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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98(Suppl. I), January 2003, pp. 103-118 Human Intestinal Parasites in the Past: New Findings and a Review Marcelo Luiz Carvalho Gonçalves, Adauto Araújo/+, Luiz Fernando Ferreira
Escola
Nacional de Saúde Pública-Fiocruz, Rua Leopoldo Bulhões 1480,
21041-210, Rio de Janeiro, RJ, Brasil Supported by CNPq, Capes/Cofecub, Papes/Fiocruz. Received
26 August 2002 Code Number: oc03016
Almost all known human specific parasites have been found in ancient feces. A review of the paleoparasitological helminth and intestinal protozoa findings available in the literature is presented. We also report the new paleoparasitologic findings from the examination performed in samples collected in New and Old World archaeological sites. New finds of ancylostomid, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Trichostrongylus spp., Diphyllobothrium latum, Hymenolepis nana and Acantocephalan eggs are reported. According to the findings, it is probable that A. lumbricoides was originally a human parasite. Human ancylostomids, A. lumbricoides and T. trichiura, found in the New World in pre-Columbian times, have not been introduced into the Americas by land via Beringia. These parasites could not supported the cold climate of the region. Nomadic prehistoric humans that have crossed the Bering Land Bridge from Asia to the Americas in the last glaciation, probably during generations, would have lost these parasites, which life cycles need warm temperatures in the soil to be transmitted from host to host. Alternative routes are discussed for human parasite introduction into the Americas.
Key words: paleoparasitology - ancient diseases - helminths - protozoa - coprolites - mummies Parasites are organisms that found their ecological niche living in organisms of distint species, called hosts. Paleoparasitology is the study of parasites in archaeological material. Paleoparasitologic findings can provide valuable information related to the antiquity of human-parasite relationship, parasite dispersion and human migrations in the past (Wilke & Hall 1975, Horne 1985, Araújo et al. 1988, Araújo & Ferreira 1997, 2000, Reinhard 1990, 1992). Fecal remains usually are found in archaeological strata during archaeological excavations, in sediment from ancient latrines and cesspits or directly collected from mummies. Specimens are preserved by dry environment or by high concentration of soluble salts (Fry 1985). When feces are desiccated or mineralized, they are called coprolites (Heizer & Napton 1969). Rehydration of desiccated coprolites is necessary to proceed to paleoparasitological analysis. Water, sodium hydroxide and EDTA solutions have been used to rehydrate specimens, but it was observed that they caused egg distortion and disintegration (Fry 1985). Only after the use of trisodium phosphate solution by Callen and Cameron (1960), rehydration techniques could obtain reliable results. They adapted the technique employed by Van Cleave and Ross (1947) and by Benninghoff (1947) to rehydrate dried zoological and herbarium specimens respectively. Since 1960, rehydration in aqueous 0.5% trisodium phosphate solution has been the standard technique. To disaggregated mineralized coprolites, 5-10% chlorydric acid solution is used (Jones 1983). Parasite remains, mainly eggs and larvae, can be identified quite easily in ancient fecal material after rehydration. This paper summarizes the available literature about intestinal parasite findings in archaeological material. We discuss some biological aspects of the parasites found and we speculate about human dispersion in the past. We also performed the examination of ancient feces belonging to the collection of the Laboratory of Paleoparasitology of the Escola Nacional de Saúde Pública-Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil. MATERIALS AND METHODS
Specimens - A total of 894 samples of probable human coprolites and organic material from latrines and cesspits, belonging to the collection of our laboratory, were examined. Their origin are listed in Table I. The samples were dated either by 14C method or by cultural context. Examination techniques - The specimens were rehydrated by immersion in a 0.5% aqueous solution of trisodium phosphate for 72 h (Callen & Cameron 1960). The rehydrated sample solutions were mixed approximately 10:1 in acetic formalin solution to avoid fungal and bacterial growth. The material was allowed to sediment following the technique proposed by Lutz (1919). A portion of each sediment was used for microscopic examination. The material was placed on a slide and covered with a coverslip and examined for the presence of parasites. Twenty slides for each sample were examined at magnification of X100 and X400. All wet preparations were examined by at least two of the authors. All eggs and larvae were photographed and the eggs were measured. RESULTS
Presented below is a summary of the paleoparasito-logical findings available in the literature as well as the unpublished findings of our material. The data are separated by parasite and by chronological order. The archaeological site or the source mummy of the coprolite, country and date are given in Table II to Table XIV. Table XV shows non-intestinal human helminths. In Table XVI human paleoparasitological finds in the New and Old Worlds are shown, if pre or post-Columbian. DISCUSSION
One of the most rich source of information about paleoecology is the analysis of ancient human droppings. The analysis of micro and macro remains like pollen, undigested seeds, fibers, small bones, scales and charcoal can reveal important aspects of diet, paleoclimate, agricultural development and prehistoric human occupation of ancient sites (Heizer & Napton 1969, Wilke & Hall 1975, Fry 1977, 1985, Reinhard & Bryant Jr 1992). Dietary habits can also be inferred when parasites with intermediate host life cycles are found in coprolites and in latrine material (Callen & Camaron 1960, Patrucco et al. 1983, Ferreira et al. 1984). Study of the coprolites in a paleontologic context can reveal interesting epidemiological patterns. Differences in parasitism between prehistorical hunter-gathering from agriculturalist population have been shown, related to sanitary patterns, type of dwelling and diet (Reinhard 1988a). According to Darwin's theory, species have their origin in only one geographical area. Therefore, the utilization of parasites as biological markers allows a new approach concerning ancient human migrations. The dispersion of parasites in time can be used to trace migrations of their human hosts (Manter 1967, Araújo et al. 1988, Araújo & Ferreira 1997). A better understanding of parasite distribution in ancient world made it also possible to speculate about the antiquity of human-parasite relationship (Araújo & Ferreira 2000). There are parasites that are specific to a host species and others that are not. Some parasites are found only in phylogenetically related host species. This kind of relationship began with a common ancient host species, early in time. There are species of parasites, however, that do not have such specificity, adapting themselves to several non-related hosts. Such parasites have been acquired by behavioral, social and biological changes, which have propitiated the host-parasite encounter, sometime during evolutionary history (Araújo et al. 2000). Enterobius vermicularis is an example of an inherited parasite, which has been present in human ancestors (Hugot et al. 1999), whereas Diphyllobothrium latum, for example, although a parasite found in ancient human populations, was acquired by food habits during the conquest of new habitats sometime in the past of mankind. Helminths such as nematodes, cestodes, trematodes and acantocephalans keep their morphological parameters almost unchanged when 0.5% Na3PO4 aqueous solution is employed to rehydrate desiccated organic remains (Confalonieri et al. 1985, Fry 1985). Protozoa cysts are identified too, although cysts suffer a faster decay, resulting in artificially low estimations of protozoa as indicated by the infrequent finding of protozoan cysts in coprolites (Gonçalves et al. 2002). In a review, the correct identification of the origin of the specimens poses special problem in the validity of data. When the coprolites are not obtained from a mummified body, it can be difficult to ascertain its origin. The same problem arises when analyzing sediment from latrines. It is possible of misdiagnosis between human and wild or domestic animal fecal material (Fry 1977, 1985, Reinhard & Bryant Jr 1982). But, if there is no absolute test to determinate the zoological fecal origin, there are some well established criteria for differentiation between human and non-human coprolites. It is possible to select human coprolites based in their size, shape, macro and micro contents, and most important, parasites. The finding of an exclusive human helminth in a sample clearly indicates their origin (Reinhard & Bryant Jr 1982, Fry 1985). When that is not the case, the parasite egg size is a valuable tool to indicate the coprolite origin (Confalonieri et al. 1985). Interpretation of some parasitic findings can be troublesome. False parasitism should always be considered when eggs from a non-human parasite are recovered in a supposed human coprolite (Taylor 1955). These eggs may have been introduced in human digestive tract by consumption of some definitive parasite host. Zimmerman (1980) reported the finding of eggs from Cryptocotyle lingua, a fish trematode, in a 1,600 year old frozen Eskimo mummy. Although eggs from this trematode have been recovered from modern Eskimos, the adult helminth has never been found in humans. Similarly, Eimeria sp., probably E. mira, a protozoa of red squirrel, has been found in the intestine of the Bog Man from Grauballe, dated between 1540 and 1740 years BP (Before Present). It appears to be due to the ingestion of contaminated meat or offal (Hill 1990). The finding of Capillaria spp., a rodent parasite, in human coprolites have been reported (Bouchet 1997). Although very rare in humans, the presence of Capillaria eggs in feces may actually indicate the ingestion of an infected liver (Roberts & Janovy Jr 2000). Coimbra Jr and Mello (1981) reported the finding of Capillaria eggs in feces of two Indians from the Amazonian region, probably resulting from the habit of animal liver ingestion. Necator americanus and Ancylostoma duodenale are the most frequent ancylostomid parasitizing humans. The former was more frequent in Southern Africa, in the Americas, and in the Pacific Islands. A. duodenale was common in northern hemisphere, mainly in southern Europe, northern Africa, in India, in China and in southeastern Asia. But now, as a result of the large human movements throughout the world, their geographic distributions are quite ubiquitous. The worms mature and mate in the small intestine of the host. Eggs, passed with feces, hatch in environment if adequate moisture, shade and warm soil are found. The newly hatched larvae go on to develop eventually into the infective filariform larvae. The life-span of N. americanus and A. duodenale varies from 4 to 5 years and from 6 to 8 years respectively (Roberts & Janovy Jr 2000, Rey 2001). The species can not be diagnosed reliably when the only remains are eggs, as most cases in coprolites or latrine sediment. Differentiation of A. duodenale from N. americanus larvae is difficult, especially in rehydrated material. Ancylostomids have been found in archaeological sites from both New and Old Worlds (Table II). Most finds are from the Americas. Human infection has been present in Amerindians far before Columbus. It strongly suggests some kind of transoceanic contact before 7230 ± 80 years ago (the oldest finding by now) (Araújo et al. 1988, Araújo & Ferreira 1997). Ancylostomids, as well as other helminths that require warm and moist conditions to complete their life cycles outside their host, could not have survived during human migration by land through Bering Strait during the last ice age. Coastal navigation along the southern coast of the Bering Land Bridge is a more feasible route (Dixon 2001). Paleoparasitological finds from that region could support this alternative pathway of peopling of the New World. Unfortunately, from a paleopara-sitological view, most ancient coastal areas are currently underwater, due to the rise of oceanic water levels after the Ice Age. Ascaris is a cosmopolitan helminth. Adult worms live in the small intestine of the host, and, as the ancy-lostomids, passed eggs need suitable environment to continue development. But Ascaris eggs can remain viable in soil for some years, even under tough conditions. The adult life time is estimated to be 2 years (Rey 2001). Table III shows a very wide distribution of A. lumbricoides in the Old World, especially in the Middle Ages. It reflects poor sanitation and high population density in enlarging villages. A. lumbricoides and A. suum are morphologically and physiologically similar. They parasitize humans and pigs respectively. It has been suggested that a common ancestor of A. lumbricoides and A. suum originally parasitized pigs. Later, this ascarid adapted to humans when pigs were domesticated. But that is still an unsolved question (Roberts & Janovy Jr 2000). The finding of A. lumbricoides eggs in France (Table III), much earlier than the time of pig domestication, nearly 9000 years ago (Giuffra et al. 2000), suggests that humans were first parasitized. After pig domestication, the parasite adapted to pigs. Similar findings in the New World in pre-European context also suggest this. Trichuris trichiura adult worms live in the colon and is also a cosmopolitan parasite. Some 70 species of Trichuris have been reported from a wide variety of mammals. T. trichiura parasitizes humans, and as an-cylostomids and Ascaris, warmth and moisture are necessary to fully develop the embryos (Roberts & Janovy Jr 2000). T. trichiura lifetime is estimated to be up to 6-8 years (Rey 2001). The egg size sometimes can be a reliable tool for identifying the species of Trichuris in coprolites of unknown origin (Confalonieri et al. 1985). Paleopara-sitological findings (Table IV a, b) show its wide distribution, including the New World in pre-Columbian times. As A. lumbricoides, the wide distribution of T. trichiura in the Antiquity and in the Middle Ages, reflects human living conditions. For unknown reasons the findings of T. trichuris in the New World are more frequent than the findings of A. lumbricoides. Enterobius vermicularis is an exclusive human parasite. Organic material containing eggs of this parasite should be of human origin. As A. lumbricoides, E. vermicularis is cosmopolitan. The adults live mostly in the ileocecal region. The eggs are passed by migrating adult females in the anus and peri-anal area. The eggs can directly infect other host, either by fecal-oral route as through airborne inhaled and swallowed eggs. Its life time is estimated to be up to 2 months (Roberts & Janovy Jr 2000, Rey 2001). For unknown reasons, the finding of E. vermicularis in archaeological material outside the New World is scarce (Table V). A hypothetical origin of this parasite in the Americas can be ruled out. E. vermicularis has a long history of coevolution with its human host. They have been coexisting together in Africa long time before human dispersion throughout the continents (Ferreira et al. 1997, Hugot et al. 1999). Strongyloides stercoralis parasitizes humans, other primates, dogs, cats, and some other mammals. It is a parasite of tropical regions, although also found in temperate areas of the world. Having a complex life cycle, with a free-living larvae stage, the female adult worm lives in the small intestine of the host. The eggs usually hatches in the intestinal host lumen. The resulting larvae are passed in feces (Roberts & Janovy Jr 2000, Rey 2001). Table VI shows the findings of S. stercoralis in archaeological material. Caution should be exerted in diagnosing this parasite in sediment or coprolites. Free-living and ancylostomid larvae can be misidentified. Many species of Trichostrongylus parasitize the small intestine of many mammals and birds. Some species can infect humans. In some areas in Asia and Africa they are very frequent. In southwest Iran and in a village in Egypt, up to 70% of human population have been found infected (Roberts & Janovy Jr 2000). Trichostrongylus eggs are remarkably similar to those of ancylostomids, but usually are larger. Human infection have been detected only in the Americas up to now (Table VI). Fasciola hepatica is a helminth of cattle and sheep. Human infection occurs occasionally, mainly in certain areas of Europe, Africa and Latin America. The adult worm lives in the bile ducts of the host, passing eggs in feces. The eggs hatch in fresh water, and the parasite completes its life cycle in a snail. Infection occurs by ingestion of metacercaria in vegetation or in water. Occasionally human infections with other Fasciola species occur (Roberts & Janovy Jr 2000, Rey 2001). Human infection with Fasciola spp. have been detected in coprolites and ancient latrines sediment after herding begun (Table VII), and until now only in the Old World paleofeces. Three species of Schistosoma have major medical importance: S. haematobium, S. japonicum and S. mansoni. Only S. mansoni is found in the New World. S. hae-matobium is found mainly in Africa and Near East. S. japonicum is found in southeastern Asia and west Pacific. S. mansoni is found mainly in Brazil, Caribbean and Africa. Adult worms of S. haematobium live in veins of urinary bladder plexus of the host, so eggs are passed in the urine. Adult worms of S. japonicum and S. mansoni live in intestinal veins, and their eggs are passed in the feces. The eggs of Schistosoma spp. hatch in fresh water and the parasite complete its life cycle in a snail. Infection occurs when the parasite penetrates through the host skin (Roberts & Janovy Jr 2000, Rey 2001). Table VIII shows that the findings of Schistosoma spp. reflect in some degree their modern distribution, except the Americas in regard to S. mansoni. The findings in medieval Europe latrines reflects imported cases from Africa, since there is no intermediate host in Europe. Dicrocoelium dendriticum is a frequent parasite of ruminants. Rarely it is found in humans. The cycle is somewhat similar to that of Fasciola, but there are two intermediate hosts, a terrestrial snail and an ant. Although cases of true parasitism occur in humans, many reported cases of human infection are actually false parasitism, as eggs can be found in feces resulting from a recent liver repast (Taylor 1955, Roberts & Janovy Jr 2000). It is virtually impossible to distinguish true and false human parasitism when the only ancient host remains are feces. Clonorchis sinensis is found in southeastern Asia. It is a parasite of human and some other mammals. The adult fluke also lives in the host bile duct. The eggs are passed in the feces and the parasite completes its life cycle in two intermediate hosts, a snail and some species of fish and crustaceans. The definitive host is infected by eating raw or undercooked fish (Roberts & Janovy Jr 2000). Ancient infection by C. sinensis has only been found in mummified corpses from China (Table IX). Two species of Taenia are frequent human intestinal parasites. T. saginata, the most frequent, is found in almost all countries where beef is eaten. T. solium is endemic in Latin America, Africa and some Asian countries. Intermediate hosts of T. saginata and T. solium are cattle and pigs, respectively. In T. solium infection, human can be both intermediate and definite host. Infection occurs when one eats infected beef or pork. Eggs are passed in the human feces (Roberts & Janovy Jr 2000, Rey 2001). In paleoparasitologic analysis, most often the only egg structure found is the oncosphere. In this case it is not possible to distinguish between the two different species of Taenia that infect humans. As expected, Taenia spp. have not been found in the New World in pre-Columbian time (Table X). Pork and beef were not available. D. latum and D. pacificum are parasites of fish-eating mammals. The former is mostly found in Europe and North America whereas D. pacificum is found mostly in the pacific coast of South America. Diphyllobothrium spp. have two intermediate hosts, copepods and fishes. Living in the host intestine, eggs are passed in feces (Roberts & Janovy Jr 2000, Rey 2001). Human infections with D. pacificum and D. latum result from the ingestion of raw or undercooked marine and fresh water fishes respectively. Table XI shows that eggs found in archaeological material reflects the modern distribution of Diphyllobothrium spp. It is related to the habits of fish consumption by humans. Although rare, seven species of the phylum Acanthocephala have been reported parasiting human hosts. This phylum accomplishes parasites of fishes, birds, amphibians, mammals, and reptiles. At least two hosts are necessary to complete their life cycle. Depending on the species involved, the first host is an insect, crustacean or myriapod. The definite host passes eggs in the feces (Roberts & Janovy Jr 2000). Ancient human infection have been detected only in the Americas, mainly in USA (Table XIII), probably reflecting insect-eating habits. The intestinal Protozoa usually live inside the host in the intestinal lumen or inside the intestinal epithelial cells. The infective stage are cysts or oocysts. They are passed in the host feces. Humans most often are parasitized with Entamoeba spp. and Giardia duodenalis (Roberts & Janovy Jr 2000, Rey 2001). Cysts are not so resistant to decay as helminth eggs are. So, reliable findings of protozoa in coprolites and cesspit material are very rare (Table XIV). But some protozoa glycoprotein antigen, detectable by immunologic test, can still be found, even centuries after these parasites have been passed in feces. Gonçalves et al. (2002) detected G. duodenalis antigen by monoclonal antibody immunosorbant assay in samples dated to about 1200 AD, 1600 AD and 1700 AD, in coprolites and latrine soil from USA and Europe. Only one sample was positive to directed microscopic examination. Paleoparasitological analysis of human mummies, human coprolites and cesspit material have been demonstrating the diversity and antiquity of human parasitism. In Africa, the following parasites have been detected in ancient human feces: S. stercoralis, S. haematobium, Taenia spp., Echinococcus granulosus, Trichinella spiralis, Dracunculus medinensis, filarial worm, and possibly A. lumbricoides and T. trichiura. In Europe, ancylostomids, A. lumbricoides, T. trichiura, E. vermicularis, Fasciola spp., F. hepatica, S. mansoni, S. haematobium, Dicrocoelium spp., D. dendriticum, Opisthorchiformes, Taenia spp., Diphyllobothrium spp., D. latum, G. duodenalis, E. granulosus, T. spiralis, and possibly S. stercoralis have been found. In Asia, A. lumbricoides, T. trichiura, E. vermicularis, S. japonicum, C. sinensis, Taenia spp., T. solium, Diphyllobothrium spp., D. latum, E. histolytica, G. duodenalis, Chilomastix mesnili, and E. granulosus have been found. In Oceania, A. lumbricoides has been found. In South America, ancylostomids, A. lumbricoides, T. trichiura, E. vermicularis, Trichostrongylus spp., Paragonimus spp., Diphyllobothrium spp., D. pacificum, Hymenolepis nana, Acanthocephala, Entamoeba spp., G. duodenalis, Cryptosporidium parvum, Cyclospora cayetanenis, Isospora belli, Sarcocystis hominis, and possibly E. coli have been found. In North America, ancylostomids, A. lumbricoides, T. trichiura, E. vermi-cularis, Trichostrongylus spp., Opisthorchiformes, Taenia spp., D. latum, D. pacificum, Hymenolepsis spp., Acanthocephala, G. duodenalis, E. granulosus, T. spiralis, and possibly S. stercoralis, Fasciola spp. and D. dendriticum have been found (Tables II-XV). Ancylostomids, A. lumbricoides, T. trichiura and E. vermicularis have been found in the Americas much earlier than colonial times (Tables II-V). It can be inferred that humans have been infected by some parasites before the peopling of the New World, as already mentioned by Darling (1920) and Soper (1927) regarding ancylostomid infection. For the above-mentioned helminths, except probably for E. vermicularis, their main gate to the Americas was not a land route through Beringia (Araújo et al. 1988, Araújo & Ferreira 1995, 1997, Reinhard 1992). To some helminths, such as ancylostomids and T. trichiura, soil temperature is crucial to evolve to an infective stage. Therefore, transmission was discontinued when infected prehistoric migrants moved through the cold northern territories, from Siberia to the Americas. The new findings presented here confirm an-cylostomid and T. trichiura infection before Columbus's arrival. Dixon (2001), based on geological and archaeological data, hypothesizes that the first settlers used a sea-route along the southern coast of the Bering Land Bridge. Humans had vessels and were able to navigate near-shore waters prior to 14,000 BP (Dixon 2001). Whether by transoceanic route or coastal navigation, prehistoric settlers brought such soil-transmitted helminths to the New World, in a journey no longer than the life-span of these helminths. As more sensitive techniques become available, as detection of parasite DNA by polymerase chain reaction and immunological antigen detection by monoclonal antibody assays, more parasitic infections will be detected. New paleoparasitological findings are been reported throughout the world, updating continuously the knowledge of parasite distribution in the past. A more complete and accurate parasitic infection understanding in antiquity will improve our knowledge about biological and social aspects of health and disease process during the evolution of human species. Coprolites, in Patrick Horne's words, one of the "least-attractive of man's relics", are helping scientist to disclose some still unclear aspects of parasitism and human dispersion in ancient times (Horne 1985). We apologize for any data omission in the review. We would appreciate any aditional paleoparasitological finding sent by colleagues. REFERENCES
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