Eimeria leuckarti in equid coprolites from the Sassanid Era (2nd-6th century CE) excavated in Chehrabad Salt Mine archaeological site, Iran.

OBJECTIVE: This study reports coccidian oocysts in an equid coprolite dated to the Sassanid Empire (2nd-6th century CE) recovered in Chehrabad Salt Mine archaeological site, Iran. METHODS: Between 2015 and 2017, an archaeoparasitological investigation led to the discovery of an equid coprolite in the Chehrabad Salt Mine archeological site, (Douzlakh), western Iran. Samples were rehydrated using trisodium phosphate solution and were examined by light microscopy. RESULTS: Seven oocysts of Eimeria leuckarti (Flesch, 1883) were identified; they were in various stages of sporulation. CONCLUSION: This is the first report of ancient coccidian oocysts from equids. The importance of this observation is discussed, and current knowledge of eimeriid oocysts at archaeological sites is reviewed. SIGNIFICANCE: The observations of E. leuckarti increases current knowledge of parasite biodiversity in ancient Iran when it rested along the Silk Road, a network of trade routes connecting the East and West that was central to economic, cultural, political, and religious interactions between these regions, and to livestock movement that could contribute to the transmission of the parasites from/to other regions. LIMITATIONS: The contextual information about animal species present in and around the Salt Mine during its working periods, including Achaemenid dynasty (6th to 4th century BCE) and Sassanid era (2nd to 6th century CE), is very limited and does not allow secure conclusions regarding the host origin of the coprolites. SUGGESTIONS FOR FURTHER RESEARCH: Application of molecular biology tools to identify the correct host origin of the coprolites and to detect more parasite species is advocated.

First paleoparasitological evidence of Muellerius sp. in sheep or goat dated to the Sasanian Empire (500 CE) in ancient Iran.

A fecal pellet was recovered in an ancient salt mine in Chehrabad located in western Iran (36.55° N, 47.51° E). Based on prior publications showing the success of the salt mine's environment to preserve various life forms, it was decided to try and ascertain whether this faex contained any parasites of paleoparasitologic interest. The rehydration involved placing the pellet in an aqueous solution of 0.5% trisodium phosphate for a week, followed by the examination of aliquots of the entire rehydrated sample on a total of 153 microscope slides. The examination of the rehydrated material revealed the presence of two larval nematodes; there were no eggs or oocysts recovered. The larvae were photographed and measured, and the decision was made not to submit the two larvae to grinding and DNA extraction for molecular diagnostics due to their excellent state of preservation. The larvae were identified as first-stage larvae that appear to represent the genus Muellerius. Herein are reported the finding of two first-stage larvae of Muellerius nematodes (Metastrongyloidea, Protostrongylidae) from the rehydrated fecal pellet collected in a Chehrabad salt mine from the era of the Sasanian Empire.

Genomic palaeoparasitology traced the occurrence of Taenia asiatica in ancient Iran (Sassanid Empire, 2th cent. CE-6th cent. CE).

Palaeoparasitology investigates parasitological infections in animals and humans of past distance by examining biological remains. Palaeofaeces (or coprolites) are biological remains that provide valuable information on the disease, diet, and population movements in ancient times. Today, advances in detecting ancient DNA have cast light on dark corners that microscopy could never reach. The archaeological site of the Chehrabad salt mine of Achaemenid (550-330 BC) and Sassanid (third-seventh century AD) provides remains of various biotic and abiotic samples, including animal coprolites, for multidisciplinary studies. In the present work, we investigated coprolites for helminth eggs and larvae by microscopy and traced their biological agents' DNA by Next Generation Sequencing. Our results revealed various helminths, including Taenia asiatica, the species introduced in the 1990s. Implementing advanced modern molecular techniques like NGS gives a paramount view of pathogenic agents in space and time.

Exceptional ancient DNA preservation and fibre remains of a Sasanian saltmine sheep mummy in Chehrabad, Iran.

Mummified remains have long attracted interest as a potential source of ancient DNA. However, mummification is a rare process that requires an anhydrous environment to rapidly dehydrate and preserve tissue before complete decomposition occurs. We present the whole-genome sequences (3.94 X) of an approximately 1600-year-old naturally mummified sheep recovered from Chehrabad, a salt mine in northwestern Iran. Comparative analyses of published ancient sequences revealed the remarkable DNA integrity of this mummy. Hallmarks of postmortem damage, fragmentation and hydrolytic deamination are substantially reduced, likely owing to the high salinity of this taphonomic environment. Metagenomic analyses reflect the profound influence of high-salt content on decomposition; its microbial profile is predominated by halophilic archaea and bacteria, possibly contributing to the remarkable preservation of the sample. Applying population genomic analyses, we find clustering of this sheep with Southwest Asian modern breeds, suggesting ancestry continuity. Genotyping of a locus influencing the woolly phenotype showed the presence of an ancestral 'hairy' allele, consistent with hair fibre imaging. This, along with derived alleles associated with the fat-tail phenotype, provides genetic evidence that Sasanian-period Iranians maintained specialized sheep flocks for different uses, with the 'hairy', 'fat-tailed'-genotyped sheep likely kept by the rural community of Chehrabad's miners.

Radiological and histological findings in ancient salt mummies from the salt mine of Douzlakh, Iran.

Computed tomography studies and histological analyses were performed on the mummified remains found in the Chehrabad salt mine in northwestern Iran. The ancient salt mummies are dated to the Achaemenid (550-330 BC) and Sassanid (3rd-7th century AD) time period and died in mining incidents. The aim of the study was to describe the radiological and histological findings of several ancient Iranian salt mummies with special interest in pathological and postmortem changes. The mummified remains show multiple traumatic alterations, such as fractures and signs of massive compression. Histological analyses can clearly differentiate soft tissue, however the preservation status is variable. These Iranian salt mummies are a rare example of the ancient Iranian population. The soft tissue and organs are well preserved, however in different degrees due to the varying conditions.

Herbivores Coprolites from Chehrabad Salt Mine of Zanjan, Iran (Sassanid Era, 224-651 AD) Reveals Eggs of Strongylidae and Anoplocephalidae Helminths.

BACKGROUND: The ancient Chehrabad Salt mine, a well-known archaeological site in Iran, has recently received increasing interest from Iranian and international archeologists. Also, the biological remains from this site have provided valuable sources for studying the pathogenic agents of ancient times. This study aimed to identify the parasitic helminth eggs preserved in the herbivores coprolites. METHODS: From 2011 to 2015, we received three coprolites belonging to herbivorous animals recovered during excavations in Chehrabad Salt mine of Zanjan, Iran. The coprolites were dated back to the Sassanid era (224-651 AD) by using radiocarbon accelerator mass spectrometry (AMS) and archeological stratigraphy methods. Following rehydration of the specimens in a 0.5% trisodium phosphate solution, the suspensions were mounted in glycerin jelly on glass slides and examined by a light microscope with 100x and 400x magnifications. RESULTS: Two coprolites belonged to donkeys and one to an unknown herbivore species. The recovered eggs belonged to members of two helminths families, Strongylidae, and Anoplocephalidae. Also, within the two coprolites, some mites, presumably of the order Oribatida, were observed. CONCLUSION: The presence of two different nematodes in the equids coprolites provide clues of the burden of helminths infection on working animal at the Sassanid time and demonstrates the appropriate preservation condition of biological remains in the ancient salt mine of Chehrabad as well.

Fasciola hepatica eggs in paleofaeces of the Persian onager Equus hemionus onager, a donkey from Chehrabad archaeological site, dating back to the Sassanid Empire (224-651 AD), in ancient Iran.

Fascioliasis is a highly pathogenic zoonotic disease caused by the liver trematodes Fasciola hepatica and F. gigantica. Within the multidisciplinary initiative against this disease, there is the aim of understanding how this disease reached a worldwide distribution, with important veterinary and medical repercussions, by elucidating the spreading steps followed by the two fasciolids from their paleobiogeograhical origins. Fasciola eggs were detected in paleofaeces of a donkey, probably the present-day endangered Persian onager Equus hemionus onager, found in the Chehrabad salt mine archaeological site, Zanjan province, northwestern Iran. The biological remains dated back to the Sassanid period, 224-651 AD. Egg characteristics allowed for their specific ascription to F. hepatica. The interest of this finding relies on the fact of being the first archaeological finding of Fasciola in Asia and the Near East. Moreover, it allows to reach many conclusions about historical, epidemiological and spreading aspects of the disease. The finding in Chehrabad indicates that, at that time, this fasciolid had already spread through the Zagros mountains eastward from the Fertile Crescent. In that region and in ancient Egypt, livestock domestication played a crucial role in facilitating the disease spread during the postdomestication period. Donkeys appear at present to be usually infected by fasciolids in countries of the Fertile Crescent - Ancient Egypt region or neighbouring that region, with prevalences from low to very high. The high pathogenicity and mortality induced by Fasciola in these equines should be considered as an additional potential factor among the causes of the extinctions of E. h. hemippus in Syria, E. h. hydruntinus in the Anatolia-Balkans area, E. h. onager in the Caucasus and maybe also its decline in Iran. Indeed, Eurasiatic wild asses were present in the region and neighbourhood of the Fertile Crescent when the domestication of the livestock reservoirs of Fasciola began.

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.

Radiological findings in an ancient Iranian salt mummy (Chehrabad ca. 410-350 BC).

OBJECTIVE: To study pathologies, peri- and postmortal alterations as well as the general preservation state of an ancient Iranian salt mummy. MATERIALS AND METHODS: Several mummified remains from two different time periods (1500-2500 BP) were found in the Chehrabad salt mine in Iran. Computed tomography was performed on Salt Man #4 (410-350 BC), the best preserved out of the six salt mummies (Siemens, Sensation 16; 512 × 512 matrix; 0.75-5 mm slice thickness, 240-mA tube current, 120-kV tube voltage, and 0.976-mm pixel size). RESULTS: Radiological analyses showed an excellent state of preservation of an adolescent body. Several normal variants such as aplasia of the frontal sinus as well as a rare congenital deformation of the 5th vertebral body (butterfly vertebra) have been observed. The individual shows multiple fractures, which is consistent with the theory that he died due to a collapse in the ancient salt mine. CONCLUSIONS: The salt preserved the soft tissue as well as parts of the inner organs remarkably well. However, further investigations including histology are needed to reveal additional details of the health status of this unique salt mummy.

Paleoparasitological Findings from Rodent Coprolites Dated At 500 CE Sassanid Era in Archeological Site of Chehrabad(Douzlakh), Salt Mine Northwestern Iran.

BACKGROUND: In this paper, paleoparasitological findings from rodent excrements obtained from Chehrabad Salt Mine archeological site located in northwest of Iran are demonstrated and discussed. METHODS: Chehrabad Salt Mine archeological site located in northwest of Iran, dated to the Achaemenid (mid 1(st) mill. BCE) and to Sassanid (3(rd) cent. - 7(th) cent. CE) period, is a unique study area to investigate parasites in the past millenniums in Iran. Rodent coprolites obtained from this archeological site were thoroughly analyzed for parasite eggs using TSP re-hydration technique. RESULTS: Specimen analyzed were attributed to juvenile and adult rats based on their apparent morphology comparing with the modern dried pellets of Muridea family. Helminth eggs retrieved from two positive pellets were identified as Trichosomoides crassicauda, yphacia sp. and Trichuris sp. CONCLUSION: The present paper discusses the first paleoparasitological findings of rodent gastrointestinal helminthes in Iran along with possible favorite items to rats in ancient Chehrabad Salt Mine.