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Biochemical detection of fecal hematin as a test for meat eating in chimpanzees (Pan troglodytes).

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American Journal of Primatology 3:327-332 (1982)
TECHNICAL NOTE
Biochemical Detection of Fecal Hematin as a Test for Meat
Eating in Chimpanzees (Pan troglodytes)
FRANK SPENCER1, NOEL T BOAZ', MEL ALLEN3, AND W C McGREW4
Deporfnwnt of Anthropology, Queens C o l l r g ~of the City linwerszty of New York, Fludzing.
New York, 'Department of Anthropology, New York [Jniuerstty, New York Clty, Neu1 York,
3Departrnent ofAnthropology, [Jnzwrsity of Oklahoma, Norman, Oklahomq and 4D~partment
of Psychology, University of Stirzing, Stirling, Scotland
Meat-eating by wild chimpanzees has been reported by a number of workers
during the last two decades. Direct observation probably underestimates the incidence, even though the behavior occurs relatively infrequently. In 1978,
Moreno-Black suggested that fecal analysis over a long period of time is probably the most effective means to determine the incidence of this behavior in
wild chimpanzees and other nonhuman primate groups. A method currently
employed by a number of fieldworkers involves the recovery of the remains of a
carnivorous meal in the animal's feces. This method, however, may also under
represent the incidence because of 1)complete digestion of mammalian parts, 2)
the unidentifiability of partially digested remains, and 3) the reingestion of
feces. This paper reports the results of a laboratory study using a fecal test not
subject t o these limitations. The test is based on the biochemical detection of
hematin, a derivative of hemaglobin which is found in all mammalian tissues.
The results of this study reveal that hematin is a reliable indicator of meat consumption. The test is available in a commercially prepared kit, namely
HEMOCULT, which was developed to detect clinically significant amounts of
blood in the feces of human patients with presumptive gastrointestinal lesions.
This kit has been evaluated with a view to its possible application in the field.
Key words: Chimpanzee, Pun, meat eating, fecal analysis
INTRODUCTION
Within the last two decades field studies of wild chimpanzees have revealed that meat
comprises a small but consistent part of the diet of the species [Goodall, 1963; Hladik,
1977; McGrew, in press; McGrew e t al., 1978,1979: Nishida e t al., 1979; Suzuki, 1975;
Teleki, 1973; Wrangham, 19751. Direct observation probably greatly underestimates
the incidence of meat eating, although this type of behavior occurs relatively rarely.
Moreno-Black [1978] has suggested that fecal analysis over a long period is the most
effective means to determine the importance of meat eating in a chimpanzee group's
diet. McGrew et al. [1978] reported results of analyses over a 2 year period of 380 fecal
samples from Mt. Assirik, Senegal, in which 2.6% of the specimens contained signs of
meat eating. This method, which relies on the recognition of the remains of a carnivorous
meal, may also under represent the incidence of meat eating because of 1)complete
Received November 24, 1981; accepted March 12, 1982.
Address reprint requests to Dr. Frank Spencer, Department of Anthropology, Queens College of the City University of New York, Kissend Boulevard, Flushing, New York 11367.
0275-2565/82/0301-04-~327$02.00 0 1982 Alan R. Liss, Inc.
328
Spencer et a1
digestion of some animal parts, 2) the unidentifiability of partially digested remains
[Harding, 19811 and 3) the occasional reingestion of feces [Wrangham, 1975; McGrew et
d., 19791. We report here a fecal test not subject to these limitations, based on the
biochemical detection of hematin, a derivative of hemoglobin. The test is available in a
commercially packaged kit, namely HEMOCULT'", which was developed to detect
clinically significant amounts of occult blood in the feces of human subjects with
presumptive gastrointestinal lesions. This kit has been evaluated with a view to its
possible application in the field.
Presuming that chimpanzees in nature do not normally eal meat daily and that the
amounts consumed vary from meal to meal depending on social and environmental factors, we designed the following simple experiment. I t seeks to simulate natural conditions and to determine: 1)if differing amounts of meat in a diet are reflected in varying
amounts of hematin in the feces; 2) if the HEMOCULT'" test can detect these predicted
changes in the concentration of hematin; 3) if the gut-passage time, namely the time
elapsed from the ingestion of the meat to the appearance of hematin in the feces, can be
measured; and 4) if the sensitivity of the HEMOCULT" test is affected by the age of the
specimen. Throughout the study, the reliability and sensitivity of the HEMOCULT'"
test was monitored by standard laboratory in vitro procedures.
METHODS
A group of communally living chimpanzees (3 males and 3 females) at the Institute for
Primate Studies of the University of Oklahoma was used in this study (Table I). This
group was considered ideal because they were accustomed to eating meat, and known
not to be coprophagous.
Over an 11-dayperiod, this group received a modified Radcliffe diet (Table 11) to which
was added on prescribed days an increasing amount of uncooked, ground beef. The living quarters of the group were routinely cleaned twice a day, namely morning and afternoon, which permitted the collection of fecal samples the approximate time of deposition
of which were known. The morning specimens were collected between 0800 and 1000
hours; the afternoon samples were collected between 1500 and 1700 hours.
From days 1 through 3 the chimpanzees received a Radcliffe meat-free diet. Fecal
samples collected during this period provided a baseline of meat-free consumption. On
day 4 each animal received approximately 93 g (0.25lb) of uncooked meat mixed in their
diet. On day 5 the subjects were returned to a meat-free diet, which was maintaineduntil
day 8, whereupon they received approximately 187 g (0.5 lb) of uncooked meat. For the
remaining 3 days of the study the animals were returned to a meat-free diet. Fecal
samples were sealed in 10 ml glass, screw-capped vials and stored at 4°C until analyzed
(see below).
In addition to the HEMOCULT'" test, the collected fecal samples were analyzed for
hematin using standard laboratory in vitro techniques. Such tests for hematin depend
upon the fact that heme proteins catalyze the oxidation of organic substances such as
benzidine and orthotolidine by hydrogen peroxide. The reaction produces a blue color,
the intensity of which is mostly a function of the concentration of hematin. The reaction
is like that catalyzed by true peroxidases, but the tests are made specific by boiling the
feces. This destroys peroxidases of bacterial or vegetable origin, leaving only the heme
compounds, which are thermostable.
In Vitro Techniques
Reagents: 1)Saturated solution of either benzidine or orthotolidine. Dissolve 1.0 g of
benzidine dihydrochloride in distilled water and dilute to 100 ml. Mix, filter, and store in
a dark bottle at 4°C. To prepare the orthotolidine solution, dissolve 1.08 g of orthotolidine in absolute methanol, and dilute to 100 ml. Store in a dark bottle at 4°C. 2) 3%
solution of hydrogen peroxide.
Test for Meat Eating in Chimpanzees
329
TABLE I. Chimpanzees Tested, Institute for Primale Studies, University of Oklahoma*
Name
Sex
McCarthy
Nim
Onan
Kelly
Lilly
Sherrv
M
M
M
F
F
F
Weight
(kg)
36
32
42
39
40
31
Date of Birth
Origin
c. 12- 7-71
11-19-73
5-25-72
C. 9-23-72
C. 1- 1-72
C. 3-14-74
wild
captivity
captivity
wild
wild
wild
*All females were nulliparous, although Lilly was approximately 7 months pregnant during the study.
TABLE 11. Individual Daily Diet*
Constituents
Amounts
Carrots
Isonicotinic acid hydrozyde‘
Vitamin B6
Ascorbic acid
Bean sprouts
Grain mix2
Water
150 g
300 mg
100 mg
6g
60 g
275 g
750 mg
*Modified Radcliffe diet.
‘Tuberculosis prophylactic.
’Constituents: maize corn, wheat, barley, oats, soybean meal, alfalfa meal, yeast, and ground oyster shell.
NOTE: The usnal diet, except for the purpose of this study contains approximately 45 g of cooked meat (skimmed
of fat) for each of the individuals used as subjects.
Procedure: 1)Thoroughly mix fecal specimen using a wooden applicator stick, and then
transfer a portion the size of a large pea to the bottom of each of 2 graduated 15 ml
centrifuge tubes. Label 1 of these tubes: “positive control.” To this tube add 0.05 ml of
(human) blood. Add 5 ml of distilled water to each tube and mix thoroughly. Add distilled water to the 10 ml mark and again mix thoroughly. 2) Centrifuge tubes at approximately 700 X g for 10 minutes. 3) Transfer 1 ml supernatant from each tube into an
appropriately labeled test tube. 4) To a third test tube labeled “negative control,” add 1
ml of distilled water. 5) Boil all three test tubes for 5 minutes. 6) Transfer test tubes to a
beaker containing water at room temperature. 7 ) When test tubes have cooled, add to
each 1 ml of benzidine or orthotolidine solution, followed immediately by 1 ml of hydrogen peroxide solution. 8) After 1 minute examine each test tube for color. If the test is
positive, the experimental tube will be distinctly blue like the positive control tube. The
negative control tube should be colorless.
Because benzidine and orthotolidine are very sensitive and prone to give false-positive
results, one must run controls with each batch of tests. Furthermore, all positive results
should be replicated. All tests in this study were done twice, and all with positive results
were repeated by another worker.
Using serial dilutions of a standard aqueous solution of crystalline human hemoglobin
mixed with human feces, the above tests were estimated to be sensitive to approximately 3 mg hemoglobin/ gm of feces.
HEMOCULT Test Kit: The HEMOCULT’” it is manufactured by SmithKline Diagnostics, PO Box 61947, Sunnyvale, CA 94086. I t consists of a specially prepared guaiac-
330
Spencer et a1
impregnated filter paper housed in a specially designed envelope which eases identification, preparation, and subsequent development of the test. The test utilizes the principle
that hemoglobin exerts a peroxidase-like activity and promotes the oxidation of the
guaiac resin by hydrogen peroxide. A positive result is signaled by the appearance of a
blue color on or at the edge of the smear.
RESULTS AND DISCUSSION
Figure 1 shows that the results of the in vitro tests and the HEMOCULT'" slide test
gave high agreement. Because the HEMOCULT'" system does not remove bacterial or
vegetable peroxidases, we expected problems with false positives. This seems to have
been unfounded. We later found that clinical trials had been done, and all reported the
HEMOCULT" system to be virtually free of false positives, even with specimens from
patients on unrestricted diets [Ostrow et al., 1973; Fleisher et al., 1977; Glober and
Peskoe, 1974; Winawer et al., 19761. This evidently has been achieved by reducing the
sensitivity of the test, which is clearly less accurate than the standard in vitro procedures (Ostrow et al., 19731. The latter authors found that the HEMOCULT" system is
sensitive to a minimum of 5 mg of hemoglobin/gm of feces. At this level of concentration
the test gives equivocal results. However, with more than 10 mg of hemoglobin/ gm of
feces, the results are reliable [Ostrow et al., 1973: 9331.
As for the possibility of false positives due to gastrointestinal bleeding, all of the subjects used in our study were known to be in good health. Also all of the obtained positive
results (Fig. I) were confined to specimens where the presence of occult blood was
expected. Because in the field an investigator may not know the physical condition of
the animal whose fecal sample is being assessed, the presence of a sick animal in a group
could confound an investigation. But while recognizing that the HEMOCULT'" system
is not sensitive to the origin of blood in the feces, it is suggested that this inherent dif-
HB4OCULT
TOLIDINE
;
0
BENZIDINE
+
-+
n
1
0
L
Fig. 1. Detection of hematin by three methods (top)compared with presence of meat in diet (bottom).0 = negative result; ? = equivocal result; = positive result.
+
Test for Meat Eating in Chimpanzees
331
ficulty may be overcome in a number of ways. For instance, the recognition of a pathological specimen: such as 1)the presence of frank blood, which generally indicates a
rectal or anal lesion, or 2) encountering a black-colored (melaena) stool which has a tarlike consistency and invariably indicates bleeding in the upper gastrointestinal tract.
Besides the macroscopic appearance of the feces, the investigator’s suspicion should
also be alerted by the continuing presence of a single positive stool over a period of time
in a group in which all other specimens deposited in the same period are negative.
I t is common knowledge among field workers that chimpanzees regularly defecate
several times daily. In the wild the chimpanzee is primarily a frugivore, consuming large
amounts of indigestible material such as pits and seeds of fruit. Hence, it seems reasonable to suggest that the frequency of bowel movements is largely a physiological
response to the accumulation of this material and the residue of digestion in the lower
intestinal tract. In this regard, Milton [1981]has noted that frugivorousprimates have a
much faster gut-passage time than do folivorous-herbivorous species -a difference that
might explain the relatively fast gut-passage time of chimpanzees compared to humans.
However, the extent to which this apparently rapid passage of food through the alimentary canal affects the ability of the chimpanzee to deal effectively with the digestion of
complex animal proteins is not known. This question has important implications for
hominid evolution and requires further investigation. From our limited study, the gutpassage time in the Oklahoma group of chimpanzees was found to be between 6 and 24
hours, which is presumed not to be significantly different from that of chimpanzees in
nature. Furthermore, a microscopic examination of the fecal specimens revealed the
presence of meat fibres in two of the three specimens with a positive occult blood test.
Also, the general consistency of the stools from days 4 through 11 were soft and loose
compared with those collected on days 1 through 3, which were firmer and more formed.
The significance of these observations remains to be seen.
In order to determine the effect of a specimen’s age on results, the specimens which had
proved positive were stored at room temperature and tested over 3 weeks at intervals of
7 days. Though they still tested positively after 2 weeks, the color was fainter. After 3
weeks the results were equivocal. These results largely confirm the findings of Ostrow et
al. [1973: 9351, but Fleisher et al. [1980]showed that a strong-positive reaction will occur
for up to 30 days with specimens applied to the guaiac impregnated filter papers. For
weakly testing positives the HEMOCULT‘“ test is reportedly unstable, and sometimes
after only 4 or 5 days tests become negative. This suggests that our initial results were
of intermediate strength.
The results obtained under the prescribed conditions of this study seem to show that
the I-IEMOCULT’” system reliably detects hematin, but several questions need to be
answered before the test can be used routinely in the field. First, it is not known if the
normal diet of wild chimpanzees would affect the specificity of the test. For example,
there is evidence that ascorbic acid in the diet might distort the results. According to
Jaffe [I9781 ingesting more than 250 mg of ascorbic acid may cause false-negative
results. Evidently the amount in the diet of the subjects of this study did not do so. Also,
it is not known how the kit will perform in a tropical climate. The manufacturer recommends that the kits be stored at “controlled room temperature 15-30°C (59-86°F)”with
“protect[ionl from heat and light.” These questions, however, can only be answered by
trials in the field.
CONCLUSION
This study indicates that hematin in the feces of chimpanzees is a reliable indicator of
meat consumption which can be detected by relatively simple biochemical procedures.
Although the present study could not evaluate the HEMOCULT” slide test under
natural conditions, the results nevertheless indicate that it should be tried in the field as
a quick and easy method of detecting meat eating. I t might also be useful in studies of
other omnivorous species [Harding, 19811.
332
Spencer et a1
ACKNOWLEDGMENTS
This study was supported by a Queens College of the City University of New York
Faculty Research Grant.
The authors are indebted to Dr. C. E. Hopla, Department of Zoology, and Dr. W. B.
Lemmon, Director, Institute for Primate Studies, University of Oklahoma, Norman, for
their assistance. Also, Andrew Sillen for initial discussions which led to the development of this approach.
REFERENCES
Fleisher, M.; Schwartz, M.K.; Winawer, S.J.
Laboratory studies on the HEMOCULT'"
slide for fecal occult blood, pp. 181-187 in
COLORRECTAL CANCER PREVENTION,
EPIDEMIOLOGY AND SCREENING. S.
Winawer and D. Schottenfeld, eds. New York,
Raven Press, 1980.
Glober, G.A.; Peskoe, S.M. Outpatient screening for gastrointestinal lesions using guaiacimpregnated slides. AMERICAN JOURNAL
OF DIGESTIVE DISORDERS 19:399-403,
1974.
Goodall, J. Feeding behavior of wild chimpanzees: A prehminary report. SYMPOSIA OF
T H E ZOOLOGICAL SOCIETY OF
LONDON 10139-47,1963.
Harding, R.S.O. An order of omnivores: Nonhuman primate diets in the wild, pp. 191-214
in OMNIVOROUS PRIMATES. R.S.O.
Harding and G. Teleki, eds. New York, Columbia University Press, 1981.
Hladik, C.M. Chimpanzees of Gabon and Chimpanzees of Gombe: Some comparative data on
t h e diet, pp. 481-501 in PRIMATE
ECOLOGY. T.H. Clutton-Brock, ed. New
York, Academic Press, 1977.
Jaffe, R.M. False-negative stool occult blood
tests caused by ingestion of ascorbic acid
(Vitamin C). ANNALS OF INTERNAL
MEDICINE 832324,1975.
McGrew, W.C. Animal foods in the diets of wild
chimpanzees: Why cross-cultural variation?
CARNIVORE, in press.
McGrew, W.C.; Tutin, C.E.G.; Baldwin, P.J.;
Sharman, M.J.; Whiten, A. Primates preying
upon vertebrates: New records from West
Africa. CARNIVORE 1:41-45, 1978.
McGrew, W.C.; Tutin, C.E.G.; Baldwin, P.J.
New data on meat-eating by wild chimpanzees. CURRENT ANTHROPOLOGY 20:
238-239,1979.
Milton, K. Food choice and digestive strategies
of two sympatric primate species. AMERICAN NATURALIST 117:496-503,1981,
Moreno-Black, G. The use of scat samples in
primate diet analysis. PRIMATES 19:
215-222,1978.
Nishida, T.; Uehara, S.; Nyundo, R. Predatory
behavior among wild chimpanzees of the
Mahali Mountains. PRIMATES 2O:l-20,
1979.
Ostrow, J.D.; Mulvaney, C.A.; Hansell, J.R.;
Rhodes, R.S. Sensitivity and reproducibility
of chemical tests for fecal occult blood with
emphasis on false-positive reactions. AMERICAN JOURNAL OF DIGESTIVE DISEASES 18:930-940, 1973.
Suzuki, A. The origin of hominid hunting: A
primatological perspective, pp. 259-278 in
SOCIOECOLOGY AND PSYCHOLOGY OF
PRIMATES. R.H. Tuttle, ed. The Hague,
Mouton, 1975.
Teleki, G. THE PREDATORY BEHAVIOR
O F WILD CHIMPANZEES. Lewisburg,
Bucknell University Press, 1973.
Winawer, S.J.; Sherlock, P.; Schottenfeld, D.;
Miller, D.G. Screening for colon cancer.
GASTROENTEROLOGY 70:783-789, 1976.
Wrangham, R. The behavioral ecology of chimpanzees in Gombe National Park, Tanzania.
Ph.D. Dissertation, University of Cambridge,
1975.
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