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Detection and cultivation of intestinal trichomonads of squirrel monkeys (Saimiri sciureus).

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American Journal of Primatology 9: 197-205 (1985)
Detection and Cultivation of intestinal Trichomonads of
Squirrel Monkeys (Saimiri sciureus)
F.F. PINDAK', M. MORA DE PINDAK', C.R. ABEE', AND W.A. GARDNER, JR2
'Departments of Pathology and 'Comparatiue Medicine, University of South Alabamq
College of Medicine, Mobile, Alabama
Routine examinations of fecal samples from squirrel monkeys suggested
that intestinal trichomonads might be common inhabitants of these animals. In pursuit of these observations, microscopic examination of fecal
suspensions and cultures have demonstrated a 100% incidence of trichomonads in 30 arbitrarily selected animals from a colony of more than 300 housed
in groups of ten. The most prominent species was Pentatrichomonas hominis. A not yet fully characterized tritrichomonad was also found on several
occasions. The main obstacle in establishing individual strains in culture
was the presence of bacterial and fungal flora in the samples. Nevertheless,
abundant cultures were obtained from 28 animals by inoculation of fecal
suspensions into tissue cultures with appropriately formulated medium and
high concentration of antibiotics. In several unattended cultures maintained at room temperature, the flagellates retained motility for at least 4
months. This long survival may explain the widespread occurrence of the
parasites within a confined animal community.
Key words: Squirrel monkey, Saimin' sciureus, intestinal trichomoniasis, trichomonads
in culture, cytopathic effect in tissue culture
INTRODUCTION
Squirrel monkeys (Saimiri sciureus) have been used less frequently in biomedical research than Old World nonhuman primates, and their "normal" intestinal
microflora is not well characterized. We have observed common infestation of these
animals with intestinal trichomonads. The current literature does not address the
frequency or significance of such parasites either in captive or feral squirrel monkeys. More thorough knowledge in this respect could have practical applicability.
Various studies, such as developmental progress of the young, nutritional requirements, and, possibly, other experimental results, could be directly influenced by the
presence of such parasites. We have determined that in our colony of squirrel
monkeys, infestation with intestinal trichomonads is more a rule than an exception.
Our study has shown that in addition to the Pentatrichomonas species, known to
inhabit both man and other primates [Hegner, 1935; Wenrich, 1935, 1944; Brooks,
Received May 1, 1985; revision accepted July 5, 1985.
Address reprint requests to Frank F. Pindak, Department of Pathology, 2451 Fillingim Street, Mobile, AL
36617.
0 1985 Alan R. Liss, Inc.
198 / Pindak et a1
1963; Kulda, 1967; Honigberg, 19781, there is a yet undetermined proportion of
squirrel monkeys that harbor an apparently new species of tritrichomonad. Methods
for detection and cultivation of these parasites are simple and reliable. Pure cultures
of individual isolates lend themselves to more thorough studies of their pathogenicity, relatedness to other trichomonads, and a number of other investigations.
METHODS
Colony Description
Animals in this study consisted of approximately 10% of the squirrel monkey
population maintained a t the Primate Research Laboratory of the University of
South Alabama. This facility was designed to provide favorable living conditions.
Cages with ample room for ten or more occupants were cleaned daily. A diet of high
protein monkey chow (Ralston Purina, St. Louis, MO), water, sucrose, electrolyte
solution, and vitamins was supplemented with fresh fruit. Before arrival a t Mobile
several years ago, the colony was maintained at the Caribbean Primate Center of
the University of Puerto Rico.
Additionally, 18 animals recently captured in Guyana were sampled on the day
of their arrival from the supplier and twice during a 6-week quarantine period.
Collection of Samples
Before collection of specimens, the perineal area was cleaned with gauze sponge
moistened in isopropyl alcohol. Fecal samples were obtained with a sterile cotton
swab inserted into the rectum approximately 2 cm beyond the anus. The collected
material was dispersed in 2 ml of growth medium (see below) in screw-cap test tubes.
Microscopic Examination and Cultivation
Samples were examined and cultured within 3 h of collection. Ten drops of each
sample were transferred into a flat-bottom well (2 cm2 area) of a tissue culture plate
(Costar, Cambridge, MA). After a settling time of 20 min, the specimens were
examined with a phase contrast inverted microscope a t x 200 magnification. Only
those samples were scored as positive in which clearly active trichomonads were
found. The remainder of each sample was inoculated into slanted test tubes (Nunc,
Vangard Internat., Neptune, NJ)containing a full monolayer of McCoy or RK-13
cells and 3 ml of growth medium. The inoculated tissue cultures were maintained
in a 37°C incubator and examined daily for the presence of flagellates. Positive
samples were transferred into small tissue culture flasks. Those showing no trichomonads during 10 days of observation were considered negative. For more definitive
morphological identification, representative specimens were prepared by standard
methods for scanning electron microscopy.
Growth Medium
Based on several preliminary attempts to establish these trichomonads in culture, the most satisfactory results were obtained with a medium of the following
composition: Medium 199, 900 ml; 1 M hepes buffer, 15 ml; glucose, 10 g; ascorbic
acid, 50 mg; pantothenic acid, 10 mg; kanamycin sulfate, 300 mg; chloramphenicol,
200 mg; amphotericin B, 5 mg; polymyxin B, 200 mg; tetracycline, 10 mg; heatinactivated (56"C/30 min) fetal bovine serum (FBS), 100 ml; final pH, 7.1.
The high antibiotic content was required for elimination of bacterial and fungal
flora. Only on rare occasions did contaminants persist. In such instances, other
antibiotics were added or the concentrations of those in use were increased up to the
limits of empirically determined tolerance (Table I).
Intestinal Trichomonads of Squirrel Monkeys I 199
TABLE I. Tolerance of Intestinal
Trichomonads to Antibiotics
Antibiotic
Chloramphenicola
Kanamycina
Streptomycin
Vancomycin
Polymyxin B"
Neomycin
Gentamicin
Amikacin
Septra
Tetracyclinea
Amphotericin B"
Tolerated concentration
(mcg/ml)
400
400
400
400
400
400
400
400
50
10
10
'Antibiotics used for routine primary culture; their
concentration was decreased to one-half or less after the
cultures were established. Other antibiotics were used
initially if contaminants persisted.
RESULTS
Although care was taken to standardize the sampling procedure, not all specimens were of equal quality. Their fecal content ranged from copious to very scant.
Nevertheless, few to numerous trichomonads were detected microscopically in 27 of
30 animals. The remaining three were positive on repeat examination. Thus, the
infestation rate was 100%.
Microscopic examination usually revealed vigorously motile organisms easily
recognizable from particles of fecal debris in the same size range. The use of phase
contrast optics offered a great advantage in instances where there were only a few
trichomonads and these appeared motionless. Under this type of illumination, the
movement of flagella and undulating membranes was more readily apparent than
under conventional (transmitted)illumination.
In general, the trichomonads appeared oblong, pear-shaped, or round, without
distinct species characteristics. Identification procedures based on morphological
features [Wenrich, 1944; Honigberg, 19781 were carried out only on enriched samples
obtained from early cultures of the organisms.
Preliminary attempts to isolate trichomonads in tissue cultures with GMP
medium (pH 6.5) previously found to be well suited for propogation of Trichomonas
uaginalis or with conventional tissue culture medium (ie, Medium 199 with 10%
FBS) were only moderately successful. The less-than-desired success was attributed,
in part, to the pH and composition of these media. Satisfactory growth conditions
were obtained with a medium described in Methods. Despite these measures, attempts to obtain pure cultures of the trichomonads were frequently hampered by
bacterial and fungal overgrowth. This difficulty was overcome by initial use of
antibiotics, which had no overt effect on the protozoa (Table I). After the cultures
were established, the antibiotic content was decreased by a t least one-half, primarily
to prevent reappearance of contamination.
The cultures of most specimens yielded organisms of diverse morphology. Characteristics typical of Pentatrichomonas were predominant (Fig. 1).Also seen were
round forms of the same species (Fig. 2). Occasionally, in luxuriantly growing
cultures, there were conspicuous giant forms (somatella) with numerous flagella and
200 I Pindak et al
Fig. 1. Scanning electron micrograph (SEMI of typical Pentatrichomonas hominis recovered after 2 days
in McCoy cell culture. Some fecal debris still present. Bar = 5 prn.
undulating membranes (Fig. 3). Several specimens yielded slender flagellates also
adhering mostly to the cell culture monolayer or to the culture vessel surface. These
were tentatively identified as a previously undescribed Tritrichomonas (Fig. 4).
Absolute separation of the individual forms by cloning was not attempted.
Nearly pure cultures of the tritrichomonads were obtained, on several occasions,
however, by a simple enrichment procedure. Owing to their propensity to become
attached, it was possible to remove and rinse out most other types of organisms.
Those remaining in the culture vessel were harvested by vigorous pipetting in fresh
growth medium and were transferred to a new tissue culture monolayer. After
several subcultures in this manner, rich cultures of the tritrichomonads were established, with only occasional pentatrichomonads remaining.
As the number of trichomonads increased, the tissue culture monolayers lost
their integrity, presumably owing to mechanical disruption by the protozoa. Preliminary attempts to produce a similar effect by culture filtrates were unsuccessful.
Cultures of RK-13 cells containing the tritrichomonads, however, often showed
Intestinal Trichomonads of Squirrel Monkeys I 201
Fig. 2. Round form of Pentatrichomonas horninis in McCoy cell culture, with clearly defined undulating
membrane. SEM. Bar = 5 pm.
distinct vacuolization (Fig. 5). Typically, the occurrence of this type of cytopathic
effect (CPE) required 3-4 days of incubation and could be observed only when the
protozoan population had not yet reached a density sufficient to disrupt the
monolayer.
Results comparable to the above were obtained from 18 feral animal samples on
arrival from their natural habitat. They all harbored at least one type of protozoon
described above. Repeated samplings in the middle and a t the end of the quarantine
period showed all to be positive.
In all instances, the trichomonads appeared to adapt readily to the artificial
growth conditions. Depending on their numbers in the original inoculum, a noticeable increase in the population was present in 2-3 days. Subcultures were usually
required in 1week, since at that time, the organisms had reached a density sufficient
to destroy the tissue culture monolayer.
On a number of occasions, cultures were left at room temperature and examined
periodically. While there was no evidence of additional proliferation, motile organ-
202 I Pindak et a1
Fig. 3. A giant organism (somatella) recovered from luxuriantly growing culture. Undulating membranes and flagella are too numerous to count. SEM. Bar = 10 pm.
isms were seen for as long as 4 months. On transfer into fresh medium with tissue
culture and incubation a t 37"C, good growth of a mixed protozoan population was
re-established.
DISCUSSION
Our survey of captive squirrel monkeys has shown that their infestation with
intestinal trichomonads may be very common, if not universal. These findings are
not peculiar to our colony, since similar results have been obtained from a comparable study of feral animals captured in Guyana and sampled immediately on arrival
from the supplier. All of these harbored the organisms and remained positive
throughout the quarantine period. We cannot say, however, that these additional
animals acquired the parasites in their natural habitat before capture. Inquiry into
their background has revealed that, before shipment to our facility, they had been
kept under quarantine conditions by the supplier for approximately 2 months.
Depending on the transhipment and the supplier's quarantine conditions, the results
from these animals may or may not be significant. Inadvertent acquisition andlor
dissemination of the parasites during that time or during transit cannot be excluded
without further studies.
Intestinal Trichomonads of Squirrel Monkeys I 203
Fig. 4. Tritrichomonads in RK-13 cell culture. Organisms are firmly adhering to the culture vessel. Cell
monolayer is mostly disrupted. The remaining cells show marked surface erosion. SEM. Bar = 5 Fm.
In cultures, typical pentatrichomonads (Fig. l), as well as their round forms
(Fig. 2>,were easily identified. The significance of the giant organisms (Fig. 3) cannot
be assessed a t present. Similar occurrences of somatella have been reported in
cultures of Trichomonas vaginalis [Bishop, 1931; Wirtschafter, 1954; Winston, 1974;
John & Squires, 19781and of other trichomonads [Stabler, 1941; Wenrich, 19441.
Very little information on intestinal tritrichomonads is available. Wenrich [ 19441
briefly mentioned having seen such a n organism in the feces of a rhesus monkey
and credited Cleveland with a single report of a similar discovery in human feces.
We were unable to find any reports on their occurrence in squirrel monkeys. In our
experience, these organisms are conspicuous by their morphology and behavior in
culture. Unlike pentatrichomonads, they have a lanceolate appearance and show a
much greater tendency toward attachment, either to the cell monolayer or to the
bare surface of the culture vessel (Fig. 4). These features are particularly obvious
when a n established culture is transferred to a monolayer of RK-13 cells with
Medium 199 and 10% FBS.
Of further interest is the occurrence of cytoplasmic vacuolization seen frequently
in RK-13 cells exposed to the tritrichomonads. The cause of the CPE remains to be
Fig. 5. Cytopathic effect in RK-13 cells associated with the presence of tritrichomonads. Left: Normal
cell control. Right: Extensive vacuolization after 5 days of incubation with the organism. Phase contrast.
Bar = 100pm.
Intestinal Trichomonads of Squirrel Monkeys I 205
determined. Because of the intimate contact of the organisms with the tissue culture,
it may be viewed as a n expression of mechanical or cytochemical damage. Similar
vacuolization was described by Kulda [1967] in monkey kidney cells inoculated with
axenically maintained strains of Tritrichomonas suis and 7: foetus. The appearance
of our cell cultures closely resembled the CPE caused by vacuolating viruses. In
addition to trichomonads, the original fecal samples may have contained a viral
agent for which the RK-13 cells were a suitable host. This possibility is being
investigated.
The relevance of the protozoa here described to the well-being of their host is
not known a t present. There appears to be no recent in-depth study on intestinal
trichomoniasis in squirrel monkeys. Older reports are limited mostly to casual
mention of occurrence in various hosts and to descriptive morphology. While the
presence of intestinal trichomonads in otherwise healthy squirrel monkeys may not
be life-threatening, their chronic persistence could have undesirable effects on the
host, and they ought to be investigated further. The remarkably long survival of
some of the organisms at room temperature (at least 4 months) would facilitate their
wide dissemination.
CONCLUSIONS
1. Both captive and recently captured feral squirrel monkeys chronically harbor
intestinal trichomonads, usually of more than one species.
2. These protozoa can be readily detected by direct phase-contrast microscopic
examination of fresh fecal samples.
3. Cultures of these organisms can be established and maintained following
elimination of bacterial and fungal flora.
4. In mammalian tissue cultures, proliferation of these organisms is accompanied by characteristic cytopathic features. Under certain circumstances, there is
a distinct foamy-virus-like cytoplasmic vacuolization.
ACKNOWLEDGMENTS
This study was supported by the South Alabama Medical Science Foundation
and NM Grant RR01254.
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Brooks, B.A. More notes on Saimiri sciureus.
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Hegner, R. Intestinal protozoa from Panama
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Honigberg, B.M. Trichomonads of imporJOURNAL OF MORPHOLOGY 74:189tance in human medicine, pp. 275-454 in
211, 1944.
PARASITIC PROTOZOA VOL. 11. J.P.
Kreier, ed. New York, Academic Press, 1978. Winston, R.M.L. The relation between size
and pathogenicity of Trichomonas uaginJohn, J.; Squires, S. Abnormal forms of Trialis. JOURNAL OF OBSTETRICS AND
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OF VENEREAL DISEASES 54:84-87,1978.
GYNECOLOGY OF THE BRITISH COMMONWEALTH 81:399-404,1974.
Kulda, J. Effect of different species of trichomonads on monkey kidney cell cultures. Wirtschafter, S.K. Giant multinucleated cells
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