apr. 2000, p. 2386–2389 Vol. 68, No.

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Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Induction of Emetic, Pyrexic, and Behavioral Effects of

Staphylococcus aureus Enterotoxin B in the Ferret
ANGELA WRIGHT,1 PAUL L. R. ANDREWS,2 AND RICHARD W. TITBALL1*
Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 OJQ,1
and Department of Physiology, St. George’s Hospital Medical School,
London SW17 0RE,2 United Kingdom
Received 15 November 1999/Returned for modification 14 December 1999/Accepted 3 January 2000

Ferrets which had been orally dosed with 5 mg of Staphylococcal enterotoxin B (SEB) responded with an
increase in subcutaneous temperature. At 75 min, the subcutaneous temperature was significantly higher (ⴙ
0.9°C ⴞ 0.38°C, P < 0.007) than in control animals. Animals dosed with 1 or 2 mg of SEB responded with a
small, but not significant, increase in subcutaneous temperature. All of the animals dosed with 5 mg of SEB
retched and vomited. The mean latency for the onset of retching was 105 ⴞ 36 min, and the mean latency for
the onset of vomiting was 106 ⴞ 34 min. The mean number of retches was 17.8 ⴞ 19.6, and the mean number
of vomits was 2.0 ⴞ 1.5. These findings indicate that ferrets can be used as alternatives to primates for the
study of the biological activities of SEB.

The enterotoxins produced by coagulase-positive strains of
Staphylococcus aureus are a major cause of symptoms of food
poisoning in humans (15, 20). It is the second most common
cause of food poisoning in the United States (5). The genes
encoding a number of Staphylococcal enterotoxins (SE) have
been cloned and sequenced: A (SEA), B, C123, D, E, G, H, I,
and J (11, 13, 15, 24). The toxins can be divided into two groups
with SEA and SEB representing each group. Despite this body
of molecular information, relatively little is known of the
mechanisms by which the toxins can produce the symptoms of
food poisoning, which in turn hampers the design of protective
measures and antitoxic drugs. A major factor contributing to
this lack of progress has been the lack of a small-animal model
which mimics the clinical features of SE intoxication, including
nausea, vomiting, abdominal cramping, and diarrhea. Low-
grade fever can occur in severe cases, but hypotension and
prostration are rare (10). The commonly used animal models
for studies of infection such as the mouse, rat, and rabbit are
tagonists now widely used to treat emesis induced by antican-
cer therapy, used the ferret as the emetic model (1). This
species has now become widely accepted for the study of
emetic mechanisms. The ferret is a small carnivore which re-
sponds to the full spectrum of agents known to induce emesis
in humans (8). A preliminary study reported that the ferret had
an emetic response to SEB when given intravenously, but the
oral route was not investigated (14) nor were other effects of
SEB monitored. A further advantage of the ferret for the study
of food poisoning is that the morphology and physiology of the
ferret gastrointestinal tract have many features in common
with the human gastrointestinal tract (23). The aim of the
present study was to investigate whether orally administered
SEB induced emesis, diarrhea, or pyrexia in the ferret and to
assess its potential as a model for studying the mechanism of
action of SEB-induced food poisoning.
Experiments were performed with adult female ferrets
(Mustela putorius furo L.; Marshall Farms, Inc., New York,

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excluded as suitable models, since they do not display an
emetic reflex. Previous animal studies have focused on the
emetic and related effects of the SEB group, although there is
less information on the SEA group. This study confined itself
to the SEB group. In the rhesus monkey, vomiting can be
induced by intravenous or intragastric (3, 17, 18) administra-
tion of SEB. In the cynomologous monkey, emesis and diar-
rhea have been reported following the intraduodenal admin-
istration of SEB (12). Intravenous administration of SEB
induced emesis in the cat, but intragastric administration cu-
riously did not (19), although pyrexia was induced via both
routes (19). Whilst nonhuman primates would appear to be the
most appropriate model, such studies are severely restricted by
the availability of animals and ethical considerations. In the
past 20 years there has been a general resurgence of interest in
emetic mechanisms because of the clinical requirement for
drugs to block the severe emetic effects of the drugs and radi-
ation used to treat cancer. These studies, which led to the
identification of selective 5-hydroxytryptamine-3 receptor an-
* Corresponding author. Mailing address: Defence Evaluation and
Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ,
United Kingdom. Phone: 44 (0)1980 613301. Fax: 44 (0)1980 613284.
E-mail: RTITBALL@dera.gov.uk.
N.Y.) with a mean body weight of 735 g (standard error of the
mean [SEM], 41 g; n ⫽ 24). They were not in estrus. SEB,
which had been purified from culture supernatant of S. aureus
S6 (Centre for Applied Microbiology and Research, Porton
Down, Wiltshire, United Kingdom) and which was greater
than 95% pure by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was used in these studies. Prior to dosing with
SEB, animals were deprived of food for 24 h but were allowed
free access to water. SEB was given to groups of five animals at
doses of 1, 2, or 5 mg into the stomach via an oral dosing tube.
The SEB was given in 10 ml of sterile 154 mM NaCl which was
also used for control experiments. Following dosing with either
saline or SEB in saline, changes in subcutaneous body temper-
ature and activity and the incidence of retching, vomiting, and
defecation were monitored over a period of 3 h by trained
observers. All values are expressed as means ⫾ SEMs. Statis-
tical comparisons were made by using unpaired sample t tests.
Activity was assessed every 5 min and was scored by using a
truncated scale of 1 to 5 (1, inactive or apparently sleeping; 2,
grooming; 3, low-level of intermittent activity; 4, medium level
of continuous activity; and 5, high level of intense continuous
activity). The scale was derived from observation of animals
under identical conditions a week prior to experimentation. In
the saline-dosed animals, there was a progressive decline in

2386
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FIG. 1. (a) Change in activity, on a scale from 1 to 5, in ferrets dosed with 1, 2, or 5 mg of SEB in comparison with control animals. The temperature of control
animals or animals dosed with 1, 2, or 5 mg of SEB is recorded in panel b. 夹, P ⬍ .01 compared to control.

activity over the first 90 min, after which it plateaued (Fig. 1a). the course of the experiment was similar to that seen in control
Although at individual times the SEB-treated animals ap- animals (Fig. 1a).
peared to have elevated levels of activity, overall this was not Fourteen days prior to experimentation, each experimental
statistically significant, and the overall decline in activity during animal was anesthetized with halothane-oxygen and a DAS5002
2388 NOTES
TABLE 1. Emetic, retching, and diarrheal responses of ferrets orally dosed with SEB
Dose of SEB No. of animals Retching
(mg) per group No. a
Mean b
None 5 0 NAe
1 5 2 8.8 ⫾ 10.6
2 5 1 10
5 4 4 17.8 ⫾ 19.6
a
Number of animals in each group showing this response.
b
Mean number of episodes (⫾ SEM).
c
Mean time (min) for response (⫾ SEM).
d
Mean score from 1 (normal) to 3 (fully mucoid).
e
NA, not applicable.
identity and temperature transponder (Plexx BV, Elst, The
Netherlands) was implanted under the dorsal skin at the junc-
tion between the thorax and the abdomen. During experi-
ments, the subcutaneous interscapular temperature was mea-
sured every 15 min by using an external receiver. The ambient
temperature for the experiments was 20°C. The subcutaneous
interscapular temperature showed a progressive decline over
the observation period in animals dosed with saline alone (Fig.
1b). This appeared to mirror the progressive decrease in the
activity score (see above and Fig. 1a). Following administration
of 5 mg of SEB, the subcutaneous temperature began to in-
crease after 45 min, reaching a peak between 60 and 75 min. At
75 min, the temperature in animals treated with 5 mg of SEB
was significantly higher (⫹ 0.9°C ⫾ 0.38°C, P ⬍ 0.007) than in
control animals. Animals dosed with 1 or 2 mg of SEB showed
a small, but not significant, increase in temperature over the
course of the experiment in comparison with control animals.
Retching and vomiting were quantified separately. Retching
was defined as the number of forceful rhythmic abdominal
contractions occurring with the animal in a chracteristic pos-
ture not resulting in expulsion of upper gastrointestinal tract
contents (22). Vomiting was defined as the forceful oral expul-
sion of upper gastrointestinal contents (22). The time of oc-
currence of each episode of retching and vomiting was noted
(Table 1). Retching and vomiting were not induced by saline in
any animal. Two out of five animals dosed with 1 mg of SEB
retched, and one of the five animals vomited. The latency of
retching was 120 and 143 min, and the mean number of retches
in the group was 8.8 ⫾ 10.6. A 2-mg dose of SEB induced
retching and vomiting in one animal which vomited eight times
and retched 50 times beginning at 160 min after dosing. Retch-
ing and vomiting was induced in all four animals tested by
using a 5-mg dose of SEB. The mean latency was 105 ⫾ 36 min
for the onset of retching and was 106 ⫾ 34 min for the onset of
vomiting (n ⫽ 4), and the mean number of retches was 17.8 ⫾
19.6 and the mean number of vomits was 2.0 ⫾ 1.5 (n ⫽ 4).
Ferrets usually defecate in one corner of their cage, and this
facilitated monitoring. As episodes of defecation are relatively
rare (see below), attention focused on the nature of the feces.
Each stool was scored as follows: 1, normal consistency and
color; 2, semisolid/partially mucoid; and 3, fully mucoid. In the
control, animals that received 1 or 2 mg of SEB and defecated
had only single episodes of defecation (Table 1), and out of
nine episodes in 15 animals, eight episodes scored 1 and the
remaining episode scored 2. In contrast, in the four animals of
the group that received 5 mg of SEB there were 10 episodes of
defecation with one scoring 1, three scoring 2, and six scoring
3. Thus, the highest dose of SEB dramatically changed the
nature of the feces produced from the normal firm type to
almost purely mucoid feces which closer inspection revealed
were either transparent or yellow/orange stained, probably in-
INFECT. IMMUN.
Vomiting Defecation
Latency c
No. a
Mean b c
Latency No. a
Appearanced
NA 0 NA NA 2 1.5
132 1 4 150 4 1
160 1 8 160 2 1
105 ⫾ 36 4 2 ⫾ 1.5 106 ⫾ 34 10 2.5
dicating the presence of bile. Similar mucoid feces have been
reported in ferrets exposed to total body radiation (7).
The dose of SEB required to cause emesis in monkeys after
intragastric dosing has been reported to be in the range of 5 to
1,000 ␮g/kg (2–4, 18), although most reports suggest the emetic
dose is between 5 and 50 ␮g/kg. In the ferret, the dose of SEB
required to elicit emesis is higher than that required to elicit it
in primates. The reason for the apparent reduced susceptibility
of ferrets to SEB is not clear. Sagrada et al. (14) have reported
that doses of 30 ␮g of SEB per kg given by the intravenous
route to ferrets reproducibly caused retching and emesis in
ferrets 45 min after administration. Taken together with our
results, this suggests that ferrets are more susceptible to SEB
given by the intravenous route. Receptors for SEB in the gut
have not been identified, but it seems likely that such receptors
do exist and that differences in the density of these receptors
might explain the different sensitivities to SEB. Alternatively, it
is possible that SEB is degraded more efficiently in the ferret
gut than in the gut of humans or other primates. The degra-
dation of SEB in the ferret gut might explain why a 5-mg/kg
dose appeared to reach critical threshold for the induction of
emesis in all tested animals. Further studies with SEB codosed
with protease inhibitors or inhibitors of gastric acid secretion,
or in foodstuffs, might allow this question to be addressed.
Previous workers have shown a febrile response (an increase
in core temperature of ⬎0.9°C) develops after the intravenous
administration of 0.1 ␮g of SEA per kg into rabbits (6, 9)
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within 5 h. It was suggested that the pyrexia was due to the
release of interferons, tumor necrosis factor, and interleukin-2
(6). Similarly, the intravenous administration of SE in cats (9)
or in goats (21) resulted in a pyrexic response. In murine
models of disease, the effects of SE on body temperature
appear to be more variable. Huang et al. (6) reported that the
intravenous administration of 0.3 ␮g of SEA per kg into C3H/
HeJ strain (endotoxin-resistant) mice resulted in a 1.15°C tem-
perature increase, whereas Stiles et al. (16) have reported that
the intraperitoneal administration of a mixture of endotoxin
and up to 10 ␮g of SEA, SEB, or SEC3 into BALB/c or
C57BL/6 mice resulted in a decline in body temperature over
a period of 12 h (16). In the latter study, the oral administra-
tion of endotoxin/enterotoxin did not elicit any change in body
temperature. To our knowledge, the experiments in ferrets
that we have reported here are the first describing a pyrexic
effect after the oral delivery of an SE.
The quantities of SEB which reliably elicit emetic, thermo-
regulatory, and diarrheal responses in the ferret are small
enough to allow the use of this model to explore the molecular
basis of SEB activity without the use of primates. Previous
studies with site-directed mutated forms of SEB in primates
have suggested that the emetic activity of SEB may not be
related to the ability of SEB to cause T-cell mitogenicity (4).
VOL. 68, 2000
However, the low numbers of animals used in this study makes
it difficult to draw firm conclusions. The finding that the ferret
responds to orally delivered SEB will also permit work to
elucidate the site and mode of action of SEB.
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