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.

Morphological and molecular differentiation between Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 (Platyhelminthes: Digenea).

Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium hospes (Looss, 1907) are recognised to affect the liver of domestic and wild ruminants. A third species, Dicrocoelium orientalis which was described from musk deer in the Baikal region of the former Soviet Union and re-named to Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 was isolated from other species of deer in Asian countries and from mouflon and roe deer in Europe. Scant information is available for D. chinensis, including the range of species that act as definitive and intermediate hosts. To provide morphological and molecular evidences differentiating D. chinensis versus D. dendriticum, 239 Dicrocoelium spp. specimens were collected from sheep, cattle and sika deer from different localities in Austria, Germany and Italy. Specimens were morphologically identified based on the testes orientation, overall size, and level of maximum body width and other morphometric measurements. From this sample, 10 specimens of D. chinensis and 25 of D. dendriticum from different hosts and geographical localities were characterized molecularly through sequencing of partial 18S rDNA (approximately 1400 bp) and ITS-2 (including the 5.8S and 28S flanking regions; approximately 600 bp). Interspecific differences between D. dendriticum and D. chinensis of 0.14% and 3.8% were recorded in 18S rRNA and ITS-2 sequences, respectively. Phylogenetic analyses via Bayesian inference were conducted using sequences of ITS-2 (276 bp) and partial 28S (221 bp) of the above species of Dicrocoelium together with 20 species belonging to the Xiphidiata within the Plagiorchiida available in GenBank. Both gene regions were strongly concordant in differentiating the Dicrocoeliidae, Gorgoderidae and Plagiorchiidae and were in agreement with their current classification. Morphological and molecular characterization clearly differentiate D. dendriticum and D. chinensis as two distinct digeneans infecting ruminants. The implications on the separate status of D. chinensis on the etiology, biology and diagnosis of dicrocoeliosis are discussed.

Ultrastructral studies on the tegument of Dicrocoelium dendriticum (Rudolphi, 1819) Looss 1899.

The tegument of Dicrocoelium dendriticum was studied by scanning and transmission electron microscopy. It consists of an anucleate surface syncytium and a deeper nucleated zone. The syncytial layer covers the entire surface of the fluke. It is connected to the nucleated zone through thin cytoplasmic strands. The syncytium is bounded by an apical plasma membrane which is highly folded. This layer mainly contains abundant mitochondria of varying size and shape, and two types of granules, dumbbell shaped and spherical. Although the apical surface shows active exocytosis, no evidence for endocytosis is observed. This is the first study from the State of Kuwait showing the presence of this liver fluke in sheep imported from other countries.

Contributions to and review of dicrocoeliosis, with special reference to the intermediate hosts of Dicrocoelium dendriticum.

An epidemiological study on dicrocoeliosis caused by Dicrocoelium dendriticum was carried out on sheep, molluscs and ants in the mountains of León province (NW Spain) between 1987-1991. The results concerning the intermediate hosts and a review of some aspects of dicrocoeliosis are summarized. Mollusc collection for the helminthological study was random throughout the study area at fortnightly intervals. Twenty-nine Gastropoda species were identified. D. dendriticum infection was only detected in 2.98%, of the 2084 Helicella itala examined and in 1.06% of 852 H. corderoi. The highest infection prevalence was detected in H. itala in September and in H. corderoi in February. Daughter sporocysts with well-developed cercariae predominated in spring and autumn. Infection prevalence increased with mollusc age and size. Ants were collected from anthills or plants to which they were attached. The behaviour of ants in tetania was followed. Twenty-one Formicidae species were identified, but only the following harboured D. dendriticum: Formica cunicularia (1158 examined specimens, 0.69% infection prevalence, 2-56 metacercariae per ant); F. sanguinea (234, 1.28%, 2-63); F. nigricans (1770, 4.97%, 1-186); F. rufibarbis (288, 6.59%, 2-107). In a flat area close to León town, 95.39% of the 2085 F. rufibarbis specimens collected in tetania contained metacercariae (1-240) in the abdomen. These were used for parasite characterization by isoelectric focusing and to infect lambs and hamsters. Only one brainworm per ant was found.

Ultrastructural investigations on the vitellaria of the digenean Dicrocoelium dendriticum.

The paired vitellaria of the parasitic plathelminth Dicrocoelium dendriticum are composed of numerous follicles each of which contains vitellocytes at different stages of maturation and is enveloped by a basal lamina-like structure and a cytoplasmic sheath. The differentiation process of vitellocytes has been subdivided into three stages on the basis of morphological and functional characteristics. Stage I vitellocytes have a high nucleo/cytoplasmic ratio and a poorly differentiated cytoplasm mainly packed with free ribosomes. Stage II vitellocytes differentiate and increase in volume. Extensive RER and small Golgi complex appear and produce vesicles with an electron-dense content which fuse and give rise to large multigranular inclusions. Stage III vitellocytes are about to enter the vitelloduct, their cytoplasm is almost completely filled with the multigranular inclusions whose content reacts positively to the test for polyphenols. The inclusions are therefore interpreted as egg-shell globules. Mature vitellocytes also contain a small number of lipid droplets which are sometimes surrounded by a few polysaccharide particles, but completely lack protein yolk globules. The role of vitellocytes of D. dendriticum in egg-shell formation and embryo nutrition is discussed.

Ultrastructural study of the spermatogenesis and mature spermatozoa of Dicrocoelium dendriticum (Plathelminthes, Digenea).

The spermatogenesis and spermatozoon of Dicrocoelium dendriticum were studied by transmission electron microscopy. Peripheric accessory cells project between germ cells. Each spermatogonium gives rise to 32 spermatozoa. The stages in spermiogenesis include development of the zone of differentiation, appearance of the intercentriolar body flanked by two centrioles from each of which a free axoneme and a striated rootlet grow, outgrowth of the differentiation zone to form the median cytoplasmic process and migration of the nucleus and mitochondria into it, and rotation of the flagella and subsequent proximodistal fusion of the three projections to form a monopartite spermatozoon. The spermatozoon possess two incorporated axonemes with the "9 + 1" pattern typical of those in trepaxonematid plathelminths. beta-Glycogen particles accumulate in the spermatozoa after they have separated from the cytophore as revealed by Thiery's method. This study confirms in a further family, Dicrocoeliidae, the constant pattern of spermiogenesis and spermatozoon structure in Digenea.