EXPERIMENTAL PARASITOLOGY 14, 23-28 (1963)

Effect of Certain Antihistamine and Antiserotonin

Agents upon Experimental Trichinosis in Mice

William C. Campbell, Robert K. Hartman, and Ashton C. Cuckler

Merck Institute for Therapeutic Research, Rahway, New Jersey

(Submitted for publication, 21 September 1962)

Diets containing an antihistamine drug, 2- [P-chloro-a- (2-dimethylamino-ethyl) benzyl]
pyridine maleate (chlorpheniramine maleate), or an antiserotonin drug, l-benzyl-l-
methyl-J-methoxytryptamine (B.A.%), or a drug possessing both properties, I-methyl-4-
(J-dibenxo-[a,el-cycloheptatrienylidine)-piperidine hydrochloride monohydrate (cypro-
heptadine), were fed to mice during the acute intestinal phase of experimental trichinosis.
In each instance the number of worms remaining in the small intestine on days 17 and 21
of infection was greater in the treated than in the untreated mice. It is suggested that
the cellular reaction responsible for the expulsion of Trichinella from the gut may be
mediated, to some extent, by histamine.

Since the early phase of severe trichinosis
is accompanied by various phenomena sug-
gestive of hypersensitivity, it was thought
to be of interest to administer to Trichinella-
infected mice a drug which might be expected
to exert a suppressive action on a wide vari-
ety of hypersensitive states. The drug used
was 1-methyl-4- (5dibenzo- [ a,e] -cyclohepta-
trienylidine)-piperidine hydrochloride mono-
hydrate (generic name: cyproheptadine) .
This compound is unusual in that it possesses
both antihistamine and antiserotonin proper-
ties (Stone et al., 1961). When cyprohepta-
dine was found to have a definite effect on
the intestinal phase of the disease, two other
drugs, one having antihistamine activity, the
other having antiserotonin activity, were
similarly tested. Data obtained with all three
drugs are presented in this report.
MATERIALS AND METHODS
Male albino mice were used; they were
approximately 5 weeks of age and weighed
20-25 gm at the time of exposure to TriGhi-
nella. Nontreated mice were fed a diet of
commercial mouse chow. Medicated diets
23
were prepared by adding drugs to finely
ground mouse chow and mixing thoroughly.
Medicated and nonmedicated diets were pro-
vided ad libitum.
Mice were exposed to T. spiralis larvae
harvested from other mice by peptic diges-
tion and washed once by sedimentation in
saline. The concentration of larvae in a
freshly-digested suspension was estimated by
a sampling technique, the aliquots being with-
drawn while the suspension was stirred. The
concentration of larvae was adjusted so that
the requisite number of larvae could be given
to mice in volumes of 0.5 or 1.0 ml. The
larvae were introduced into the stomach of
the mice by means of a syringe and dosing
needle; the suspens&n was stirred while the
inocula were withdrawn.
The following method was used to estimate
the average number of adult Trichinella per
mouse. The mice of a group or subgroup were
killed, and their small intestines were slit
open and cut into lengths of a few centi-
meters. These fragments were incubated at
37’C for about 1 hour in saline. Sufficient
0,40J0 NaOH was then added to give a final
24 CAMPBELL, HARTMAN, AND CUCKLER

concentration of approximately 0.04%, and ginning on day 7 of infection and continuing

the mixture was refrigerated overnight. The until the end of the experiment. The second
intestinal fragments were washed on a 10 group was held as untreated controls. It had
mesh wire screen, the worms being washed been planned to kill mice from each group at
through by spraying the preparation with intervals in order to count the adult worms,
strong jets of tap water. About a liter of tap but heavy mortality made this impracticable.
water was used for every 10 intestines. The In the treated group 3 mice died on day 8,
filtrate was then passed through a 200 mesh 8 died on day 9, and all 30 were dead on day
screen,which retained the worms. The worms 18 of infection. In the control group, the first
were washed off the screen, collected, and death occurred on day 7, but 12 (40%) of
counted by repeatedly sampling the stirred the mice were still alive after 5 weeks. A
suspensionof worms. condensedrecord of mortality for both groups
To estimate the number of larvae in the is given in Table I. In an ancillary experi-
musclesof mice, the skinned carcassesof each ment the feeding of diet containing 0.025%
group were eviscerated and ground twice in cyproheptadine to noninfected mice caused
a meat grinder. The ground meat was then a slight depression in weight gain but no
stirred and a 15 gm sample taken for diges- mortality.
tion. Each sample was digested at 37°C for TABLE I
Mortality of Mice in Experiment la
4-5 hours in 0.7% pepsin, containing 1.0%
HCI. The digest was filtered through gauze, Cumulative number
allowed to stand, and brought to a volume of deaths at
convenient for counting. The larvae were Number Weeks after infection
counted in aliquots which were removed while Treatment exposed 2 3 4 5
the larval suspensionwas stirred. None 30 10 13 18 18
Many investigators have drawn attention Cyproheptadine
to the great variation in parasite numbers 0.025% 30 2.5 30 - -
encountered among individuals of a given a Mice in both groups infected with Trickinelh.
host strain exposed to T. spiralis. Becauseof
In the second experiment a group of 50
this variation, emphasis was placed on the
mice was fed a diet containing 0.025%
average number of adults or larvae recovered
cyproheptadine, beginning 4 days after ex-
from relatively large groups of mice. To in-
posure of the mice to 575 larvae each. A
crease the reliability of the method of es-
secondgroup, of 49 mice, exposedat the same
timating the average parasite burden, all
time, received no medication. Medication was
groups were divided into subgroups (usually
continued for 13 days, at the end of which
of 9 or 10 mice each), each subgroup being
period the mice were killed and their in-
handled separately throughout the necropsy
testines examined for Trichindla.
proceedings.
The results are summarized in Table II.
EXPERIMENTAL PROCEDURE AND RESULTS During the period of medication, 21 of the
In the first experiment mice were dosed treated mice died, while none of the control
orally with 875 T. spiralis larvae each. On mice died. The average number of worms
day 7 of infection, 60 of these mice were per mouse,harvested from the five subgroups
divided randomly into two equal groups. At into which the controls were divided, was
this time, the mice were visibly ill; but it is 221. The surviving treated mice yielded an
not known how many deaths, if any, had average of 374 worms per mouse. The differ-
occurred. The mice in one group were fed a ence in worm burden between the two groups
diet containing 0.025% cyproheptadine, be- is highly significant (p < 0.001).
 ANTIHISTAMINES IN TRICHINOSIS 25

TABLE II
Results of Experiments 2 and 4”
Initial
Worms per mouse, day 17
number Number mice
Treatment mice necropsied Rangea Mean”
Experiment 2
None 50 50 198-239 221(+ 8.4)
Cyproheptadine, 0.025% 49 28 317-424 374(f 9.8)
Experiment 4
None 31 21a 59-79 69(+ 10.0)
Cyproheptadine, O.O2S% 19 18 104-127 116(k 11.5)
B.A.S., 0.2% 19 13 111-148 130(+ 18.5)
Chlorpheniramine, 0.2% 19 13 133-152 143 (-c 9.5)
a Numbers of Trichtnek recovered from the intestine of mice on day 17 after exposure to infection. Mice
unaccounted for in necropsy figures died during the experiment.
b Range of the means of the sub-groups into which each group was divided.
C Mean of the means of the sub-groups; Standard Error in parentheses.
d The other 10 mice necropsied on-day-7f see text.

In a third experiment, two groups of 92 On day 21 of infection it was evident that

mice each were exposed to 550 larvae per
mouse. On day 7 of infection 12 mice from
each group were killed and examined. In one
group the remaining 80 mice were fed a cy-
proheptadine diet for 14 days, beginning on
day 7. In order to minimize mortality, the
drug was fed at a concentration of 0.0125%,
except on days 10-14, when the concentra-
tion was increased to 0.025%. In the second
group, the 80 remaining mice were held as
untreated controls.
On day 21 of infection roughly half of the
mice in each group were killed and examined
for adult Trichinella. On day 28 the remain-
ing mice in each group were killed and
examined both for adult and larval Trichi-
nella.
The results of this experiment are sum-
marized in Table III. During the course of
the experiment, 1 of the control mice and 6
of the treated mice died. The number of
adults recovered on day 7 of infection from
the mice in the control and treated groups
was approximately the same, viz., 280 and
3 15 adults per mouse, respectively; this in-
dicates that before treatment the general
severity of the infection was similar in the
two groups of mice.
a substantial decline had occurred in the
adult worm population of both groups of
mice; but the average number of worms
recovered from 39 untreated mice was rough-
ly half of the average number recovered from
39 treated mice. On day 28 of infection the
great majority of adult worms had been
eliminated from both groups; but the average
number in 40 untreated mice was about one-
third of the average number in 3.5 treated
mice. On day 28 of infection the treated mice
yielded 1570 more larvae per gram of muscle
than did the untreated mice; the difference
is statistically significant (p < 0.0250/o).
A fourth experiment was conducted to
determine whether an effect similar to that
of cyproheptadine could be obtained by the
use of the antihistamine drug 2-[p-chloro-a-
( 2-dimethylamino-ethyl) benzyl] pyridine ma-
leate (chlorpheniramine maleate) and the an-
tiserotonin drug 1-benzyl-2-methyl-S-meth-
oxytryptamine (B.A.S.). The latter drugs
were tested at a concentration of 0.2% in the
diet. No quantitative comparison with cy-
proheptadine was sought, but cyproheptadine
was tested concurrently at 0.0125% to pro-
vide qualitative comparison of the effect of
all three drugs under identical conditions
26 CAMPBELL, HARTMAN, AND CUCKLER

TABLE III
Results of Experiment 3a
Treatment
Cyproheptadine
Day None 0.0125-0.025%

Initial number mice 92 92

Day 7
Number mice necropsied 12 12
Worms per mouse, mean 280 319

Day 21
Number mice necropsied 39 39
Worms per mouse, range* 53-87 124-147
Worms per mouse, meant 70(+- 8.1) 137(& 5.1)

Day 28
Number mice necropsied 40 35
Worms per mouse, rangeb 5-17 30-73
Worms per mouse, mean0 13(& 2.6) 42(-1- 10.5)
Larvae per gram, range* 4262-5439 5039-6993
Larvae per gram, mean0 4668(+-262) 6238(*425)

a Numbers of Trichinella recovered from intestine and musculature of mice on days 7, 21, and 28
after exposure to infection. Mice unaccounted for in necropsy figures died during the experiment.
* Range of the means of the sub-groups.
0 Mean of the means of the sub-groups; Standard Error in parentheses.
6 Necropsy performed before start of medication,

(Table II). The mice were exposed to 850
larvae each. Administration of each drug was
begun on day 4 of infection and continued
until day 17.
The results of the fourth experiment are
summarized in Table II. Ten untreated mice,
killed on day 7 after exposure, harbored an
average of 199 worms each. By day 17 of
infection the three groups of treated mice
were in poor physical condition, and a con-
siderable number had died. The control mice
weighed an average of 24.7 gm; the average
weight of the mice treated with cyprohepta-
dine, B.A..%, and chlorpheniramine maleate,
was 22.2, 16.5, and 19.1 gm, respectively. The
mice treated with cyproheptadine yielded 1.7
times as many worms, on average, as did the
control mice; the difference, in this particular
experiment, was of borderline statistical sig-
nificance. The mice treated with B.A.S.
and chlorpheniramine maleate, respectively,
yielded 1.9 and 2.1 times as many worms as
did the controls; the difference, in each case,
is statistically significant (p < 0.05 and p <
0.01).
DISCUSSION
One of the characteristics of T. spiralis in-
fections is the rather sudden elimination of
most of the adult worms from the intestine
of the host within a few weeksafter infection.
In mice a substantial loss of worms generally
occurs at the end of the second week of in-
fection. The occurrence of an acute cellular
process in the intestinal mucosa shortly be-
fore the expulsion of Trichinella from the
intestine of the mousehas been described by
Larsh and Race, 1954. This processis inflam-
matory in nature and can be reduced by ad-
ministration of cortisone, under which condi-
tions the worms remain longer in the intestine
(Coker, 1955). As might be expected, such
prolongation of the intestinal phase results
in an increasein the number of larvae in the
host musculature (Markell, 19.58). Adreno-
cortical steroids are capable of blocking anti-
body synthesis (Robinson, 1960), and cor-
tisone might therefore suppress the inflam-
matory process indirectly by blocking a pre-
requisite antigen-antibody reaction.
In the present study, cyproheptadine, and
two other drugs, significantly reduced the
number of adult Trichinella which were ex-
pelled from the gut of mice by day 17 or 21
of infection. It is presumed that this effect
was due either to a delay in the onset of the
cellular reaction which is responsible for the
expulsion of the worms, or to a cellular reac-
tion of diminished intensity. In view of the
well-known anti-inflammatory property of
antihistamine drugs, the present findings add
strength to the belief that the expulsion of
Trichinella is caused by the inflammatory
response of the host’s intestinal mucosa. In
the mouse, serotonin has been shown to have
a histaminelike action (Page, 1958). The
activity of B.A.& in Experiment 4, was
therefore not unexpected.
The inflammatory reaction of mouse in-
testinal mucosa occurs much earlier in pre-
viously infected mice than in mice with a first
infection, and Larsh and Race (1954) have
proposed that the response is, in both cases,
stimulated by the interaction between the
worms and the humoral antibodies which
they, or their by-products, have elicited. If
this be true, the very rapid onset of the
response in immunized rats and mice (within
3-12 hours after challenge, according to
McCoy, 1940, and Larsh and Race, 1954)
could be attributed either to the immediate
availability of circulating antibody or to the
hypersensitivity of the host’s mucosa. Indeed,
both factors may well be involved, i.e., in
immune mice the mucosa may be hypersen-
sitive, not only to the challenge worms or
their by-products, but to a complex formed
by the combination of antigen from the chal-
lenge infection with residual antibody from
a previous infection.
Conditions of hypersensitivity are well
known in helminthic infections and have
formed the basis of many immunodiagnostic
techniques. Most of these responses could
be considered to be of the “immediate” vari-
ety (Andrews, 1962). Stewart (1955) has
reported that the phenomenon of “self-cure”
in hemonchosis in sheep results from hyper-
sensitivity of the abomasal mucosa and can
be suppressed by antihistamine drugs. Mar-
kell and Lewis (1957) suggested that the
cellular reaction of mice to the presence of
intestinal Trichinella represented a state of
“delayed” hypersensitivity and was inde-
pendent of circulating antibodies. There is
abundant evidence, however, that humoral
antibodies play a role in providing protection
against reinfection with Trichinella (Culbert-
son, 1942; Larsh, 1953).
The prolongation of the intestinal phase of
trichinosis, following administration of anti-
histamine or antiserotonin compounds, does
not indicate whether the cellular response is
an allergic phenomenon. The observation
does suggest that the cellular response is
mediated, to some extent, by histamine. How-
ever, in view of the weight loss recorded in
all groups of treated mice in Experiment 4,
the experimental findings might reflect a
reduced antibody production, resulting from
stress and/or lack of protein resources.
In Nippostrongylus infections in mice,
inflammation of the mucosa was associated
with an increase in the number of mast cells
but no increase in the level of free histamine
in the tissue; this was attributed to the
destruction of histamine by substances re-
leased from the eosinophils which were pre-
sent in large numbers (Wells, 1962). Perhaps
the eosinophilia characteristic of trichinosis
similarly serves to diminish histamine activ-
ity.
In Experiment 2, since the majority of
worms had been expelled from both the cy-
proheptadine-treated and the untreated mice
within 21 days after exposure, it is not sur-
prising that there was so little difference be-
tween the estimated number of larvae in the
muscles of treated and nontreated mice. In
28 CAMPBELL, HARTMAN, AND CUCKLER

preliminary experiments, in which very high LARSH, J, E. 1953. Studies in old mice to test the
mortality occurred in infected cyprohepta- hypotheses of local and general immunity to
Trichinella spiralis. Journal of Infectious Dis-
dine-treated mice, the few surviving treated eases 93, 282-293.
mice harbored fewer larvae in their muscles LARSH, J. E., AND RACE, G. J. 1954. A histopatho-
than did nontreated mice. This may simply logical study of the anterior small intestine of
be due to the fact that these few mice sur- immunized and non-immunized mice infected
vived because their infections had been ex- with Tn’chinella spiralis. Journal of Infectious
Diseases 94, 262-272.
ceptionally light.
MARKELL, E. K. 1958. The effect of cortisone
Inflammatory processes are important in treatment upon the longevity and productivity
the elimination, not only of infecting orga- of Trichinella spiralis in the rat. Jourmzl of In-
nisms, but also of their noxious by-products. fectious Diseases 102, 158-161.
MARKELL, E. K., AND LEWIS, W. P. 1957. Effect
The unexpectedly high mortality of the of cortisone treatment on immunity to subse-
treated mice in these experiments may be quent re-infection with Trichinella in the rat.
due to decreased tolerance of the infected American Journal of Tropical Medicine and
mice to the drugs used, but may on the other Hygiene 6, 553-561.
hand reflect the failure of the host to elimi- MCCOY, 0. R. 1940. Rapid loss of Trichinella
larvae fed to immune rats and its bearing on
nate toxins produced by the parasites.
the mechanism of immunity. American Journal
of Hygiene 32, 105-116.
PAGE, I. H. 1958. Serotonin. Phwioloaical
_ - Re-
The authors thank Dr. R. W. Schayer for his &ems 36, 277.
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analysis of the data was performed by Mr. A. G. therapy 7, 199.
Itkin. STEWART, D. F. 1955. “Self-cure” in nematode
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