Are immunoenzymatic tests for intestinal protozoans reliable when used on archaeological material?

Intestinal protozoans found in ancient human samples have been studied primarily by microscopy and immunodiagnostic assays. However, such methods are not suitable for the detection of zoonotic genotypes. The objectives of the present study were to utilize immunoenzimatic assays for coproantigen detection of Cryptosporidium sp., Giardia duodenalis, and Entamoeba histolytica/Entamoeba dispar in sixty ancient human and animal samples collected from 14 archaeological sites in South America, and to carry out a critical analysis of G. duodenalis according to results obtained from three diagnostic methodologies: microscopy, immunodiagnostic tests (immunoenzymatic and immunofluorescence), and molecular biology (PCR and sequencing). More than half (31/60) of the samples analyzed using immunoenzymatic tests were positive for at least one of the intestinal protozoans, with 46.6% (28/60) corresponding to G. duodenalis, 26.6% (16/60) to Cryptosporidium sp., and 5% (3/60) to E. histolytica/E. dispar. Cryptosporidium sp. and G. duodenalis coinfection was observed in 15% (9/60) of the samples, whereas all three protozoans were found in 5% (3/60) of samples. In the Northeast Region of Brazil, by immunoenzymatic tests there is evidence that G. duodenlais and Cryptosporidium sp. have infected humans and rodents for at least 7150 years. However, for G. duodenalis, the results from the three diagnostic tests were discordant. Specifically, despite the efficiency of the molecular biology assay in the experimental models, G. duodenalis DNA could not be amplified from the ancient samples. These results raise the following question: Are all ancient samples positive for coproantigen of G. duodenalis by immunoenzymatic tests truly positive? This scenario highlights the importance of further studies to evaluate the sensitivity and specificity of the immunoenzymatic method in the archaeological context.

Recovering parasites from mummies and coprolites: an epidemiological approach.

In the field of archaeological parasitology, researchers have long documented the distribution of parasites in archaeological time and space through the analysis of coprolites and human remains. This area of research defined the origin and migration of parasites through presence/absence studies. By the end of the 20th century, the field of pathoecology had emerged as researchers developed an interest in the ancient ecology of parasite transmission. Supporting studies were conducted to establish the relationships between parasites and humans, including cultural, subsistence, and ecological reconstructions. Parasite prevalence data were collected to infer the impact of parasitism on human health. In the last few decades, a paleoepidemiological approach has emerged with a focus on applying statistical techniques for quantification. The application of egg per gram (EPG) quantification methods provide data about parasites' prevalence in ancient populations and also identify the pathological potential that parasitism presented in different time periods and geographic places. Herein, we compare the methods used in several laboratories for reporting parasite prevalence and EPG quantification. We present newer quantification methods to explore patterns of parasite overdispersion among ancient people. These new methods will be able to produce more realistic measures of parasite infections among people of the past. These measures allow researchers to compare epidemiological patterns in both ancient and modern populations.

Paleoparasitological analysis of the extinct Myotragus balearicus Bate 1909 (Artiodactyla, Caprinae) from Mallorca (Balearic Islands, Western Mediterranean).

Myotragus balearicus (Artiodactyla, Caprinae) is an extinct caprine endemic of the Eastern Balearic Islands or Gymnesics (i.e., Mallorca, Menorca and surrounding islets, Western Mediterranean Sea). In spite of its small size, c. 50cm height at the shoulder, it was the largest mammal inhabiting these islands until the human arrival, and it had peculiar short legs and frontal vision. It disappeared between 2830 and 2210calBCE. The coprolites here studied were recovered from Cova Estreta, in Pollença, Mallorca. The samples were subjected to microscopic examination and enzyme-linked immunosorbent assays (ELISA) for E. histolytica/E. dispar, Giardia intestinalis and Cryptosporidium parvum. This study provides new paleoparasitological data from an extinct animal species of the Holocene period. The microscopy revealed one sample containing uninucleated-cyst of Entamoeba sp., whereas ELISA detected nine positive samples for Cryptosporidium sp. The finding of these protozoans can help in the discussion of its extinction cause and demonstrates the antiquity and the evolutionary history of host-parasite relationships between protozoa and caprines since the Messinian.

Prehistoric Pathoecology as Represented by Parasites of a Mummy from the Peruaçu Valley, Brazil.

Paleopathologists have begun exploring the pathoecology of parasitic diseases in relation to diet and environment. We are summarizing the parasitological findings from a mummy in the site of Lapa do Boquete, a Brazilian cave in the state of Minas Gerais. These findings in context of the archaeology of the site provided insights into the pathoecology of disease transmission in cave and rockshelter environments. We are presenting a description of the site followed by the evidence of hookworm, intestinal fluke, and Trypanosoma infection with resulting Chagas disease in the mummy discovered in the cave. These findings are used to reconstruct the transmission ecology of the site.

Temporal and Spatial Distribution of Enterobius vermicularis (Nematoda: Oxyuridae) in the Prehistoric Americas.

Investigations of Enterobius sp. infection in prehistory have produced a body of data that can be used to evaluate the geographic distribution of infection through time in the Americas. Regional variations in prevalence are evident. In North America, 119 pinworm positive samples were found in 1,112 samples from 28 sites with a prevalence of 10.7%. Almost all of the positive samples came from agricultural sites. From Brazil, 0 pinworm positive samples were found in 325 samples from 7 sites. For the Andes region, 22 pinworm positive samples were found in 411 samples from 26 sites for a prevalence of 5.3%. Detailed analyses of these data defined several trends. First, preagricultural sites less frequently show evidence of infection compared to agricultural populations. This is especially clear in the data from North America, but is also evident in the data from South America. Second, there is an apparent relationship between the commonality of pinworms in coprolites and the manner of constructing villages. These analyses show that ancient parasitism has substantial value in documenting the range of human behaviors that influence parasitic infections.

Past Intestinal Parasites.

This chapter aims to provide some key points for researchers interested in the study of ancient gastrointestinal parasites. These few pages are dedicated to my colleague and friend, Prof. Adauto Araújo (1951-2015), who participated in the writing of this chapter. His huge efforts in paleoparasitology contributed to the development and promotion of the discipline during more than 30 years.

Recovery of Toxoplasma gondii DNA in experimentally mummified skin and bones: Prospects for paleoparasitological studies to unveil the origin of toxoplasmosis.

Paleoparasitology studies parasite infections by finding the parasites' remains in preserved organic remains such as natural or artificial mummy tissues, skeletons, teeth, and coprolites, among others. However, some currently important infections like toxoplasmosis have not been studied by paleoparasitology. The reasons include this parasite's complex life cycle, the resulting difficulties in locating this protozoan in the intermediate host tissues, and the limitation of coprolite studies to felines, the protozoan's definitive host. The current study thus aimed to produce an experimental model for molecular diagnosis of toxoplasmosis, prioritizing its study in bones and skin, the most abundant materials in archeological collections and sites. The study demonstrated the feasibility of recovering Toxoplasma gondii DNA from desiccated material, including bones and skin, in experimental models both with circulating tachyzoites (RH strain), characteristic of acute infection, and with cysts (ME49 cystogenic strain), characteristic of chronic infection. At present, most individuals with T. gondii infection are in the chronic phase, and the same was probably true in the past. The current study thus expands the odds of finding the parasite in archeological material, enhanced by the nature of the material in which the diagnosis was made. Finding the parasite may help answer questions that are widely debated in the literature on this protozoan's origin (Old World versus New World). In addition, when conditions do not allow ideal storage of samples for molecular tests, the methodology creates the possibility of testing oven-dried samples transported at room temperature.

THE PROCESS OF Leishmania INFECTION - DISEASE AND NEW PERSPECTIVES OF PALEOPARASITOLOGY.

Species of the genus Leishmania (Kinetoplastida, Trypanosomatidae) are causative agents of leishmaniasis, a complex disease with variable clinical spectrum and epidemiological diversity, constituting, in some countries, a serious public health problem. The origin and evolution of leishmaniasis has been under discussion regarding some clinical and parasitological aspects. After the introduction of paleoparasitology, molecular methods and immunodiagnostic techniques have been applied allowing the recovery of parasite remains, as well as the diagnosis of past infections in humans and other hosts. The dating of archaeological samples has allowed the parasitological analysis in time and space. This manuscript presents the state of the art of leishmaniasis and prospects related to paleoparasitology studies and their contribution to the evolutionary and phylogenetic clarification of parasites belonging to the genus Leishmania, and the leishmaniasis caused by them.

Evidence of Helminth Infection in Guanche Mummies: Integrating Paleoparasitological and Paleogenetic Investigations.

The Guanches, ancient inhabitants of the Canary Islands, Spain, practiced mummification of their dead. A paleoparasitological and paleogenetic analysis was conducted on mummified bodies (n = 6) (AD 1200, Cal BP 750) belonging to the Guanche culture from Gran Canaria Island. Coprolite and sediment samples (n = 19) were removed from below the abdominal region or sacral foramina. The samples were rehydrated in 0.5% trisodium phosphate solution for 72 hr at 4 C, and the paleoparasitological investigation was conducted by spontaneous sedimentation method and microscopic examination. The results revealed the presence of well-preserved eggs of Ascaris sp., Trichuris trichiura , Enterobius vermicularis , and hookworms. Ancient DNA was extracted from sediment samples to elucidate the ancestry of the mummies and for molecular detection of Ascaris sp. infection. Results of paleogenetic analysis demonstrated Ascaris sp. infection using 2 molecular targets, cytb and nad1. The mtDNA haplotypes U6b, U6b1, and HV were identified, which confirmed records of Guanche ancestry. The excellent preservation of Guanche mummies facilitated the paleoparasitological and paleogenetic study, the results of which contribute to our knowledge of Guanche culture and their health status.

Palaeoparasitology - Human Parasites in Ancient Material.

Parasite finds in ancient material launched a new field of science: palaeoparasitology. Ever since the pioneering studies, parasites were identified in archaeological and palaeontological remains, some preserved for millions of years by fossilization. However, the palaeoparasitological record consists mainly of parasites found specifically in human archaeological material, preserved in ancient occupation sites, from prehistory until closer to 2015. The results include some helminth intestinal parasites still commonly found in 2015, such as Ascaris lumbricoides, Trichuris trichiura and hookworms, besides others such as Amoebidae and Giardia intestinalis, as well as viruses, bacteria, fungi and arthropods. These parasites as a whole provide important data on health, diet, climate and living conditions among ancient populations. This chapter describes the principal findings and their importance for knowledge on the origin and dispersal of infectious diseases.

Potencialidades da Amazônia para a paleoparasitologia / The potentialities of the Amazon for paleoparasitology

Uma falácia científica muitas vezes leva à falta de interesse em estudar determinadas regiões geográficas no que diz respeito a áreas específicas do conhecimento científico. Este é o caso da Paleoparasitologia na região amazônica, que pretendemos desmitificar com esta revisão. Não é incomum encontrarmos afirmações de que na Amazônia seriam raras as possibilidades de recuperação de vestígios orgânicos preservados, o que limitaria o estudo da Paleoparasitologia. Isso pelo fato de a região haver sido habitada no passado por populações pequenas, o que resultaria em pouco material para o estudo, ou em razão dos baixos índices de preservação do material. Porém, isso não é verdade, uma vez que a região apresenta grande potencial de estudo, sobretudo de vestígios ósseos, mas também do solo arqueológico. A pesquisa deste material traria ganho inestimável para a área e poderia elucidar questões acerca da origem de infecções, se no Velho ou Novo Mundo, do caminho percorrido por elas, das datações e explicações para sua entrada nas Américas. A maioria das infecções que poderiam ser pesquisadas continua presente na região e acomete a população local constituindo, em alguns casos, importantes problemas na área da saúde coletiva. É preciso alertar os pesquisadores e moradores da região que têm conhecimento da existência deste material arqueológico sobre o fato de que, com as técnicas atuais, o menor fragmento ou quantidade do material possibilita seu estudo.

The identification of malaria in paleopathology-An in-depth assessment of the strategies to detect malaria in ancient remains.

The comprehensive analyses of human remains from various places and time periods, either by immunological or molecular approaches, provide circumstantial evidence that malaria tropica haunted humankind at least since dynastic ancient Egypt. Here we summarize the "actual state-of-the-art" of these bio-molecular investigations and offer a solid basis for the discussion of the paleopathology of malaria in human history.

Macracanthorhynchus hirudinaceus Eggs in Canine Coprolite from the Sasanian Era in Iran (4(th)/5(th) Century CE).

Present paper is the second publication introducing the paleoparasitological findings from animal coprolites obtained from archeological site of Chehrabad salt mine in northwestern Iran. The current archeological site is located in northwest of Iran, dated to the Sassanian Era (4(th)/5(th) century CE). In the summer 2012 the carnivore coprolite was obtained within the layers in the mine and were thoroughly analyzed for parasites using TSP rehydration technique. Eggs of 0 were successfully retrieved from the examined coprolite and were confidently identified based on reliable references. Identifying of M. hirudinaceus eggs in paleofeces with clear appearance as demonstrated herein, is much due to appropriate preservation condition has been existed in the salt mine .The present finding could be regarded as the oldest acanthocephalan infection in Iran.

Trichuris trichiura in a post-Colonial Brazilian mummy.

Trichuris trichiura is a soil-transmitted helminth which is prevalent in warm, moist, tropical and subtropical regions of the world with poor sanitation. Heavy whipworm can result either in Trichuris dysenteric syndrome - especially in children - or in a chronic colitis. In heavy infections, worms can spread proximally and may cause ileitis. Here we provide first microscopic evidence for a T. trichiura adult worm embedded in the rectum of a post-Colonial Brazilian adult mummy. During Colonial and post-Colonial times, many European chroniclers described a parasitic disease named Maculo whose symptomatology coincides with heavy helminthiasis. Based on our findings and on comparison of ancient textual evidence with modern description of heavy whipworm, we feel confident in considering that the two syndromes are expressions of the same pathological condition.

Leishmania tarentolae molecular signatures in a 300 hundred-years-old human Brazilian mummy.

BACKGROUND: L. tarentolae, the lizard-infecting species of Old World geckos, has been classified as non-pathogenic to man. While it has been demonstrated that L. tarentolae is capable of infecting human phagocytic cells and to differentiate into amastigote-like forms, there is no clear evidence for its efficient replication within macrophages. Here we provide first evidence for L. tarentolae ancient DNA sequences from bone marrow and intestines of a 300yo adult male. METHODS: We identified molecular signatures of Leishmania tarentolae, the lizard-infecting species of Old World geckos, in hard and soft tissue biopsies from a Brazilian mummy (A74) uncovered in Itacambira (Brazil) and dating to the Colonial Period (end of 18th/beginning of the 19th century). RESULTS: Our results imply that efficient replication of the parasite occurred within human macrophage and to lead to a systemic spread and visceralization in this individual. The ancient sequences show a 100% similarity with those of isolated L. tarentolae parasites grown on artificial nutrient media and a 99% similarity with two modern sequences isolated from reptiles. CONCLUSIONS: De facto, our findings re-open the debate about the potential survival of ancient L. tarentolae strain within human macrophage and its ability to spread systemically. They also raise ecological issues since it is unknown whether this parasite circulates in the reptilian reservoir in modern day Brazil or not. Investigations on fossil fauna and arthropods are needed to shed light on the interactions between saurian Leishmania and lizards in Brazil's remote and recent past.

Trichuris trichiura in a post-Colonial Brazilian mummy

Trichuris trichiura is a soil-transmitted helminth which is prevalent in warm, moist, tropical and subtropical regions of the world with poor sanitation. Heavy whipworm can result either in Trichuris dysenteric syndrome - especially in children - or in a chronic colitis. In heavy infections, worms can spread proximally and may cause ileitis. Here we provide first microscopic evidence for a T. trichiura adult worm embedded in the rectum of a post-Colonial Brazilian adult mummy. During Colonial and post-Colonial times, many European chroniclers described a parasitic disease named Maculo whose symptomatology coincides with heavy helminthiasis. Based on our findings and on comparison of ancient textual evidence with modern description of heavy whipworm, we feel confident in considering that the two syndromes are expressions of the same pathological condition.

Discovery of a 240 million year old nematode parasite egg in a cynodont coprolite sheds light on the early origin of pinworms in vertebrates.

BACKGROUND: We report the discovery of a nematode parasite egg (Nemata: Oxyurida) from a coprolite closely associated with the remains of several species of Cynodontia, dated to 240 million years old. This finding is particularly significant because this is the oldest record of an oxyurid nematode yet discovered, and because the cynodonts are considered a stem-group of the mammals. METHODS: We extracted material from a fully mineralized coprolite by both scraping the surface, and removing fragments from its interior with clean dental instruments used a single time. A single drop of glycerol from a new vial was added as a clearing reagent. Each slide was sealed with wax and examined with an optical microscope at 100× to 400× magnification. RESULTS: From one coprolite, 550 slides were examined; from 275 of these slides, sediment was examined that was scraped from the surface of the coprolite, and from the other 275 slides, material was examined that was extracted from the interior of the coprolite. All microscopic structures encountered were photographed, measured, and identified when possible. CONCLUSIONS: From the coprolite examined, we discovered an egg representing a new species of pinworm that, based on the egg structure, clearly places it in the family Heteroxynematidae. Nematodes of the order Oxyurida have very constrained life-histories, occurring only in animals that are not strictly carnivorous and also ingest large amounts of plant material. This fact enabled us to determine which species of cynodont, from several collected at the site in Brazil, are most likely the depositors of the coprolite, and therefore were the putative host of the parasite.

Helminths in feline coprolites up to 9000 years in the Brazilian Northeast.

The identification of parasites in animal coprolites has been an important tool to promote knowledge about parasites infecting different zoological groups in the past. It also helps the understanding of parasites causing zoonoses, which is especially important for animals that were part of the diet of prehistoric human groups. Nevertheless, the study of feline coprolites is still scarce. This study analyzed 30 feline coprolites from southeastern Piauí taken from archeological sites used by human groups in the past. Eggs of Spirometra sp., Toxocara cati, Spirurida, Oxyuroidea Calodium cf. hepaticum, Trichuris cf. muris, Trichuris sp., and other Trichuridae, Oncicola sp., and nematode larvae were found. Some of these findings reflect the consumption of infected prey. The role of felines in the transmission of helminthes causing zoonoses in the region is discussed.

Paleoparasitological studies on mummies of the Joseon Dynasty, Korea.

Paleoparasitology is the application of conventional or molecular investigative techniques to archeological samples in order to reveal parasitic infection patterns among past populations. Although pioneering studies already have reported key paleoparasitological findings around the world, the same sorts of studies had not, until very recently, been conducted in sufficient numbers in Korea. Mummified remains of individuals dating to the Korean Joseon Dynasty actually have proved very meaningful to concerned researchers, owing particularly to their superb preservation status, which makes them ideal subjects for paleoparasitological studies. Over the past several years, our study series on Korean mummies has yielded very pertinent data on parasitic infection patterns prevailing among certain Joseon Dynasty populations. In this short review, we summarized the findings and achievements of our recent paleoparasitological examinations of Joseon mummies and discussed about the prospects for future research in this vein.