Comparative recruitment, morphology and reproduction of a generalist trematode, Dicrocoelium dendriticum, in three species of host.

Epidemiological rate parameters of host generalist parasites are difficult to estimate, especially in cases where variation in parasite performance can be attributed to host species. Such cases are likely common for generalist parasites of sympatric grazing mammals. In this study, we combined data from experimental exposures in cattle and sheep and natural infections in elk to compare the recruitment, morphology and reproduction of adult Dicrocoelium dendriticum, a generalist trematode that has emerged in sympatric grazing hosts in Cypress Hills Provincial Park, Alberta. Overall, there were no significant differences in the recruitment of metacercariae and in the pre-patency period of adults in experimentally exposed cattle and sheep. All flukes reached reproductive maturity and the degree of reproductive inequality between individual flukes within each infrapopulation was moderate and approximately equal among the three host species. Neither fluke size nor per capita fecundity was constrained by density dependence. Thus, fitness parameters associated with growth and reproduction were approximately equivalent among at least three species of definitive host, two of which are sympatric on pastures in this Park. The generalist life-history strategy of this trematode, which is known to extend to other stages of its life cycle, has likely contributed to its invasion history outside its native range in Europe.

A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium.

DNA primers were designed from the 18S rRNA sequence from the relevant digenean trematode Dicrocoelium dendriticum to evaluate a polymerase chain reaction-based diagnostic method of this parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get DNA from D. dendriticum eggs, several hatching mechanisms were studied. Successful results were obtained when the eggs were frozen to -80 °C and/or in liquid nitrogen and then defrosted. This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification single band of 1.95 kb was observed using primers designed for the total 18S rRNA sequence in both cases as well as when the template DNA was from adults of the closely related parasite Fasciola hepatica; in addition, a single and specific 0.8-kb band was obtained when primers based on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in samples coming from adults, but in eggs from gall bladder and faeces as well. F. hepatica internal 18S rRNA primers were also designed and used as a negative control to prove that the eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been developed for the first time and could provide a useful molecular tool to improve the conventional coprological diagnosis for detecting D. dendriticum eggs.

Spatiotemporal Patterns of Infection for Emerging Larval Liver Fluke ( Dicrocoelium dendriticum) in Three Species of Land Snail in Southern Alberta, Canada.

The control of emerging parasites requires a fundamental knowledge of where and when rates of transmission are high. Data on spatiotemporal patterns of infection are challenging to obtain, particularly for complex life cycle parasites that involve transmission into multiple obligate hosts. The lancet liver fluke, Dicrocoelium dendriticum, has a long history of colonization outside its native host and geographical range in continental Europe. Infection patterns involving adult and metacercarial stages have been characterized for this trematode in a region of emergence in western Canada within co-grazing herbivores and ants, but infection patterns in snail intermediate hosts in this region are unknown. We combined spatiotemporal prevalence surveys with sequence analyses of the cytochrome c oxidase subunit 1 ( COI) barcoding gene from samples of sporocyst tissue in infected snails to confirm that D. dendriticum utilizes 3 sympatric species of Oreohelid land snail ( Oreohelix subrudis, Oreohelix sp., and Oreohelix cooperi) as first intermediate host. Mean prevalence within a total sample of 900 adult snails collected over 1 field season from 6 sites was 9.9 ± 2.4%. For each species of snail, prevalence ranged between 5-30% within monthly samples, with peaks in mid-summer followed by declines in fall. Between-site variation in prevalence was low and non-significant, implying that rates of transmission of D. dendriticum miracidia from domestic stock and wildlife into snails are similar within localized sites, despite high variation in local habitat characteristics and in the structure of the definitive host community.

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.

3D virtual histology at the host/parasite interface: visualisation of the master manipulator, Dicrocoelium dendriticum, in the brain of its ant host.

Some parasites are able to manipulate the behaviour of their hosts to their own advantage. One of the most well-established textbook examples of host manipulation is that of the trematode Dicrocoelium dendriticum on ants, its second intermediate host. Infected ants harbour encysted metacercariae in the gaster and a non-encysted metacercaria in the suboesophageal ganglion (SOG); however, the mechanisms that D. dendriticum uses to manipulate the ant behaviour remain unknown, partly because of a lack of a proper and direct visualisation of the physical interface between the parasite and the ant brain tissue. Here we provide new insights into the potential mechanisms that this iconic manipulator uses to alter its host's behaviour by characterising the interface between D. dendriticum and the ant tissues with the use of non-invasive micro-CT scanning. For the first time, we show that there is a physical contact between the parasite and the ant brain tissue at the anteriormost part of the SOG, including in a case of multiple brain infection where only the parasite lodged in the most anterior part of the SOG was in contact with the ant brain tissue. We demonstrate the potential of micro-CT to further understand other parasite/host systems in parasitological research.

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.

Relative Effects of Temperature, Light, and Humidity on Clinging Behavior of Metacercariae-Infected Ants.

The lancet fluke, Dicrocoelium dendriticum, is perhaps the best-known example of parasite manipulation of host behavior, which is manifested by a radically changed behavior that leaves infected ants attached to vegetation at times when transmission to an herbivore host is optimal. Despite the publicity surrounding this parasite, curiously little is known about factors inducing and maintaining behavioral changes in its ant intermediate host. This study examined the importance of 3 environmental factors on the clinging behavior of red wood ants, Formica polyctena , infected with D. dendriticum . This behavior, hypothesized to involve cramping of the mandibular muscles in a state of tetany, was observed in naturally infected F. polyctena under controlled temperature, light, and humidity conditions. We found that low temperature significantly stimulated and maintained tetany in infected ants while light, humidity, ant size, and infection intensity had no influence on this behavior. Under none of the experimental conditions did uninfected ants attach to vegetation, demonstrating that tetany was induced by D. dendriticum . Temperature likely has a direct impact on the initiation of clinging behavior, but it may also serve as a simple but reliable indicator of the encounter rate between infected ants and ruminant definitive hosts. In addition, temperature-sensitive behavior manipulation may protect infected ants from exposure to temperatures in the upper thermal range of the host.

Dicrocoelium dendriticum induces autophagic vacuoles accumulation in human hepatocarcinoma cells.

The relationship between Dicrocoelium dendriticum and cancer has been poorly investigated so far, but a large amount of findings suggest that other trematodes can favour cancer in both animals and humans. In this study, the effects of D. dendriticum on cell proliferation, cell death mechanisms and oxidative stress induction were evaluated in hepatocellular carcinoma (HCC) cell lines (HepG2 and HuH7). Results showed that short time exposure to low concentrations of somatic antigens from D. dendriticum caused slight proliferation in both HepG2 and HuH7 cells while high concentrations and long exposure time to extracts from D. dendriticum caused a significant growth inhibition. This effect was, however, not paralleled by apoptosis but it occurred with an about 40% increase of the formation of autophagic vacuoles. In the same experimental conditions, a strong oxidative stress was recorded with an about 100% increase of the intracellular O(2-). These data suggest the occurrence of an escape anti-apoptotic mechanism in HCC cells. In conclusion, these results suggest a role for D. dendriticum in the chronic oxidative stress and in the regulation of transformation processes in HCC warranting additional investigations in this specific area of research.

Survival and infectivity of Dicrocoelium dendriticum eggs under field conditions in nw Spain.

A study was made of the survival of Dicrocoelium dendriticum eggs in sheep faeces in an area with a temperate climate (NW Spain). It appears that mortality is independent of the age of the eggs and that there is a marked seasonality within the period of time considered (20 months). A study was also carried out on the infectivity of D. dendriticum eggs by experimental infections of suitable intermediate hosts. The data show that no loss took place in infectivity during the period of study (15 months).

Effect of some abiotic factors on the survival of dicrocoelium dendriticum eggs in laboratory conditions.

In the present work the results obtained from the study of the effect of temperature, RH, UV radiation and day-night thermal variations, in controlled laboratory conditions, on the survival of D. dendriticum eggs in sheep faeces are described. Mortality was seen to be maximum with a RH of 75-80% and temperatures higher than 18 degrees C. The results are compared with those obtained in natural conditions. It is suggested that the final cause of the mortality of the eggs both in laboratory and natural conditions could be the action of some microorganisms.

[Developmental periods and adaptation of parthenites of Eurytrema pancreaticum (Dicrocoeliidae, Trematoda) to environmental conditions]. / O srokakh razvitiia i adaptatsii partenit Eurytrema pancreaticum (Dicrocoeliidae, Trematoda) k usloviiam vneshnei sredy

Data are given on the developmental periods of parthenites of E. pancreaticum (Janson, 1889) from the Far East of the USSR. The growth and formation of parthenites were found to take place from May to October and to coincide with the active period of the intermediate host's life cycle. With the decrease of autumn temperatures to 8 degrees the growth of parthenites slows down and ceases completely in October. The pause in the development of parthenites from October to April causes longer developmental periods of parthenites as compared to those in the regions of tropical and subtropical climate. Under laboratory conditions at a temperature of 8 to 22 degrees the development of parthenites proceeds without any intervals within 6.5 months.

Oxygen inhibits--even though in traces--the hatching of the eggs of the liver fluke Dicrocoelium dendriticum R.

A chamber was constructed to investigate the influence of gaseous surroundings on the hatching of Dicrocoelium eggs (Fig. 1). It was found that the exclusion of even traces of oxygen is the prerequisite for successful hatching. Oxygen was removed by baker's yeast suspensions as well as by dithionite solutions. Hatching rates reached a maximum when the carbon dioxide content of the chamber ranged between 0.1% and 4%. Under natural hatching conditions within the intestine of the pulmonate intermediate hosts, bacteria ensure oxygen consumption and carbon dioxide production. The consequence is the hatching of Dicrocoelium eggs. Carbon monoxide also stimulates hatching in the presence of oxygen and therefore seems to block the inhibition mechanism, which is based on oxygen acting. The hatching of eggs started about 5 min after exposure to hatching conditions and continued over a period of 60-90 min.

Dicrocoelium hospes Looss, 1907 (Digenea, Dicrocoeliidae): spermiogenesis, mature spermatozoon and ultrastructural comparative study.

This work describes the first ultrastructural results on spermiogenesis and on the mature spermatozoon of Dicrocoelium hospes (Trematoda, Digenea) collected in Bos indicus from Senegal (Africa). Examination of this species was processed by TEM. Spermiogenesis follows the general pattern found in the digenean, but reveals a particularity consisting of the appearance of glycogen granules in the late spermatids within the testes. The mature spermatozoon possesses five distinct regions and presents all features found in Digenea gametes: two axonemes, mitochondria, nucleus and parallel cortical microtubules. However, several characters allow us to distinguish D. hospes from other digenetic trematodes within the Dicrocoeliidae family. In fact, we observed several structures that are absent in the other species of Dicrocoeliidae studied until now, such as: a cytoplasmic expansion, extramembranar ornamentation, spine-like bodies and two parallel mitochondria in the mature sperm. Moreover, additional particular characteristics were observed in this species in both extremities of the spermatozoon. This work produced new data on the ultrastructure of this trematode family which may be useful for phylogenetic purposes.

[Function of the acetabulum of digenetic trematodes, as exemplified by Dicrocoelium dendriticum]. / Die Arbeitsweise des Bauchsaugnapfes digenetischer Trematoden am Beispiel des Dicrocoelium dendriticum.

The suckers of animals adhere to the substratum either in air or in aqueous fluids. The varying compressibility of these media causes differences in function, the principles of which are described. The ventral arch of the acetabulum of Dicrocoelium dendriticum, like the remaining body, is limited by the integument, basal lamina and skin muscles. The dorsal arch covers a basal lamina, which is close to a plexus of diagonal, longitudinal and circular muscles. The radical muscles, attached at the ventral basal lamina by thin connective tissue, continue in relatively thick contractile fibers, which split up into several fibrils, which also attached by thin connective tissue at the dorsal basal lamina. In this way the tension of the muscles is likewise distributed over the dorsal and ventral arches of the acetabulum. After contact with the substratum the sucker creates a partial vacuum and attachment by means of the pressure of the radial muscles against the wall of the hole. Because of the fluid content of the hole, the volume does not change much. The dorsal arch of the sucker withstands the pressure of the radical muscles, because its surface area is six times greater than that of the ventral arch and consequently the load is six times less. The sucker, covered with basal lamina, has a constant volume; its layer of muscles resists deformation and supports the stability of the arch.

[The modification of Helicella obvia by Dicrocoelium parthenitae]. / Zur Beeinflussung von Helicella obvia durch Dicrocoelium-Parthenitae.

During the pasture period in 1990 the snail species Helicella obvia, collected in a dicrocoeliosis area in the east of Brandenburg, was investigated for Dicrocoelium sporocysts. Simultaneously in adult snails the condition of albumen gland was examined. Investigations showed that Dicrocoelium infection in the snail host impairs their reproduction ability and life expectancy. The term "parasite caused sterilisation" is suggested for changes which lead to infertility in the case of parasite infection.

Larval development of Dicrocoelium dendriticum in Cernuella (Xeromagna) cespitum arigonis under controlled laboratory conditions.

The larval development of Dicrocoelium dendriticum (Digenea: Dicrocoeliidae) in experimentally infected Cernuella (Xeromagna) cespitum arigonis (Schmidt, 1853), a species of mollusc important in the epidemiology of dicrocoeliosis in Spain, has been studied. A total of 948 specimens of this mollusc, distributed in five batches, were tested with individual doses of 50 to 150 parasite eggs, obtained from sheep, after 4 days without food. After infection these molluscs and control specimens were kept in an environmental simulation chamber at 20 degrees C, 50% relative humidity and 7 h of light per day. To detect the parasite, a minimum of six molluscs were examined every 20 days from day 1 post-infection (p.i.). The eggs of D. dendriticum were eliminated in the molluscan faeces 48 h post infection. The percentages of molluscs harbouring the parasite ranged between 17.53% and 75%. Daughter sporocysts with undifferentiated germinal masses and occupying very reduced areas of the hepatopancreas were observed 50 days p.i. and in the period immediately following. After 110 days p.i. sporocysts with cercariae at different stages of development were found although slimeball emission was never observed.