176 V. PARASITOLOGICAL EXAMINATIONS OF PREHISTORIC HUMAN COPROLITES FROM LOVELOCK CAVE, NEVADA F. L. Dunn and R. Watkins Department of International Health and George Williams Hooper Foundation. University of California Medical Center, San Francisco This paper summarizes the findings in two series of parasitological and other biomedical investigations of prehistoric (i.e., pre-contact) human copro- lites collected by Professor R. F. Heizer and his colleagues from several localities in Lovelock Cave, a Great Basin dry cave site in Churchill County, Nevada. Our studies were initiated in 1965-1966 when we examined a group of 52 specimens. Some of the findings of these preliminary studies have been noted briefly elsewhere (Dunn 1968; Heizer 1967; Heizer and Napton 1969). Studies of 116 additional specimens were undertaken during 1969 in an effort to provide further substantiation for some of the earlier observations. For this report we have combined the data for the two series of studies; thus we describe findings for samples from 168 Lovelock Cave coprolites. We shall also report briefly on the examination of a single sample of dry fecal material taken from the pelvic cavity of a mummified human, excavated from a cave site near Pyramid Lake, Nevada. SPECIMENS AND METHODS The localities in Lovelock Cave from which the coprolites of this report were collected are listed in Table 1, together with the numbers of samples from each locality, the numbers of samples that were parasitologically "posi- tive", and the types of samples. In the 1965-1966 study we examined coprolites from two localities in Lovelock Cave: those from the "entrance" or "crevice lot" (from a rock cre- vice "latrine" in the ancient entrance to the cave) and those from the "interior" or "cache lot" (from a concentration of specimens in the interior of the cave) (Heizer 1967:1-2, 12). A coprolite from the interior lot has been dated at A.D. 756+60 (UCLA-1071-F) while a specimen from the entrance lot has been dated at A.D. 1805+80 (UCLA-1071-E). In further discussion of these dates Tubbs and Berger (1967:89-92) indicate that the age of the interior lot coprolite was actually about 680, 800, or 880 A.D. (depending upon interpretation of the re- lationship between radiocarbon and calendar years at the time in question) and that of the entrance lot coprolite was either about 250 or 300 years old for similar reasons. The specimens for the 1969 studies came from three localities in the western end of the cave: the West Alcove (near the shelter line), the West End (in the interior), and the West Crevice (running between the West Alcove 177 and the West End). Five localities within the cave are thus represented in the series of samples examined for parasites (see Napton and Heizer, this volume). Forty-three samples were received in the dry state; these were reconsti- tuted in 0.5 percent aqueous solution of trisodium phosphate in the laboratory in San Francisco. The remainder of the samples were previously reconstituted fine screenings, received from the laboratory in Berkeley in small vials. The dry samples from the entrance lot (examined in 1965-1966) were fairly large frag- ments of whole coprolites. These large samples were required so that uncontami- nated material could be removed from the core of each fragment under sterile conditions for a bacteriological investigation. Small quantities of each vial sample or reconstituted dry sample were examined microscopically as direct smears under 22 x 22 mm. coverslips. In the 1965-1966 studies at least two direct smears were prepared from each specimen and examined completely and systematically under several magnifications. In the 1969 study one smear per specimen was searched. Many smears were, of course, examined for those speci- mens with positive findings on initial screening. RESULTS Bacteriological Studies Core samples were removed with sterile techniques from each of the 16 dry specimens from the entrance lot. These were studied by Drs. Vera L. Sutter and Valerie Hurst of the Department of Microbiology, School of Medicine, Univer- sity of California Mecical Center, San Francisco. In their report of 28 October 1965 on this investigation they state that the specimens were tested for the presence of coliform bacteria, Salmonella, Shigella, and coagulase positive Staphylococcus aureus "with negative results for all of these bacteria. Our methods were as follows: 1. Specimens were emulsified in a small amount of water. 2. 0.1 ml of the heavy suspension was inoculated to EMB, SS, and Tellu- rite Glycine Agars. 3. All cultures were incubated at 370 C. for 72 hours before being discarded as negative." Crystals of the Charcot-Leyden Type Specimen SF-13 from the entrance lot--a semi-formed fecal mass--was found to contain rare crystals resembling those known as Charcot-Leyden crystals which are often noted in diarrheal or dysenteric fecal specimens in association with the intestinal protozoan Entamoeba histolytica, the agent of amoebiasis. Cry- stals of this type were detected only in this single specimen. Protozoan cysts, if present in SF-13 or other samples, were not recognized as such. Mites Fourteen sarcoptiform mites were recovered from the fine coprolite screen-' ings in vial B-29 of the 1965-1966 entrance lot series. These well-preserved 178 specimens are described in a separate report by Radovsky (1970). No other Lovelock Cave coprolites have yielded mites. Rhabditoid Nematodes Specimens of rhabditoid nematodes, mainly larvae but also several mature forms, were encountered in six coprolites from four of the five cave locali- ties (table 1). Some of these specimens are illustrated (figs. 1-3). At least two species seem to be represented but specific and generic identifica- tions have not been made and may not be feasible unless additional mature specimens can be recovered. If these worms are plant parasites they must have been ingested with plant foods; if, on the other hand, they are free- living worms they must have penetrated the fecal material before desiccation occurred, i.e., very soon after excretion of the feces. Helminth Eggs One of the most interesting observations in the initial study (1965-1966) was the apparent absence of the common parasitic helminth eggs of man, espe- cially those of the hookworms (Necator americanus and Ancylostoma duodenale), the whipworm (Trichuris trichiura), and the large roundworm (Ascaris lumbri- coides). Nor were the eggs and/or larvae of other less common parasitic helminths of man detected. Our additional studies in 1969 were carried out in an attempt to confirm or disprove this observation of apparent freedom from intestinal helminth infection, and the recent examinations have indeed failed to reveal eggs or larvae of the common parasitic helminths. However we have encountered eggs of two species of presumed helminths that remain to be iden- tified. The first of these is a single egg, apparently that of a fasciolid trematode (a Fasciola or Fasciolopsis-type egg), found in a smear of material from a West Crevice coprolite (WC-20). This thin-walled and rather distorted egg, whose identification even at the familial level must remain tentative, measured 60 microns in greatest breadth and 100 microns in length. Unfortu- nately, satisfactory photographs of this egg were not obtained. The thick- walled eggs of a second helminth species are beautifully preserved; details of embryonic structure are visible within the egg (fig. 4). It has not as yet been possible to identify these eggs. The average dimensions are 60 microns in length and 40 in breadth. Two of these eggs were seen in smears from a West End coprolite (WE-22) and three were found in specimen WE-28, also from the West End. Examination of Fecal Material from a Pyramid Lake Mummy Fecal material collected from the pelvic cavity of a mummified human, Burial #2, excavated at Pyramid Lake site 26-WA-525 in Nevada was also made available to us for parasitological study. A dry sample of the intestinal contents was reconstituted in San Francisco and many direct smears were searched 179 for parasites. None was found, but a single fragment of a rhabditoid nematode and one very well-preserved hypopal stage sarcoptiform mite were recovered. The mite, which has proven to be of exceptional interest, is described in the separate contribution by Radovsky (1970). DISCUSSION The lack of success of Drs. Sutter and Hurst in their 1965 effort to cul- tivate pathogenic bacteria from Lovelock Cave coprolites was experienced also by Tubbs and Berger (1967) who have described a similar bacteriological attempt using two coprolites from the same cave site. Many smears prepared from freshly reconstituted dry samples contained yeast cells but we were not able to culti- vate these either. No protozoan cysts were recognized as such although small objects in some of the smears were cyst-like and provoked speculation about their possible protozoan nature. Protozoa appear to have been recognized in coprolites in the Americas only once, i.e., cysts of Entamoeba sp. from a mummified Inca child from a tomb in the Andes (Pike 1967). The presence of well-preserved rhabditoid nematodes in several coprolites of considerable antiquity--notably those from the West End locality which may be more than 2000 years old (R. F. Heizer and L. K. Napton, personal communica- tion)--indicates that parasitic worms and eggs, if present in the feces at the time of excretion, should also have been well-preserved. The rhabditoids may have been ingested with plant materials or may have penetrated the feces from the soil prior to desiccation, but in any case they are ancient, dating from the time when the feces was formed or was still freshly passed and moist. Samuels (1965:176) has observed similar nematodes in Weatherill Mesa coprolites and has reached the same conclusion about their antiquity. Since we should have been able to detect parasitic helminth eggs or worms if present in the coprolites, and since we have now examined an adequate num- ber of specimens (168), we may conclude with some assurance that the inhabitants of Lovelock Cave at the times represented by these coprolites were free from infection with the common parasitic helminths of the human intestinal tract. We cannot be quite so certain that the inhabitants of the cave were actually free of pinworm (Enterobius vermicularis) infection because pinworm eggs are commonly shed by the female worm outside the intestinal tract on the peri-anal skin. Pinworm infection without pinworm eggs in the stool is a normal occur- rence; thus these infections are often overlooked upon routine stool examination for eggs. That Enterobius infection could have occurred in Lovelock Cave inhabi- tants is at least suggested by the discovery of such eggs in a prehistoric copro- lite from Wetherill Mesa, Colorado (Samuels 1965). The presumed fasciolid egg from a West Crevice coprolite may represent human parasitism but it could also represent pseudo-parasitism resulting from 180 ingestion of a parasitized host animal. The second, unidentified egg (from a West End coprolite and therefore perhaps several thousand years old) is probably a pseudo-parasite. The egg is not identifiable with one of the familiar parasitic helminths of man; in all likelihood it was ingested with infected animal viscera. With the addition of these eggs, apparently shed by two kinds of hel- minths, the literature for the Western Hemisphere provides evidence for pre- contact human parasitism or pseudo-parasitism by six kinds of helminths. The four others are: 1) Enterobius vermicularis, the pinworm of man, recog- nized in a 1000 year old coprolite from Wetherill Mesa, Mesa Verde National Park, Colorado (Samuels 1965). 2) A thorny-headed worm, Moniliformis sp. (probably Moniliformis clarki), that may have been parasitic or pseudo- parasitic in man at Danger Cave, Utah 2-4000 years ago (Moore et.al. 1969). 3) The whipworm, Trichuris trichiura, represented by eggs found in a mummi- fied eight or nine year old Inca child from a tomb in the Andes. The age of the body is said to be about 450 years (Pike 1967). 4) "objects that might have been the eggs of a species of tapeworm belonging to the genus Diphyllo- bothrium" (Callen and Cameron 1960:40) found in a Peruvian coprolite from Huaca Prieta dated between 3000 and 5000 years old. The record for Diphyllobothrium has been noted in several reviews as though it were fully confirmed, but Cameron himself was hesitant about the identification. How- ever he concludes that "there is a strong presumption that these objects actually are the eggs of a species of Diphyllobothrium." We have noted elsewhere that the apparent absence of common intestinal parasites in those who used Lovelock Cave is not surprising (Dunn 1968:222). The ecosystem that the occupants of Lovelock Cave exploited was, and remains today, relatively "simple" (in contrast to a tropical rain forest ecosystem, for example) and we have shown that one should expect to find very few species of parasitic organisms in man in such an ecosystem. This is the case for the Australian Aborigine and the African Bushman and it seems also to have been the case for those who occupied Lovelock Cave. But perhaps the apparent absence of Ascaris and hookworm at the cave also reflects total freedom from infection by these parasitic helminths for pre- Columbian man in the Western Hemisphere? Perhaps even whipworm was absent from man in the Americas prior to 1492, for the status of the Trichuris infec- tion in an Inca child is uncertain. The body of the child dates to about the time of first European contact in western South America; the child could have acquired the infection, indirectly, from one of the newcomers. At this stage we can only speculate. Many additional parasitological surveys of coprolites are needed to help us unravel the story of prehistoric human parasitism in the New World. 181 ACKNOWLEDGEMENTS We should like to express our appreciation to Dr. Vera L. Sutter and Dr. Valerie Hurst of the Department of Microbiology, School of Medicine, Univer- sity of California Medical Center, San Francisco for their contribution of a bacteriological study to this series of investigations. We should also like to thank Mr. Frank L. Lambrecht for his participation in the examination of the dry samples from the entrance lot. Finally we record our deep apprecia- tion to Prof. Robert F. Heizer and Mr. Lewis K. Napton for providing us with the samples and thus giving us the opportunity to conduct these studies. 182 4i '0 '.0L '.0m ct a' a'~~~~~M. a' , 7-A T-4 '4-4 I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~:410$ '.0 '.0 '.0 '.0 '.0 '.~~~~~~~~~~~~~~~~~~~~~~~~~~0 0 c P-4 co co -4-4 -4v-Iv- co C u 44 co~~~~~~~~~~~4. .r4 0 p~~~~~~~~~~~~~~.-CO .- M) bo (L)~~~~~~~~~~~~~~~o a v4C 0 -4Q . 4 P t 0 > -,4 $4 r4 r4 Q)~~~C U bO CU :4.J E -4 . > > > > > Q)~~~CU c ~ C 4. co CU U .7 co '44 C .74 0>.' 1). ~ o ) T- .3 -4 '-4 'I-4 ,- 4 4 . L r. 1-4 4- p4 0 0 .1-4 la) CU .rO L140'N Lt 0 a' .74 a) '- 04 I-40 '.o 4IJ 0 a) ( ) - 1)c U ) U) CU~~~~~~~~ Q z Q) 40 4.c a) ~ ~ ~~ ~~~~~Q A- O 4- 14)4Jc 4- '-4 a)) c I) U) (L 1 - T aE af CU - l 4 %)Q) Q Q .a) '-f3% C/ cc cc .0O c 14 0 C )0 0a '4-4 4'e bL) bO 1 4-J 4- 1-r4 I 1 4 c c -4 4U ) Q cc cc~~~~~~p COaU) En a),:3 3:4 ~74 : e CO'o ~~o 0- C) C4 1-4a C)4 ~ 4c -0 0 ' p.4 g I I I I I I I I 0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~r C CIO r~~~~~~~~~-z- l )).7 . a) 4-4 Cl.-4 I I I I I I I 4-J~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~9 44 o co . -. 4. 0)0 04 0 44 )-4 CD 0 a ) c c4 - - o ~~~~~~~~~~~~~~~-I .74 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~V- 4. 4- V -I a) 0 4.1~~~~~~~~~~~~~ . 34~ - >U'- >~~~~~~~3 41 0 C > X X X X S S ~ ~~ ~ ~~z ~4 , .74 z 4-4 Z- x ~ rl a > ' > X CU X XU FX X HX H H H . A c) H H: Hq X? ut c n . r o zo zo z .U . ?3 Explanation of Plates Plate 1 Anterior portion of a rhabditoid nematode larva from coprolite WC-8, West Crevice, Lovelock Cave, Nevada. Plate 2 Rhabditoid nematode larva from coprolite WE-22, West End, Lovelock Cave. About 170 microns in length. Plate 3 Coiled adult male rhabditoid nematode from coprolite WC-14, West Crevice, Lovelock Cave. Plate 4 Egg, presumably that of a helminth, from coprolite WE-28, West End, Lovelock Cave. Length 60 microns; breadth 40 microns, 184 4 * Bibliography Callen, E. 0. and T. W. M. Cameron 1960 A Prehistoric Diet Revealed in Coprolites. The New Scientist 8:35-40. Cowan, R. A. 1967 Lake-margin Ecologic Exploitation in the Great Basin as Demon- s-trated by an Analysis of Coprolites from Lovelock Cave, Nevada. Univ. Calif. Archaeol. Survey, Report 70:21-35. Dunn, F. L. 1968 Epidemiological Factors: Health and Disease in Hunter-gatherers. In R. B. Lee and I. DeVore, Man the Hunter, 221-228, Aldine Publishing Company, Chicago. Heizer, R. F. 1967 Analysis of Human Coprolites from a Dry Nevada Cave. Univ. Calif. Archaeol. Survey, Report 70:1-20. Heizer, R. F. and L. K. Napton 1969 Biological and Cultural Evidence from Prehistoric Human Copro- lites. Science 165:563-568. Moore, J. G., G. F. Fry and E. Englert, Jr. 1969 Thorny-headed Worm Infection in North American Prehistoric Man. Science 163:1324-1325. Pike, A. W. 1967 The Recovery of Parasite Eggs from Ancient Cesspit and Latrine Deposits: An Approach to the Study of Early Parasite Infections. In D. Brothwell and A. T. Sandison, Diseases in Antiquity, 184-188, Charles C. Thomas, Springfield, Illinois. Radovsky, F. J. 1970 Mites Associated with Coprolites and Mummified Human Remains in Nevada. Univ. Calif. Archaeol. Survey, (this volume). Samuels, R. 1965 Parasitological Study of Long-dried Fecal Samples. Soc. Amer. Archaeol., Memoirs 19:175-179. Tubbs, D. Y. and R. Berger 1967 The Viability of Pathogens in Ancient Human Coprolites. Univ. Calif. Archaeol. Survey, Report 70:89-92.