Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan.

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.

Zoonotic parasite infection from a funerary context: A Late Antique child case from Cantabrian Spain.

OBJECTIVE: To evaluate the presence of Dicrocoelium sp. in a child from a Late Antique funerary context from Cantabrian Spain and discuss whether the infection is true infection or pseudoparasitosis. MATERIALS: Four skeletons, including one from a 5-7 year old child, have been analysed from the archaeological site of El Conventón, dated between the sixth and seventh centuries AD. METHODS: The paleoparasitological study was conducted through the analysis of soil samples from different parts of the skeleton and funerary context using the rehydration, homogenization, and micro-sieving method, and visualized through brightfield microscopy. RESULTS: A soil sample from the pelvic region tested positive for Dicrocoelium sp. (possibly D. dendriticum). CONCLUSIONS: The child was infected with Dicrocoelium dendriticum, which based on archaeological and historical contexts may be related to hygiene or dietary behaviour. SIGNIFICANCE: We present one of the few cases of the identification of a Dicrocoelidae parasite directly associated with a human skeleton that provides historical knowledge of a zoonotic disease. LIMITATIONS: The diagnosis of a zoonosis through the identification of ancient parasites is complex. In addition, Dicrocoelium sp. in association with skeletal human remains is rare due to the potential low prevalence of this parasite. SUGGESTIONS FOR FURTHER RESEARCH: Highlight the importance of paleoparasitological analysis to link parasitic infection diseases with socioeconomic issues by using funerary contexts with skeletal remains.

A survey of sheep dicrocoeliosis in Sulaymaniyah slaughterhouse, northern Iraq in 2013-2014.

Dicrocoelium dendriticum is a helminth which lives in the bile ducts and gall bladder of mammalian definitive hosts. Dicrocoeliosis is thought to be endemic in some countries and it has an increasing prevalence in Northern Iraq, potentially due to increased importation of infected animals, particularly sheep, in high numbers from neighboring countries. The parasite's ability to infect rodents, wild animals, livestock and humans means this parasite is of significant interest in veterinary and human medicine. While D. dendriticum causes relatively mild clinical disease in animals, infection leads to liver condemnation at slaughter and subsequent economic losses to farmers. In this study, the livers of 91,486 sheep slaughtered at Sulaymaniyah New Slaughterhouse (Northern Iraq) were visually inspected for D. dendriticum infection between November 2013 and March 2014, with 1269 livers rejected due to D. dendriticum in this period, representing 29.4% of all condemned livers. The highest rate of rejection due to D. dendriticum infection was seen in December, possibly linked to increased numbers of intermediate host snails during the wet season. Routine inspection of condemned livers revealed adult flukes of D. dendriticum and eggs were seen in the faeces of imported sheep, indicating these animals may be a vector for introduction of D. dendriticum to this region. Due to the complex life cycle and wide range of animals which it infects, we cannot establish the exact route of introduction into Iraq; however, our study suggests that slaughterhouse workers, farmers and local health authorities should be aware of the presence of D. dendriticum, and the potential risks it represents to both human and animal health. Our data also suggest that some level of quarantine or border checks may be useful to prevent further introduction of D. dendriticum or other pathogens into Iraq, although this may prove difficult until accurate diagnostic assays are developed.

Parasitic causes of meat and organs in cattle at four slaughterhouses in Sistan-Baluchestan Province, Southeastern Iran between 2008 and 2016.

This 8-year (from 2008 to 2016) retrospective study calculated the percentage of carcass and organ (lung and liver) condemnations and estimated the direct financial costs at four slaughterhouses in Sistan-Baluchestan Province, Southeastern Iran. Each carcass and organ (lung and liver) was thoroughly examined through inspection, palpation and incision following the standard protocol. Identification of the parasites was performed macroscopically. The total direct economic loss due to meat's condemnation was estimated by adding weights of each organ or carcass part and multiplying individual organ totals by their 2016 market unit price. A total of 857,039 cattle were slaughtered during this period, 64,497 livers (7.5%), 31,401 lungs (3.6%) and the carcasses of 1,171 cattle (0.1%) were condemned due to lesions caused by parasites. The main parasitic lesions in the condemned livers were attributed to Echinococcus granulosus (4.2%), Fasciola spp. (3.1%) and Dicrocoelium dendriticum (0.1%). All the condemned lungs were due to E. granulosus (3.6%). Taenia saginata cysticerci were detected in 0.1% of inspected animals. Liver condemnation due to cystic echinococcosis was the highest in fall (4.7%, p < 0.001); while lung condemnation was the highest during spring (3.98%, p < 0.001). Liver condemnation due to Fasciola spp. was the lowest in winter (2.99%, p < 0.001). Carcass condemnation as a result of cysticercosis was the highest in summer (p < 0.001). Considering the 2016 market prices, condemnations due to the studied parasites caused direct costs estimated U.S. $ 3,191,879. To the best of our knowledge, this is the first report estimating the monetary losses due to parasitic infections in the slaughterhouses of this province. Due to the high financial impact of the studied parasites, a control programme should be implemented to decrease this impact.

Dicrocoelium spp. in cattle from Wa, Ghana: prevalence and phylogeny based on 28S rRNA.

Dicrocoeliosis is a trematode infection in cattle, sheep and goats caused by the small liver fluke, Dicrocoelium spp. Though endemic in Ghana, its disease situation is poorly understood. In the present study, the prevalence, distribution and worm load of Dicrocoelium spp. in cattle at slaughter in Wa were determined. A total of 389 cattle were screened during meat inspection for liver flukes, and polymerase chain reaction accompanied by DNA sequencing of the 28S rRNA gene was used to identify Dicrocoelium spp. Generally, prevalence of bovine dicrocoeliosis (small liver fluke) stood at 19.54 % with prevalence in males and females being 17.62 % and 21.43 %, respectively. Animals under 2 years suffered more infection than older ones (23.08 % vs. 16.80 %). Dicrocoelium infection was recorded in animals from all the communities where slaughtered cattle came from. On average, 31 flukes per infected animal were recorded. A molecular confirmatory test on seven flukes identified them as D. hospes. This preliminary study highlights the importance of bovine dicrocoeliosis in Ghana and has identified D. hospes as a causal agent. The data provides basis for further studies to appraise the trematode disease situation in animals and phylogeny of Dicrocoelium spp. circulating in Ghana.

Gastrointestinal parasite burden in 4th-5th c. CE Florence highlighted by microscopy and paleogenetics.

The study of ancient parasites, named paleoparasitology, traditionally focused on microscopic eggs disseminated in past environments and archaeological structures by humans and other animals infested by gastrointestinal parasites. Since the development of paleogenetics in the early 1980s, few paleoparasitological studies have been based on the ancient DNA (aDNA) of parasites, although such studies have clearly proven their utility and reliability. In this paper, we describe our integrative approach for the paleoparasitological study of an ancient population from Florence in Italy, dated to the 4th-5th c. CE. The first stage consisted in the study of sediment samples from the pelvic area of 18 individuals under light microscopy. This allowed us to detect Ascarid-type eggs belonging very probably to the human-infesting roundworm Ascaris lumbricoides. Ten subsamples were selected corresponding to five individuals, and we extracted their whole DNA following sediment aDNA protocols. A targeted approach allowed us to detect two nematodes and one trematode aDNA fragments, namely Ascaris sp., Trichuris trichiura, and Dicrocoelium dendriticum. Among the five individuals tested for microscopic eggs and aDNA, three of them showed the remains of eggs (only Ascarid-type), but all of them tested positive to the presence of at least one parasite aDNA. Microscopic diagnosis first guided our research for the selection of promising samples while the targeted aDNA approach significantly improved our knowledge in terms of parasitic diversity and frequency in this population subgroup. These results enabled us to discuss the possible impact of latent parasitism in this past population at the time of an epidemic, as suggested in Florence. In particular, the singular case of D. dendriticum detection is discussed in light of the present-day scarcity of genuine human infections. Nevertheless, actual infections are known in the paleoparasitological record, and food habits may have led to false parasitism in this historical context. aDNA leaching from overlying strata may also explain this detection. This study strongly pleads for a systematic integrative approach combining microscopy and aDNA in paleoparasitology.

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings.

Molecular Characterization of Fasciola and Dicrocoelium Species Isolated from Ruminant Livestock in Qazvin, Iran.

INTRODUCTION: Fascioliasis and dicrocoeliasis are the most frequent zoonotic diseases with increasing human health problems in different parts of Iran. Two species, Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica), are spread in the country. Molecular approaches have a decisive role in identifying both the species. The aim of this study was to detect Fasciola spp. and Dicrocoelium spp. by amplifying the ITS-2 and 28S rDNA gene sequence. METHODS: Overall, 30 infected liver samples were collected from the livestock of Qazvin, Iran. The adult flukes were collected from different livestock. DNA extraction and PCR amplification of ribosomal RNA gene region (ITS2) and 28S rDNA gene fragment were conducted and a phylogenetic tree was constructed. RESULT: All the isolates obtained from the cattle (No: 7) and 82.6% (No: 19) of sheep isolates were infected with F. hepatica species, whereas 17.4% (No: 4) of sheep isolates were infected with F. gigantica. It was also shown that F. hepatica was the predominant species of Fasciola present in the region. All the specimens were infected with Dicrocoelium dendriticum (D. dendriticum). CONCLUSION: Both the species of Fasciola were found in Qazvin. D. dendriticum was the sole infecting species of the Dicrocoelium genus in the livestock of the city of Qazvin. Further research studies are needed to determine the intermediate host of the parasites in the region.

Predicting environmental suitability and geographical distribution of Dicrocoelium dendriticum at littoral of Caspian Sea: An ecological niche-based modeling.

Dicrocoeliasisis caused by the small liver fluke (Dicrocoelium spp.), mainly Dicrocoelium dendriticum in domestic and wild ruminants. The small liver fluke is the probable predisposing cause of economic burden. The impact of geographic and climatic factors on the incidence of dicrocoeliasis has been severely ignored in different geographical areas. Due to the lack of data regarding dicrocoeliasis in Iran, this study was aimed to investigate the prevalence and intensity of ovine and bovine Dicrocoelium infection in the coastal strip south of the Caspian Sea. Fecal samples were obtained from the cattle and sheep in three provinces of Guilan, Mazandaran and Golestan at the littoral of the Caspian Sea. All collected samples were then tested by flotation methods for determining the number of eggs per gram of feces (EPG). Moreover, we applied maximum entropy niche-based modeling (MaxEnt), coupled with remote sensing and the Geographical Information System (GIS) to visualize the spatial distribution and risk factors of Dicrocoelium dendriticum at the littoral of Caspian Sea. A total of 2688 stool samples were collected from cattle (n = 1344) and sheep (n = 1344) in coastal provinces of the Caspian Sea including Guilan (n = 1280), Mazandaran (n = 768) and Golestan (n = 640) provinces. Based on the data presented here, the highest rate of infection was observed in Guilan and Mazandaran provinces. The results revealed the prevalence rates of 36.72% and 6.09% for sheep and cattle in Guilan province, respectively. This rate was 22.4% for sheep and 3.91% for cattle in Mazandaran province. However, the rate of sheep infection was 90% in some point locations. Dicrocoelium infection was found to be significantly different between three provinces in sheep (P < 0.00001, Chi = 111.633). Our findings exhibited a high reliability of the MaxEnt model, and area under the curve (AUC) values of the training and test data sets were determined to be 0.852 and 0.818, respectively. Jackknife analysis showed the relative variable contribution to the model performance, where four variables were found as key influential factors that highly affected the habitat suitability of the presence of the lancet fluke including the precipitation of driest quarter (Bio17), altitude, temperature seasonality (Bio4), and precipitation of driest month (Bio14). The findings of this study demonstrated a high presence rate of Dicrocoelium infection at the littoral of Caspian Sea, Iran. Moreover, climatic variables can be considered as important predictive factors affecting the distribution of infection in the studied areas. Further studies based on the findings of the GIS are also very important in the country for planning control programs.

Importance of Land Snails in Dicrocoeliosis Epidemiology.

Dicrocoeliosis is a helminthosis caused by the small liver fluke Dicrocoelium spp. (Trematoda, Dicrocoeliidae) parasitizing in the bile ducts and gall bladder of ruminants as well as many other animal species including humans. In the biological life cycle of Dicrocoelium, land snails are first intermediate hosts and ants are second intermediate hosts. Sporocysts and cercaria, which are larval stages, live in the hepatopancreas of land snails and metacercaria, which is also the larval stage, lives in the abdomen and brain of ants. Land snails, which are the first intermediate host of this parasite in Turkey, include Helicopsis derbentina, Helicopsis protea, Helicopsis krynickii, Cernuella virgata, Trochoidea pyramidata, Cochicella acuta, Monacha carthusiana, Helicella candicans, Helix aspersa, Helix lucorum, and Chondrus tournefortianus. Dicrocoeliosis is widespread in ruminants and affects their liver, which can lead to weight loss and reduced milk production. The number of reports on dicrocoeliosis is increasing due to the expansion of dry habitats and parasites becoming resistant to antihelminthic drugs. This study provides information on the epidemiology and control methods of Dicrocoelium.

Dicrocoelium Dentriticum in Explanted Liver: Report of an Unusual Finding.

Dicrocoelium dentriticum, a member of trematode type helminths, is a liver parasite of ruminants. Humans are infected accidentally by ingestion of intermediate host, through infected ants via eating of raw vegetables or drinking of contaminated water. Infection is often asymptomatic or results in subtle symptoms; therefore, infections are usually unrecognized. However, it can produce chronic cholangitis and swelling or adenomatous proliferation in the bile ducts and lead to abdominal pain, diarrhea, fatigue, jaundice, and other symptoms. We report a 49-year-old female patient with end-stage hepatic cirrhosis from viral hepatitis B and D coinfection who underwent liver transplant. Shortly after transplant, she developed symptoms suggesting an obstructed biliary duct. Liver needle biopsy was done 24 hours after transplant to rule out rejection. Biopsy of her explanted liver was also examined pathologically. Microscopic examination of the liver needle biopsy ruled out rejection. Prepared sections of explanted liver revealed a helminth in the common bile duct. Morphologic reconstruction of helminth by microscopic findings and consultation with an expert parasitologist supported the diagnosis of Dicrocoelium dentriticum.

Evaluation of molecular methods for the field study of the natural history of Dicrocoelium dendriticum.

There is a need for improved methods for the study of the impacts of climatic and livestock management change on the epidemiology of production-limiting helminth parasitic diseases. In this study we report the application of molecular methods to describe the natural history of the small lancet fluke, Dicrocoelium dendriticum on Machair pastures on the Inner Hebridean Isle of Coll. Our results build upon those of the only previous historic field study of D. dendriticum in the British Isles that had been undertaken on the same study site. We demonstrate the value of combining conventional parasitological methods with PCR amplification of a mitochondrial DNA fragment for the detection of D. dendriticum in ants and snails, and PCR amplification of ITS2 and 28S ribosomal DNA fragments to support the species identity of the intermediate hosts, to improving understanding of the epidemiology of D. dendriticum. We report the presence of D. dendriticum infection in cattle, sheep and rabbits grazing on Machair pastures. D. dendriticum infection was identified in a high percentage of the snails, identified as Cochlicella acuta and Cernuella virgata, and in a high percentage of Formica fusca and Myrmica ruginoides ants that were collected from, or clinging to, the tops of flowers. We have identified the involvement of different intermediate host species and higher prevalences of snail and ant infection than previously reported, in part reflecting differences between the sensitivity and specificity of morphological and molecular speciation methods. Overall, our results highlight the complex life history of dicrocoeliosis and illustrate the parasite's generalist host strategy that confers potential to exploit new niches created by climatic change or grazing management for habitat conservation.

Life Cycle, Host Utilization, and Ecological Fitting for Invasive Lancet Liver Fluke, Dicrocoelium dendriticum, Emerging in Southern Alberta, Canada.

The expansion of parasite distributions outside of their native host and geographical ranges has occurred repeatedly over evolutionary time. Contemporary examples include emerging infectious diseases (EIDs), many of which pose threats to human, domestic animal, and wildlife populations. Theory predicts that parasites with complex life cycles will be rare as EIDs due to constraints imposed by host specialization at each life-cycle stage. In contrast to predictions of this theory, we report 2 new intermediate hosts in the 3-host life cycle of the liver fluke Dicrocoelium dendriticum in Cypress Hills Provincial Park, Alberta, Canada. Results of sequence analysis of the cytochrome oxidase 1 (cox1) mitochondrial gene identified the terrestrial snail Oreohelix subrudis and the ant Formica aserva as first and second intermediate hosts, respectively, in the region. Neither of these intermediate hosts, nor their suite of domestic and wild mammalian grazers used in the life cycle, occurs within the native range of D. dendriticum in Europe. Our results from host surveys show that the prevalence of D. dendriticum in samples of O. subrudis varied between 4% and 10%, whereas mean metacercariae intensity in F. aserva varied between 33 and 41 (n = 163, mean ± SD = 38 ± 35). These results are the first to describe the complete life cycle of emerging lancet fluke in western North America. The process of multi-level ecological fitting, in which the lancet fluke possesses pre-existing traits to utilize host resources, rather than host species, at each life-cycle stage provides a mechanism for the establishment of this complex life cycle in a novel habitat and in novel hosts.

Study on the Relationship between Liver Parasitic Infections and Serum Vitamin A and ß-Carotene Status in Cattle.

OBJECTIVE: This study was conducted to evaluate the relationship between serum levels of vitamin A and ß-carotene in the liver of cattle. METHODS: A total number of 150 samples were selected according to the type of parasitic infections of the liver after postmortem examination and confirmation. Parasitic lesions in the liver were subdivided into three major parasites, including Fasciola hepatica, Dicrocoelium dendriticum, and hydatid cysts. Fifty samples were obtained from cattle without any parasitic infection as a control group. Serum levels of vitamin A, ß-carotene concentrations (µg/dL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity were assayed. RESULTS: Naturally infected cattle with D. dendriticum, F. hepatica and hydatid cyst showed lower vitamin A levels. Serum ß-carotene levels were significantly decreased in all groups (p<0.05). ALT and AST activities in animals with parasitic diseases were statistically higher than in control group (p<0.01). A significant negative correlation was determined between the ß-carotene, vitamin A levels and enzyme activities of the liver in all the three types of liver infections (p<0.001). CONCLUSIONS: This study indicated that serum levels of vitamin A and ß-carotene decline was present in cattle with liver parasite infection and vitamin supplements should be supplied.

On the presence and immunoregulatory functions of extracellular microRNAs in the trematode Fasciola hepatica.

Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune system and live inside a hostile environment such as the mammalian liver and bile ducts. Recently, microRNAs, a class of short noncoding gene regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction, and recent data illustrate that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe) and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions that we review here.

Characterization of nine microsatellite loci for Dicrocoelium dendriticum, an emerging liver fluke of ungulates in North America, and their use to detect clonemates and random mating.

This study characterizes polymorphic microsatellite loci from adults of the liver fluke Dicrocoelium dendriticum sampled from a population of sympatric beef cattle and wapiti in a region of emergence in southern Alberta, Canada. We also scrutinized the markers to validate their use in studying the population genetics of this complex life cycle parasite. Among the nine loci described, four deviated significantly from Hardy Weinberg Equilibrium (HWE) due to technical artefacts. The remaining five loci were in HWE. These five provided sufficient resolution to identify clonemates produced from the obligate asexual reproduction phase of the life cycle in snails and to assess the impact of non-random transmission of clonemates on measures of FIS, FST and genotypic disequilibrium. Excluding clonemates, we show that the sub-population of worms was in HWE, that average FIS within hosts was 0.003 (p=0.4922) and that there was no population genetic structure among hosts FST=0.001 (p=0.3243). These markers will be useful for studies of Dicrocoelium dendriticum ecology, transmission, and evolution.

Dicrocoelium dendriticum found in a Bronze Age cemetery in western Iran in the pre-Persepolis period: The oldest Asian palaeofinding in the present human infection hottest spot region.

Dicrocoeliasis of animals and humans is caused by trematode species of the genus Dicrocoelium, mainly Dicrocoelium dendriticum in ruminants of the Holarctic region. D. dendriticum may be considered an old parasite, probably related to the appearance and diversification of Eurasian ovicaprines, occurred 14.7-14.5 million years ago. The oldest palaeoparasitological findings of Dicrocoelium in domestic animals and humans date from more than 5000 years BC in Europe. Eggs of D. dendriticum have been found in a burial of a Bronze Age cemetery (2600-2200 BC) close to Yasuj city, southwestern Iran. This is the oldest finding of D. dendriticum in the Near East, where present human infection reports are more numerous than in other world regions where human dicrocoeliasis is rare and sporadic. This palaeofinding in the Zagros mountainous chain area is of interest by its location close to Persepolis, suggesting a narrow relationship between humans and herbivorous animals in these highlands. Domestic ruminant populations of these highlands were following a repeated contact with those of the western flat lowlands of the Fertile Crescent thanks to annual altitudinal transhumance migrations of the nomadic pastoral tribes with their herds living throughout Zagros Mountains in the several millennium period BC. It is concluded that D. dendriticum spread together with sheep and goats westward throughout Europe from the Fertile Crescent during the 8000-6000 year BC period and somewhat later southward into Africa, both spreads facilitated by the low specificity of that trematode species regarding the snail and ant intermediate hosts.

Pulmonary Arterial Lesions in New World Camelids in Association With Dicrocoelium dendriticum and Fasciola hepatica Infection.

In Switzerland, dicrocoeliasis is regarded as the most significant parasitic infection of llamas and alpacas. Fasciola hepatica infestation is also a problem but less common. The aim of the present retrospective study was to evaluate the lungs of New World camelids (NWCs) for evidence of arterial hypertension in association with liver changes due to liver fluke infestation. The lungs of 20 llamas and 20 alpacas with liver fluke infestation were histologically evaluated. The hematoxylin and eosin and van Gieson (VG)-elastica stains as well as immunohistology for the expression of α-smooth muscle actin (α-SMA) were used to visualize the structures of arterial walls. Parasitology of fecal matter (11 llamas and 17 alpacas) confirmed that most of these animals were infested with both Dicrocoelium dendriticum and other gastrointestinal parasites. In most cases (10/12 llamas, 4/6 alpacas), liver enzyme activity in serum was elevated. Histologically, arteries in the lungs of 9 of 20 llamas (45%) and 3 of 20 alpacas (15%) showed severe intimal and adventitial and slight to moderate medial thickening, which was confirmed with α-SMA and VG-elastica staining. All animals exhibited typical liver changes, such as fibrosis and biliary hyperplasia, in association with the presence of liver flukes. This study shows that liver flukes can induce proliferative changes in lung arteries in NWCs that resemble those seen with pulmonary arterial hypertension due to liver parasites in humans. However, the degree of liver fluke infestation was not correlated with the extent of liver damage, or with the amount of thoracic or abdominal effusion or pulmonary arterial changes.

Characterization of Dicrocoelium dendriticum isolates from small ruminants in Shaanxi Province, north-western China, using internal transcribed spacers of nuclear ribosomal DNA.

The genetic variations in internal transcribed spacers (ITS) spanning ITS-1, 5.8S and ITS-2 rDNA of Dicrocoelium dendriticum, isolated from sheep and goats in four geographical regions in Shaanxi province, were examined. The lengths of ITS-1, 5.8S and ITS-2 rDNA sequences for D. dendriticum were 749 bp, 161 bp and 234 bp, respectively. Intra-specific sequence variations of D. dendriticum were 0-0.5% for ITS-1 and 0-1.3% for ITS-2 rDNA, while the inter-specific variations among species in genus Dicrocoelium in ITS-2 rDNA were 3.4-12.3%. Phylogenetic analysis based on sequences of ITS-2 rDNA showed that all D. dendriticum isolates in the present study were grouped with reference D. dendriticum isolates from sheep and goats, and D. dendriticum isolates from cattle and Japanese serow were clustered in a sister clade. However, the phylogenetic tree could not reveal geographically genetic relationships of D. dendriticum isolates in different origins and hosts. These findings provided basic information for further study of molecular epidemiology and control of D. dendriticum infection in Shaanxi province as well as in the world.