Carnivores as zoonotic parasite reservoirs in ancient times: the case of the Epullán Chica archaeological cave (Late Holocene, northwestern Patagonia, Argentina)

Zoonoses are currently considered as one of the most important threats for Public Health worldwide. Numerous zoonoses known today have occurred since antiquity. Carnivores act as definitive hosts for many intestinal parasites; some of them are responsible for several zoonotic diseases. The aim of this work was to study the parasite remains found in coprolites assigned to carnivores from the archaeological site Epullán Chica (ECh) and to discuss the results from a zoonotic point of view. ECh is located in northwestern Patagonia, Argentina and was occupied since the end of the Late Holocene (∼2200 years B.P.). Nine coprolites were examined for parasites. Samples were processed by rehydration in a 0.5 % water solution of trissodium phosphate, followed by homogenization, filtered and processed by spontaneous sedimentation. The macroscopic remains were separated and dried at room temperature and were examined for diet analysis. Six out of 9 coprolites examined were positive for parasites. Representatives of at least 10 parasite taxa were registered. Results are in line with the reconstruction of the scenario of zoonoses in the past and the diseases that the human populations and animals from Patagonia could be exposed. The present study provides the first palaeoparasitological report of carnivore coprolites recovered from the archaeological site Ech and reflects contamination of the cave used by hunter-gatherers with different parasites causative of zoonotic diseases.


Introduction
Zoonoses are defined as any disease or infection that is naturally transmissible from vertebrate or invertebrate animals to humans and vice versa.Zoonoses are currently considered as one of the most important threats for Public Health worldwide.Viruses, parasites, bacteria and fungi can cause zoonotic infections (Bueno-Marí et al. 2015).Numerous zoonoses known today have occurred since antiquity.
Palaeoparasitology is the study of parasite remains from archaeological and palaeontological sites (Ferreira 2014), focussed on the knowledge of parasite-induced illness of humans in the past and on the palaeoecological knowledge of the environment, ecology, settlement, diet, hygiene and health in the antiquity (Reinhard 1992).Previous palaeoparasitological studies on South American ancient populations have shown the presence of the zoonotic helminths Echinostoma sp., Paragonimus sp., Diphyllobothrium spp., Capillaria spp., Trichostrongylus sp. and Acanthocephala (Sianto et al. 2009).
Carnivores act as definitive hosts for many intestinal parasites; some of them are responsible for several zoonotic diseases such as paragonimiasis, sarcocystosis, toxoplasmosis and cysticercosis (Jay 1996, Acha andSzyfres 2003).Current diversity of South American terrestrial carnivores is relatively high, with 40 out of 245 species of the order Carnivora (Hunter 2011).The palaeontological record exhibits that a rich diversity of carnivores also inhabited South America in the past (e.g.Soibelzon and Prevosti 2007).
From ancient times, a close interaction between humans and carnivores was present.This interaction may have favoured the transmission of zoonotic parasitic diseases.The study of carnivore parasites can improve the knowledge of human zoonotic parasitic diseases in the past.
The archaeological site Epullán Grande cave is located in the middle Limay River basin (northwestern Patagonia, Argentina) and yielded remains from ca. 10,000 years B.P. (before present) (Crivelli Montero et al. 1996).About 100 m east from this cave is located a smaller archaeological cave site, Epullán Chica (ECh), with a rich zooarchaeological record, occupied since the end of the Late Holocene (Fernández et al. 2016).The zooarchaeological material present in both caves evidences the interaction between hunter-gatherers and animals from Pleistocene-Holocene transition to historical times.
The aim of this work was to study the parasite remains found in coprolites assigned to carnivores from the archaeological site Epullán Chica and to discuss the results from a zoonotic point of view.

Material and methods
The archaeological site named Epullán Chica (ECh) is a cave located at the southern of Neuquén Province, Argentina, 5 km north of the Limay river valley (40°23′ 10″ S, 70°11′ 44″ W, 680 m) (Fig. 1).It is 5 m wide at the mouth and 3.5 m long and covers 11 m 2 .Maximum depth of the fill was 1.40 m.The archaeological sequence was dated around 2220 ± 50 years 14 C B.P. (from a charcoal sample recovered from the deeper layers) to the twentieth century (from a metal knife collected from the upper layers).ECh is located in the Monte-Patagonia ecotone of the three major vegetation units that occur in Patagonia: forest, Patagonian steppe and Monte desert.The annual precipitation is around 300 mm.Archaeological materials were recovered, including lithic artefacts, plant remains, owl pellets, freshwater mollusc shells (Diplodon chilensis), feathers of Cyanoliseus patagonus (burrowing parakeet), bones and egg shell fragments of Rheidae birds (Rhea americana [greater rhea] and R. pennata [lesser rhea]) and bones and teeth from large, medium and small-sized mammals, such as Lama guanicoe (guanaco), Chaetophractus villosus (large hairy armadillo), Leopardus geoffroyi (Geoffroy's cat), Lycalopex griseus (grey fox or chilla), Galictis cuja (lesser grison), Conepatus chinga (Molina's hog-nosed skunk), Microcavia australis (southern mountain ca vy) and nu m erou s sig modon tine rode nts (Fernández et al. 2016).
Nine coprolites collected from unit II from ECh (1980 ± 80 to 1740 ± 60 years 14 C B.P.) were examined for parasites.The coprolites were described, measured and weighted.The smaller samples were whole processed by rehydration in a 0.5 % water solution of trissodium phosphate (TSP) in a glass tube for at least 72 h, followed by homogenization, filtered and processed by spontaneous sedimentation (Lutz 1919).In the case of bigger samples, 0.5 g from the surface and the interior of coprolites was rehydrated.Samples were preserved in 70 % ethanol.Twenty slides of each sample were made with the aid of a drop of sediment mixed with one drop of glycerin and examined at 100× and 400× using a light microscopy.The measurements are based on those taken from well-preserved eggs.Egg dimensions and morphologies were compared with data from the literature in order to identify the parasites at the lowest taxonomic level.The macroscopic remains were separated and dried at room temperature and were examined for diet analysis.

Results
Table 1 presents measurements, microscopic and macroscopic remains and parasitological records of each coprolite.Macroscopic remains found in coprolites were indicative of an omnivorous diet.Coprolites number 1, 2 and 3 showed a mixture of inclusions such as small bone fragments, diminutive vertebrae and ribs, scales of reptiles, vegetal remains and chitin of insects.Coprolites showed dark colouration; they were very light and easily broken.Coprolites 4 to 9 were brown and cylindrics.Macroscopic observation of coprolite 4 displays light brown hairs, vegetal remains and fragments of insect exoskeletons.Coprolites 5 to 9 showed vegetal and insect remains.Coprolite 7 also displays diatoms.After rehydration, all samples exhibited a dark colouration (typical of carnivore faecal material) and the coprolite number 3 presented an intense smell.
Microscopic observations revealed that 6 coprolites examined contained parasite remains.At least representatives of 10 species of helminths were recovered.Helminth eggs of nematodes, cestodes and acanthocephalans were found (Table 1).In all cases, eggs were very well-preserved.
Three different parasite species were found in pellet number 2. Eggs identified as acanthocephalan (Archiacanthocephala, Oligacanthorhynchidae), probably Prostenorchis sp.(Travassos 1915), were brown-coloured and thick-shelled, with four membranes.The outer membrane was thick, granular and with hyaline poles; the inner membrane thin and the embryos with hooks in one extremity (Fig. 4).Eggs were found in internal and external samples.Egg measurements (N = 18) ranged from 90.0 to 100.0 μm (96.20 ± 3.16) long and 47.5 to 57.5 μm (50.38 ± 3.06) wide; they were found from both internal and external samples.
One egg compatible with the capillariid Eucoleus sp.(Nematoda: Capillariidae) (Fig. 7), probably E. aerophilus, was found in coprolite number 3 (internal sample).The surface of the wall exhibited a network of ridges.Egg measurements were 50.0 (without plugs) and 62.5 (with plugs) in length and 27.5 in width respectively.
The faeces of carnivores are characterized by their cylindrical shape, with subdivisions and tappered at one of the extremities (Chame 2003).However, in C. chinga, these subdivisions are not easy to distinguish (Medina et al. 2009).Hog-nosed skunks (Conepatus) are carnivores widely distributed, ranging from Texas to the outhernmost areas of South America.The skunk C. chinga is a mephitid whose distribution ranges from northeastern Perú to southern Chile and to certain areas of Bolivia, Paraguay, Uruguay, Argentina and Brazil.Studies on the feeding habits of C. chinga suggest a generalist consumer character, in which arthropods are the most frequent prey, although small mammals are also a substantial part of its diet (Castillo et al. 2014;Donadío et al. 2004;Medina et al. 2009).Other preys include annelids, amphibians, reptiles and carrions.The morphology, diet and measurements of the examined coprolites 1, 2 and 3 were similar to those of C. chinga.
Faeces 4 to 9 exhibited the typical carnivore characteristics.Their measures and morphology correspond to those of a small canid or feline.However, the diets of Felidae tend to be more specific than those of other Carnivora, generally consisting of mammalian prey of sizes commensurate with their own body size, with little or no fish, vegetation or invertebrates (Kruuk 1986;Walker et al. 2007).In general, foxes tend to be omnivorous and opportunistic.The grey fox is both diurnal and nocturnal and occurs in a broad range of habitats from grasslands to forests, although in Argentina, it typically inhabits arid and semiarid temperate portions of Patagonia and the Andes.L. griseus is a generalist omnivore.The diet may contain plant material, insects, birds, rodents, lizards and even a high amount of fruit (Núñez and Bozzolo 2006;Zapata et al. 2005).Measurements of studied coprolites were similar to that  Little is known about the parasite fauna of C. chinga.Spirometra erinacei and Atriotaenia sanmarci (cestodes) and A. conepati, Physaloptera cahuide and Physaloptera maxillaris (nematodes) were reported (Gomez-Puerta et al. 2009, 2012;Sarmiento et al. 1999;Stein et al. 1994;Vega et al. 2014;Vieira et al. 2008).
Species of Physaloptera are parasitic nematodes with the adult stages living in the digestive tract of amphibians (3 species), reptiles (45 species), birds (24 species) and mammals (over 90 species) and more than 82 cases were described from humans (Mohamadain and Ammar 2012).Their life cycle includes intermediate hosts (orthopterans and coleopterans) or paratenic hosts that harbour larval forms in the outer intestinal wall (Quadros et al. 2014).Domestic and wild carnivores (cougar, lynx, badger, raccoon, fox, striped hog-nosed skunk and coyote) may be infected after eating arthropods containing third-stage larvae.The development into the adult form takes place in the definitive host and the worms lodge, preferably in the oesophagus, in the gastric mucosa and in the small intestine (Ortlepp 1922).Frequent movement of these nematodes results in erosions and ulcers in the gastrointestinal tract (Naem and Asadi 2013).Physaloptera maxillaris was the most common helminth reported from striped skunks (Mephitis mephitis) from USA (Dyer 1970;Lincoln and Anderson 1972) and from hog-nosed skunks (Conepatus leuconotus), striped skunks (M.mephitis) and spotted skunks (Spilogale gracilis) from an area of sympatry from Texas (Neiswenter et al. 2006).Humans on rare occasions have been infected with this parasite.Physaloptera was identified in other prehistoric coprolites, which belonged to a canid (Fugassa et al. 2006), to a prehistoric human from Patagonia, Argentina (Fugassa et al. 2007) and to a human from the BLa Cueva de los Muertos Chiquitos^archaeological site, México (Cleeland et al. 2013).
Paragonimus species are extremely successful parasites with more than 40 species described (Blair et al. 2005).Paragonimus spp.exhibit a complex life cycle that includes a mammal as the definitive host and snails and crustaceans as intermediate hosts (crabs and crayfish).Definitive hosts usually become infected by eating raw or undercooked tissues from crustaceans.Paragonimiasis is a disease of humans and other mammals caused by several species of Paragonimus.Symptoms and signs mimic those of tuberculosis (Diaz 2013;Procop 2009).Human infections are present in Africa, Asia and America, including USA, Mexico, Costa Rica, Guatemala, Honduras, Nicaragua, Panamá, Colombia, Perú, Venezuela and Brazil (Acha and Szyfres 2003).Paragonimus has not been reported from Argentina.There only one previous palaeoparasitological record by Hall (1976) (in Horne who reported eggs of Paragonimus, from human coprolites from the Atacama Desert of northern Chile dated to 5900 B.P. Exoskeletal fragments of freshwater crayfish or shrimp were found in the same coprolites.Eggs were found in a coprolite probably assigned to the skunk.Presidente and Ramsden (1975) found P. kellicotti infection in striped skunks (M.mephitis) and also in minks (Mustela vison), red foxes (Vulpes vulpes) and coyotes (Canis latrans).
Individuals belong to the phylum Acanthocephala are exclusively parasitic, including approximately 1150 described species with indirect life cycles, always involving arthropods as intermediate hosts and vertebrates as definitive hosts (Kennedy 2006).A main characteristic of the phylum is a protrusible proboscis armed with recurved hooks.Worms are attached to the intestine walls of the definitive host.The family Oligacanthorhynchidae includes 12 genera (Amin 2013).One of them, Prosthenorchis, has been reported from South America, as P. elegans from primates and P. cerdocyonis from the crab-eating fox Cerdocyon thous; from Africa, as P. lemuri from lemurs in Madagascar, P. pardalis from Felis pardus in Sierra Leone and P. fraterna from felid Panthera pardus in Congo; and from Asia as P. sinicus, from a dog in China (Amin 2013;Gomes et al. 2015).Eggs identified as Macracanthorhynchus sp. or an unidentified species were found in raptor pellets from the same layer of ECh (Beltrame et al. 2015).There is scarce information of Oligacanthorhynchids from South America.Although very rare, acantocephalan human infection does occur.The earliest discovery of infection was from the coprolite of a prehistoric man in Utah with a significant number of Moniliformis eggs (Moore et al. 1969).The finds of acanthocephalans from North American archaeological sites were summarized by Fugassa et al. 2011.Most patients with acanthocephalan infection remain asymptomatic, but others experience abdominal pain, diarrea and anorexia with weight loss (Andres et al. 2014;Salehabodi et al. 2008).
Parasites of the superfamilies Strongyloidea, Trichostrongyloidea and Ancylostomatoidea are nowadays of major importance in human and veterinary medicine.The strongylid species found in carnivores belong to the suborder Strongylida (Ancylostomatidae) (Anderson et al. 2009).The most common species found are Ancylostoma spp.and Uncinaria spp.The life cycle of these hookworms begins with the larvae hatching from eggs that passed in the host's faeces, developing to the infective stage (filariform larva) in soil and accessing to the definitive host through skin penetration or via ingestion.Infective larvae can also establish infections if ingested.Larvae that penetrate the skin travel through various organs, including the respiratory tract, before entering the intestines and developing into mature hookworms.Hookworms can cause anaemia, abdominal pain and diarrhoea when they reside in the intestines or lungs with dermatologic and other signs during their migration through the body (Bowman et al. 2010;Traversa et al. 2014).Zoonotic hookworms in humans may cause different pictures of skin, enteric and pulmonary diseases, the cutaneous larva migrans being the most important.
From South America, Ancylostoma and Uncinaria are usually found in several species of domestic carnivorous as dogs and cats, as well as wild carnivorous as Darwin's fox (Lycalopex fulvipes) from Chile (Jiménez et al. 2012), L. griseus from Argentina (Zanini et al. 2006), L. geoffroyi from Argentina (Beldoménico et al. 2005), stray cat (Felis catus) from Brazil (Uchôa et al. 1998) and A. braziliense and P. concolor from Brazil.U. stenocephala was found in L. culpaeus from Tierra del Fuego, Argentina (Aguilera 2001), from the Magellanic and Artic regions of Chile (Alarcón Navarro 2005) and from Perú (Moro et al. 1998).Ancylostoma conepati was found in C. chinga from Perú and Brazil (Sarmiento et al. 1999;Vieira et al. 2008) and from Chubut Province, Argentina (Vega et al. 2014).
Trichostrongylus spp.are primarily parasites of herbivores with a worldwide distribution.Human infections are found mainly in warm areas.T. colubriformis adults live in the intestines of the host.The female lays eggs with faeces.Eggs hatch and mature into infectious larvae.Humans become infected, ingesting unwashed vegetables contaminated by animal faeces or contaminated water containing strongyloid larvae.Larvae mature into adults in the intestines.The zoonotic tricostrongylosis is produced by several species of Trichostrongylus.Sporadic cases of T. colubriformis in humans have been reported (Lattès et al. 2011;Sato et al. 2011).Their presence in carnivores is accidental.T. colubriformis was also found in L. griseus from Chile (Alarcón Navarro 2005).
Another nematode frequently associated to wildlife is E. aerophilus (syn.Capillaria aerophila).It is a trichuroid nematode affecting the respiratory system of dogs, cats, wild carnivores and occasionally humans (Traversa et al. 2011).Adult stages live embedded underneath the epithelium of trachea, bronchi and bronchioles of the infected host.Females produce non-larvated eggs, which are coughed up and swallowed, reaching the environment via the faeces, where eggs mature.Eggs may also mature within earthworms (facultative intermediate hosts).Animals become infected by ingesting the larvated eggs or, rarely, the invertebrates.After ingestion, eggs hatch; larvae penetrate the intestinal wall and migrate via the bloodstream to the lungs and moult and reach their sexual maturity after 3 to 6 weeks post-infection (Anderson 2000;Traversa et al. 2011).Clinical signs include coughing and sneezing and in severe cases cause chronic respiratory disease.E. aerophilus can infect humans, causing bronchitis, coughing, mucoid sputum, blood in the mucous, fever, dyspnea and pulmonary carcinoma-like masses (Lalosević et al. 2008).
Previous studies on carnivores from Patagonia (Argentina and Chile) stated that species found were Toxocara spp.and Toxascaris leonina (Aguilera 2001;Alarcón 2005;Jiménez et al. 2012;Sánchez Thevenet et al. 2003;Stein et al. 1994;Zanini et al. 2006).Species belong to genus Toxocara and Toxascaris which are zoonotic.Toxocarosis is a major helminth zoonosis.These parasites have an oral-faecal transmission cycle, and humans can be infected by ingestion of larvae in undercooked infected organ or muscle tissues (rare); infective eggs from contaminated soil (gardens, sandpits and playgrounds); from unwashed hands or raw vegetables or by direct contact with pets (Overgaauw and van Knapen 2013).A number of different syndromes have been attributed to Toxocara spp.and Toxascaris spp.infection: visceral larva migrans (VLM), ocular larva migrans (OLM) and covert toxocarosis (CT).In addition, associations with neurological and atopic symptoms have also been described.
The main faunal resources exploited by humans from ECh were L. guanicoe, C. villosus and eggs of Rheidae.Carnivores also seem to have been an occasional resource, possibly not only for nutrients (e.g.defleshing marks in Lycalopex sp. and G. cuja) but also for skins, as evidenced by skinning marks in the mandible of L. geoffroyi.In addition, evidences of L. griseus died naturally in situ were found, reinforcing the idea of the use of the cave as a shelter by carnivores (Fernández et al. 2016).Zooarchaeological studies of Epullán Grande revealed a human subsistence similar to that recorded in ECh, mostly focussed on the consumption of large-sized mammals (L.guanicoe), and an increase through time of a complementary source of food integrated by largesized birds (R. pennata) and medium-sized (L.griseus and L. culpaeus, C. chinga and C. villosus) and micro-sized mammals (the tuco-tucos Ctenomys spp., and the cavies G. leucoblephara and M. australis) (Cordero 2009;Crivelli Montero et al. 1996).These evidences show the close interaction of human with carnivores and therefore the exposition to the zoonoses previously mentioned.
Zooarchaeological evidence from ECh also includes freshwater mollusc shells (D. chilensis) reflecting that the freshwater ecosystems were part of the subsistence patterns of the Late Holocene hunter-gatherers of the middle Limay River basin (Fernández et al. 2016).Hunther-gatherers from ECh could be infected by Paragonimus if they have the practice of hunting crustaceans in the close rivers.
There are ∼1400 known species of human pathogens, including viruses, bacteria, fungi, protozoa and helminths (Taylor et al. 2001).Of these, 65 % are zoonotic and 177 species are responsible for emerging infectious diseases (Dutour 2013).Results displayed in the present study are in line with the reconstruction of the scenario of zoonoses in the past and the diseases that the human populations and animals from Patagonia could be exposed.The present study constitutes the first palaeoparasitological report of carnivore coprolites recovered from the archaeological site ECh and reflects contamination of the cave used by hunter-gatherers with different parasites causative of zoonotic diseases.

Fig. 1
Fig. 1 Map of the archaeological site Epullán Chica cave, northwestern Patagonia, Argentina