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Enteric trichomonads of squirrel monkeys (Saimiri sp) Natural infestation and treatment.

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American Journal of Primatology 1455-71 (1988)
Enteric Trichomonads of Squirrel Monkeys (Saimiri sp):
Natural infestation and Treatment
ALAN G. BRADYl, FRANK F. PINDAK2,CHRISTIAN R. ABEE',
AND WILLIAM A. GARDNER JRZ
'Department of comparative Medicine and 2Department of Pathology, College of Medicine,
University of South Alabama, Mobile
In a large breeding colony of squirrel monkeys, a previous study demonstrated apparent universal infestation of adult animals with enteric trichomonads. The potential of these organisms to act as a source of experimental
variability and the potential pathogenic effects of parasitism in this species
stimulated this study of organism acquisition and treatment. Age of natural
infestation with trichomonads was determined from results of microscopic
examination and culture of fecal samples from infants of different ages. A
majority of squirrel monkey infants showed first evidence of trichomoniasis
at 2 to 4 weeks of age, with apparent 100%infestation by 8 weeks of age.
Treatment of adult monkeys was investigated. In vitro techniques were
utilized to determine sensitivity to metronidazole of a number of isolates.
An effective regimen for treatment of adult monkeys was determined to be
25 mgkg body weight of metronidazole given orally, twice daily for 5 days.
Key words: metronidazole, parasite, primate, lkichomonas
INTRODUCTION
Trichomonads are flagellate protozoa found in a variety of locations within the
many species that act as their hosts. Characteristically, they possess three or more
anterior flagella, a central axostyle, and an undulating membrane [Noble & Noble,
19723. The trichomonads described in this study are normally found in the lower
intestinal tract of squirrel monkeys. There is no published information available on
the survival of this organism outside of the host or on its dissemination. A previous
study from our laboratory [Pindak et al, 19851 described the wide prevalence of
intestinal trichomonads in a breeding colony of Bolivian (Saimiri boliviensis) and
Guyanan (Saimiri sciureus) squirrel monkeys. In the course of that study, a new
species of Tritrichomonw was isolated and subsequently characterized [Culberson
et al, 19861 (Fig. 1). Interest in the possible health significance of these protozoa as
well as in their potential influence of investigations utilizing squirrel monkeys has
stimulated efforts to define the natural history of infestation and to develop methods
Received December 18,1986;revision accepted July 23,1987.
Address reprint requests to Dr. Alan Brady, Department of Comparative Medicine, 1040 Medical Sciences
Bldg., College of Medicine, University of South Alabama, Mobile, AL 36688.
0 1988 Alan R. Liss, Inc.
66 I Brady et al.
for therapeutic eradication of these organisms. To our knowledge, this is the first
report to describe age of acquisition of enteric trichomonads in colony-born squirrel
monkeys and to provide a treatment regime supported by in vitro and in vivo evidence.
MATERIALS AND METHODS
Colony Description
The animals included in this study were members of a breeding colony of
squirrel monkeys maintained at the University of South Alabama. Animals were
housed in indoor pens of 10-15 animals each. Husbandry procedures were in accordance with NIH guidelines [NIH, 19851. Diet consisted of a pelleted high protein
primate diet (High Protein Monkey Chow No. 5045; Ralston Purina, St. Louis, MO)
fed ad libitum and supplemented with fresh fruit and a fruit-flavored electrolytevitamin-enriched drinking solution. Cages were cleaned daily with high-pressure
water hoses and thoroughly scrubbed with detergent solution (Ultra Blue; UNX
Chemical Co., Greenville, NC) at 4-week intervals.
Sample Collection
Samples contained as much fecal material as could be obtained with sterile
swabs inserted 2 cm beyond the anus. This method of collection was selected to
assure freshness of samples (an important factor in culture and identification of
trichomonads) and to preclude any possibility of sample contamination. The specimens were dispersed in 2 ml of GMP medium [Pindak et al, 19861. Microscopic
examination of wet mounts and initiation of cultures described previously [Pindak
et al, 19851 were performed within 4 hours of collection.
To investigate organism acquisition, a study was performed during the 1985
delivery season on all infants living at the time of sampling. All samples were taken
during a 2-day period. This group included 30 infants. The age of the infants ranged
from 1t o 6 months with the following distribution: 1month, 16 animals; 2 months,
4 animals; 3 months, 2 animals; 4 months, 2 animals; 5 months, 5 animals; 6
months, 1animal. In a second study, 11infants were sampled during their first week
of life and at 1-to 3-week intervals thereafter for as long as was necessary to obtain
the first trichomonad-positive sample. The study included one additional infant (No.
1412)that was sampled during weeks 3,4, 5,6, and 8. Data from the cross-sectional
study done in 1985 were used to predict the best time period for sampling. Previous
experience had shown that animals that became positive for trichomonads did not
revert to a negative state unless treated.
Culture Methods
Initial recovery of trichomonads from stool samples was done by cocultivation
with tissue culture (RK-13 or McCoy cells) in GMP medium. Methods used for that
purpose have been published previously [Pindak et al, 19851. Established cultures
were further propagated in GMP medium only.
In Vitro Determinations of Sensitivity to Metronidazole
Isolates from 17 juvenile and adult animals were grown in GMP medium.
Organisms from 3-day-old cultures were harvested by centrifugation and resuspended in GMP medium to contain approximately 5 x lo4 trichomonads per milliliter. GMP medium containing 2, 4, 6, 8, 10, and 15 mcgiml of metronidazole (Flagyl
IV RTU; G.D. Searle and Co., Chicago, IL)was dispensed in 1.8-ml amounts in flatbottomed wells (24-well cluster; Costar, Cambridge, MA). Each well, and a Flagylfree control well, was innoculated with a 0.2-ml aliquot of the organism suspension.
Enteric Trichomonads in Squirrel Monkeys I 67
Fig. 1. Scanning electron micrograph (SEM) of typical enteric trichomonads obtained from squirrel
monkeys. X5,640.
I.D.
1407
1408
1412
1414
1415
1416
1418
1410
1423
1429
1430
1435
1
/
0 1
2
\
I
3
Weeks of Age
/ 4 1 5 /
/ + I
1
6
(
I
7
1
S
I
Notes
1 .o.indicates
test negative for Trlchomonada
2 '+'indicates
lest positive for Trichornonads
Fig. 2. Results of direct microscopic examinations and cultures from infant squirrel monkeys during the
1986 delivery season. 0, test negative for trichomonads; t, test positive for trichomonads.
Cultures were maintained in a COa incubator at 37°C and examined daily for
motility of the trichomonads.
In Vivo Effectiveness of Metronidazole
Determination of a therapeutic regimen for metronidazole was based on results
from five adult animals. These individuals were confirmed as carriers of trichomonads by microscopic examination and by culture of fecal samples collected five times
during the 8 weeks prior to treatment. The dose and daily treatment frequency for
metronidazole were modified from methods published previously [Swenson et al,
19791 for eradicating ciliates with this agent. Duration of therapy was based on
results from the in vitro study described above. Animals were lightly anesthetized
68 I Brady et al.
with ketamine (Ketaset; Bristol Laboratories, Syracuse, NY) and given 25 mgkg
body weight of Flagyl by stomach tube twice daily.
After the first treatment, animals were bathed with mild soap and water and
moved to individual decontaminated cages in a previously sanitized room. Thereafter, they were kept in strict isolation; all housing and handling equipment was
kept separate from equipment used in the remainder of the colony. The efficacy of
treatment was monitored by microscopic examination and by culture of fecal samples collected by rectal swab daily during treatment and six times over the 7 weeks
posttreatment. Methods used for posttreatment testing were identical to those used
in all other parts of the study.
RESULTS
The purpose of the study at the end of the 1985 breeding season was twofold: 1)
to determine if infants born in captivity under known conditions acquire the trichomonads; 2) to determine at what age infestation is likely to be found.
Direct microscopic examination revealed trichomonads in all samples, and cultures were established from most infants. Occasionally the culture attempts were
abandoned because of persistent bacterial or fungal contamination. These results
demonstrated that acquisition of the trichomonads by the young can occur in a wellmaintained colony under what would be considered adequate and acceptable sanitary conditions. No change in stool consistency was noted between negative and
positive animals; however, this was a subjective evaluation.
These findings also indicated that infestation is likely to take place at an early
age, possibly not exceeding 1 month. This hypothesis was tested during the 1986
birth season. The results of this second study are summarized in Figure 2. In all
instances, the first sample was negative. Infant No. 1435 was sampled at 1 day of
age and at the end of the first week of life; the second sample was positive. No firstweek sample was obtained from infant No. 1415; its 2-week sample was positive.
Four other infants became positive during the second week (Nos. 1419, 1423, 1429,
and 1430). Two additional conversions were documented in the third week (Nos.
1416 and 1418). One infant (No. 1414) tested negative during the third week; this
infant and two others (neither of which were tested during the second and third
weeks) were positive during the fourth week. Thus, all 11 infants harbored the
organisms at the end of the fourth week of life. Infant No. 1412 was sampled during
weeks 3, 4, 5, 6, and 8; the first four samples yielded negative results, but the 8thweek sample was positive. Quantitation of trichomonads in samples was not attempted due to the fact that any meaningful standardization of sample size could
not be done.
In vitro testing of metronidazole against trichomonad isolates from the colony
demonstrated that a certain minimal continuous exposure time was necessary to
achieve a toxic effect, as indicated by the steady decrease in the minimum concentration of metronidazole required to render the organism nonviable (Table I). Thus, on
the first day of exposure, the concentration needed to render the organism nonmotile
ranged from 4 to 15 mcg (mean of all strains tested was 13.35 mcg). On the second
day the mean decreased to 7.82 mcg, and on the third day it decreased to 6.0 mcg.
The lowest concentration of metronidazole was determined on the fourth day, at
which time the mean for samples was 4.82 mcg/ml; after 4 days, there was no decline
in culture viability. Untreated control cultures showed no decline in viability during
or after the treatment period. To the extent that we were able to determine by
microscopic examination, isolates used in the in vitro study consisted of only one
species of tritrichomonad. Based on this observation a 5-day regimen of Flagyl
therapy was established.
Enteric Trichomonads in Squirrel Monkeys I 69
TABLE I. Sensitivity of Trichomonad Isolates to Metronidazole (Flagyl)
Isolate
25
47
48
49
52
72
81
546
816
820
834
846
856
898
1,039
1,060
1,063
Daily mean
Day 1
8
15
> 15
> 15
15
15
> 15
15
10
4
> 15
15
15
15
15
15
10
13.35
Flagyla
Day 2
6
8
15
10
10
10
8
8
6
4
8
6
8
6
8
6
6
7.82
Day 3
Day 4
4
6
10
10
8
8
8
4
4
4
6
4
6
4
6
6
4
6.0
4
4
8
6
6
6
6
4
4
2
6
4
6
4
4
4
4
4.82
'Concentration (mcgtml) required to inhibit motility on days of observation.
In the in vivo therapeutic studies, all culture specimens taken prior to treatment
were positive for the organism. All animals were negative for the organism when
treatment was completed on the fifth day and remained negative in six tests performed over the next 7 weeks, at which time testing was discontinued. No obvious
change in stool consistency was noted between the pre- and posttreatment period.
DISCUSSION
The early acquisition and wide prevalence of infestation observed in infants in
this study is in agreement with previously published observations [Pindak et al,
19851that enteric trichomoniasis appears to be essentially universal among captive
squirrel monkeys. Subsequent studies (unpublished)have produced similar results
from two unrelated squirrel monkey colonies. All 30 animals in the previous study
were adults, and all were found to be infested. In this study, all infants tested in the
cross-sectional study (ages 28-181 days) were also infested. The only animals to test
negative were less than 8 weeks of age in the final, intermittent sampling study; by
8 weeks, all of these animals were also positive for trichomonads.
Although collection of standard-sized fecal samples would have been desirable,
it was not practical in this work; the small size of these animals and the relatively
fluid consistency of their normal stools made rectal collection of standard samples of
any reasonable size impractical. Sample collection from animal housing surfaces
risks contamination and can result in collection of samples of questionable freshness
(and organism viability). Although it would have been of interest of quantitative
relative numbers of trichomonads observed at different ages, this was not possible
given these constraints. The age at which infestation is acquired might be explained
by the interaction of these infants with their environment and with other animals.
These infants spend almost all of their first 2 weeks of life sleeping and nursing
from the mother. At ages 2 4 weeks, they begin to leave their mothers' backs and
70 I Brady et al.
have been observed licking fluids from cage walls, sucking their fingers, and contacting the fecal material of other animals. Since all older animals are likely to be
infested, the onset of these behaviors undoubtedly contributes to infestation.
Our experience indicates that the use of a combination of immediate microscopic
examination and culture provides greater sensitivity than microscopic examination
alone for the detection of trichomonads. The use of both tests allowed each test to
act as a check on the other; we found that, if trichomonads were discovered in a
sample by only one of the tests, a repeat of both tests would show both to be positive.
In addition, our observations have shown that animals who are positive for enteric
trichomonads remain positive unless treated; this conclusion is based on serial
testing experience with a large number of animals.
An earlier attempt at metronidazole therapy called for administration of a
single dose of metronidazole, then removal to a n isolated, uncontaminated pen that
would house all five animals. In that experiment, only three of five animals showed
elimination of motile forms of the organism 1 day posttreatment. By 3 days posttreatment, all five animals had motile trichomonads observed in fecal samples,
suggesting that single-dose therapy is not sufficient and that therapy of a period of
days is required for eradication. The in vitro studies with metronidazole reported
here provide further experimental evidence that therapy of over 4 days is required.
These studies do not, however, rule out the possibility that a shorter treatment
period with a higher dose would achieve the same effect; such a possibility awaits
further study. Extension of therapy over a period of 5 days produced the lasting
absence of organisms that was desired in animals included in the in vivo studies. In
the in vivo therapeutic study, each animal was uniformly and repeatedly positive
during the 8 weeks prior to treatment and uniformly and repeatedly negative during
the 7 weeks after treatment; thus, each animal effectively functioned as its own
control for this study.
As noted here and previously [Pindak et al, 19851, the clinical significance of
enteric trichomonads in squirrel monkeys is uncertain. Although reviews on the
subject have considered the organism as a nonpathogen in primates [Burrows, 19721,
the lack of any apparent, published comparisons between squirrel monkeys with
and without the organism suggests that more study in this area is needed before
such a conclusion can be made with confidence. Enteric trichomonads of squirrel
monkeys do cause cytopathic effects in tissue culture [Pindak et al, 19851 and have
demonstrated virulence and invasiveness in subcutaneous mouse innoculation, a
standard measure of protozoan pathogenicity [Culberson et al, 19871. The role of
Tritrichononas foetus in abortion problems of cattle has been well established, and
a few case reports have suggested enteric trichomonads as etiologic agents of disease
in primates. In one report, an adult Titi monkey (Cullicebus moloch) became depressed, hypothermic, and incoordinated 4 weeks after being isolated because of
diarrhea, dehydration, and depression. At necropsy, culture attempts failed to isolate
any pathogenic bacteria. Microscopic examination revealed large numbers of trichomonads in inflamed and necrotic areas of the colon, in colonic crypts, and in mesenteric lymph nodes [Bunton et al, 19831. Another report describes a large, trichomonadinduced peritoneal granuloma as the only lesion found at necropsy of a rhesus
monkey that became moribund 3 months after a n experimental laparotomy. The
lesion was apparently caused by inadvertent innoculation of trichomonads from
feces or vaginal discharge during the laparotomy [Migaki et al, 19781. The techniques described here for eliminating infestation and maintaining trichomonad-free
squirrel monkeys will aid in the performance of controlled studies to investigate the
host-organism relationship. These studies may include investigations of the dose of
trichomonads necessary to establish infestation, prepatent period duration, and
Enteric Trichomonads in Squirrel Monkeys I 71
determination of the minimum daily dose of metronidazole required for efficacious
treatment (the daily dose in this study was adapted from a regimen established for
Balantidium coli [Swenson et al, 19791).
CONCLUSIONS
1. Infant squirrel monkeys first show evidence of enteric trichomoniasis between 2 to 8 weeks of age. Animals older than this appear to be universally
infested.
2. In vitro studies showed that organism viability is most affected by metronidazole by the fourth day of treatment.
3. A 5-daytreatment regimen consisting of twice-daily administration of 25 mgl
kg of metronidazole solution was sufficient to clear animals of any evidence of
infestation.
4. The 5-day metronidazole regimen described appears to clear squirrel monkeys of the organism permanently, provided that animals are not reexposed.
ACKNOWLEDGMENTS
The expert technical assistance of Mr. Robert Ricker is gratefully acknowledged.
This project was supported in part by NIH grant RR01254 and the South Alabama
Medical Science Foundation.
REFERENCES
Bunton, T.E.; Lowenstine, L.V.; Leininger, R.
Invasive Trichomoniasis in a Callicebus
moloch. VETERINARY PATHOLOGY
20:491-494, 1983.
Burrows, R.B. Protozoa of the intestinal tract.
Pp.2-28 in PATHOLOGY OF SIMIAN PRIMATES. R.N.T.-W. Fiennes, ed. Munich,
Germany, S. Karger Basel, 1972.
Culberson, D.E.; Pindak, F.F.; Gardner, W.A.,
Jr.; Honigberg, B.M. Tritrichomonas mobiIensis n. sp. (Zoomastigophorea: Trichomonadida) from the Bolivian squirrel Monkey
Saimiri boliviensis bolivensis. JOURNAL
OF PROTOZOOLOGY 33:301-304,1986.
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Migaki, G.; Bernirschke, K.; McKee, A.E.;
Casey, H.W. Trichomonal granuloma of the
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NARY PATHOLOGY 15:679-681.1978.
National Institutes of Health. GUIDE FOR
THE CARE AND USE OF LABORATORY
ANIMALS, 2nd ed. Bethesda, Maryland,
U.S. Public Health Service, 1985.
Noble, E.R.; Noble, G.A. Introduction, Sarcomastigophora. Pp. 17-57 in PARASITOL
OGY, THE BIOLOGY OF ANIMAL PARASITES. Philadelphia, Lea & Febiger, 1971.
Pindak, F.F.; Mora de Pindak, M, Abee, C.R.;
Gardner, W.A., Jr. Detection and cultivation of intestinal trichomonads of squirrel
monkeys (Saimiri sciureus). AMERICAN
JOURNAL OF PRIMATOLOGY 9:197-205,
1985.
Pindak, F.F.; Mora de Pindak, M.; Gardner,
W.A., Jr. Growth and cytopathogenicity of
Trichomonas vaginalis in tissue cultures.
JOURNAL OF CLINICAL MICROBIOLOGY 23:672-678,1986.
Swenson, B.; Strobert, E.; Orkin, J., eds.
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