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Amphistomes, commonly referred to as 'stomach' or 'rumen' flukes because of the localization of these flukes in the stomach of ruminants, are digenetic trematodes distinguished by the absence of an oral sucker and the position of the ventral sucker or acetabulum at the posterior end of the body. The body is characterized by a leaf-like fleshy structure, pink or red with a large posterior sucker. Amphistomes are an important group of parasites since they cause 'amphistomiasis' (variously known as paramphistomosis/amphistomosis), a serious disease of great economic importance in ruminants worldwide. These parasites have a broad spectrum of definitive hosts together with a wide geographical distribution. Though they form a continuous evolutional lineage from fishes to mammals, amphistomes mainly inhabit the rumen and reticulum of ruminant mammals, while some species occur in the large intestine or parenteric sites of ruminants, pigs, equines and man., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In order to ensure global food security a rationale approach is required to control all those factors which directly or indirectly affect the food productivity. The neglected helminthic diseases alone are responsible for huge economic losses to the agrarian stakeholders. The problem is further compounded by the emerging drug resistance in flukes against the commonly used anthelmintics like triclabendazole. Therefore, the search for alternatives including the nano-based approaches has become a necessity to develop future control strategies. In the present study the effect of biologically synthesized silver nanoparticles (AgNPs) was investigated on an economically important amphistome parasite, Gigantocotyle explanatum, obtained from the infected liver of the Indian water buffaloes, Bubalus bubalis. In vitro treatment of the adult worms with different doses of AgNPs severely affected the worm motility and caused ROS mediated damages in the treated flukes. The antioxidant system and the detoxification ability of the worms appeared to be disrupted along with pronounced DNA damage in the treated worms as compared to the controls. Following the treatment of worms with different concentrations of AgNPs there was a significant (p < 0.05) increase in lipid peroxidation and protein carbonylation levels which are the key oxidative stress markers. The tegumental surface which is metabolically active, was severely damaged as evident from the loss of papillae, severe blebbing, shearing and erosion of the surface structures. Such topographical disruptions would facilitate the penetration of the nanoparticles deep within the tissues that might greatly reduce the invasive potential of the flukes as evident from the decreased motility. Taken together our findings suggest that the AgNPs posses great anthelmintic potential and could be further exploited for the development of anthelmintic formulations which may be tested in vivo., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Fasciola hepatica has always represented a threat to Irish livestock because the Irish climate is highly suitable for the main local intermediate host of the parasite, the snail Galba truncatula. The recent clinical emergence of infections due to Calicophoron daubneyi has raised the question of whether the two parasites, which share a niche during part of their life-cycles, interact in some way. Here, we used geographical information systems (GIS) to analyse the distribution of both parasites in cattle and sheep. We also developed the first predictive model of paramphistomosis in Ireland., Results: Our results indicated that, in cattle, liver fluke infection is less common than rumen fluke infection and does not exhibit the same seasonal fluctuations. Overall, we found that cattle had a higher likelihood of being infected with rumen fluke than sheep (OR = 3.134, P < 0.01). In addition, infection with one parasite increased the odds of infection with the other in both host species. Rumen fluke in cattle showed the highest spatial density of infection. Environmental variables such as soil drainage, land cover and habitat appeared to be the most important risk factors for C. daubneyi infection, followed by rainfall and vegetation. Overall the risk of infection with this parasite was predicted to be higher in the west of the country., Conclusions: This study shows differences between the infection rates and spatial patterns of bovine and ovine infections with F. hepatica and C. daubneyi in Ireland. Whether the reasons for this are due to susceptibility, exposure and/or management factors is yet to be determined. Furthermore, the rumen fluke model indicates distinct risk factors and predicted distribution to those of F. hepatica, suggesting potential biological differences between both parasite species.

Paramphistomosis, caused by Calicophoron daubneyi, is an emerging infection of ruminants throughout Western Europe. Despite its prevalence, many questions remain regarding the basic biology of this parasite and how it interacts with its host. Consequently, there is a need to develop methods to study C. daubneyi in vitro to improve our understanding of rumen fluke biology. Towards this, we aimed to identify a suitable protocol for in vitro excystment of C. daubneyi metacercariae. Six methods that have been used to excyst metacercariae from a number of trematode species were tested with C. daubneyi metacercariae. Three of these achieved an average of >50% excystment whilst one method, which included an acid-pepsin treatment, incubation in reducing conditions and an alkaline/bile salt solution to activate the larvae, consistently gave >80% excystment. The latter protocol also showed no detrimental effect on the motility of newly excysted juvenile (NEJ) parasites when observed for up to 24 h in RPMI 1640 medium post-excystment. The successful production of C. daubneyi NEJs in vitro is a significant step forward, and will enable the discovery of infective stage-specific parasite antigens and facilitate drug screening trials, to aid the development of much needed diagnostic and therapeutic options for paramphistomosis.

Whilst historically regarded as being of minor importance in European livestock, recent evidence suggests that the prevalence of paramphistomosis is greater than that of fasciolosis in parts of the UK. In order to address this emerging threat to ruminant farming systems, and associated risks for food security posed by rumen fluke infection, it is imperative that we develop a better understanding of the basic biology of this parasite and how it interacts with its ruminant host. In this Opinion article we review recent progress in tracking the spread of rumen fluke infection in Europe, and propose some research questions that should be addressed if we are to develop tools to diagnose and treat paramphistomosis more effectively in the future., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected with P. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL-1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected with P. gracile, Fasciola gigantica, Moniezia benedeni, Trichuris sp., Strongyloides sp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

There is concern about the probable recent introduction, increased prevalence and potential economic impact of rumen fluke infection of United Kingdom cattle. A study of 339 cattle slaughtered in a Scottish red meat abattoir was undertaken with the aims of describing the prevalence and geographical distribution of rumen fluke infection, estimating its effect on production, and evaluating faecal egg counts (FECs) as a tool to diagnose infection in live animals and study the epidemiology of the disease. The overall proportion of cattle consigned to the abattoir from northern United Kingdom with rumen fluke infection in the forestomachs was 0.29. Rumen flukes were distributed predominantly in the cranial sac of the rumen and adjacent to the reticular groove. Overall, a mean of 213 and median of 44 rumen flukes was identified in the forestomachs of rumen fluke-positive cattle. The mean and median FECs of animals were 26.01 and 5.20 eggs per gram (epg), respectively. There was a significant difference between the mean FECs per rumen fluke of 0.08 and 0.13 epg during summer/autumn and winter sampling periods, respectively. The overall correlation between rumen fluke FECs and the number of flukes in the forestomach was high, albeit lower in the summer/autumn than in the winter period. The sensitivities of rumen fluke FECs for the identification of flukes in the forestomach during the summer/autumn and winter sampling periods were 0.65 and 0.85, respectively. These results will aid in the interpretation of rumen fluke FECs when monitoring cattle health and production and studying the parasite's epidemiology in a temperate environment, thereby informing rational, precise and sustainable disease control., (Copyright © 2016. Published by Elsevier B.V.)

Aim of present study was to screen medicinal plants for flukicidal activity in vitro to develop alternative sources of treatment for trematodes infection. For this purpose, crude methanolic extracts (CME) of Cymbopogn jwarancusa and Conyza canadensis were prepared and live adult flukes viz; Fasciola gigantica, and Paramphistomum cervi isolated from liver and bile ducts of slaughtered buffalo were subjected to different drug concentrations as well as positive and negative control. Motility inhibition and paralysis leading to the death of parasites was considered as flukicidal activity of plants extracts. The results revealed that CME of C. jwarancusa and C. canadensis showed significant (P<0.05) flukicidal activity compared to positive control. Also there was a significant effect of different concentrations (P<0.05) and exposure of time on the flukes (P<0.05). Furthermore, ED50 for C. jwarancusa and C. canadensis against F. gigantica were 13.1 and 41.4 mg/ml, respectively. In the case of P. cervi, it was 10.8 and 29.0 mg/ml. It can be concluded that both tested plants showed greater flukicidal activity as compared to positive control with Albendazole till the 8(th) hr. These potent plants needs further studies invivo to elucidate their mode of action.

Cross-species infection among humans, chimpanzees (Pan troglodytes) and baboons (Papio spp.) is potentially a significant public health issue in Africa, and of concern in the conservation of P. troglodytes. However, to date, no statistical comparisons have been made between the prevalence, richness and composition of parasite communities in sympatric populations of baboons and P. troglodytes. We compared parasite communities in sympatric P. troglodytes and Papio papio living in a wilderness site, in the Republic of Senegal, West Africa. We asked whether, in the absence of humans, there are significant differences between these hosts in their interactions with gastrointestinal parasites. We tested whether host, location, or time of collection accounted for variation in prevalence, richness and community composition, and compared prevalence across six studies. We concluded that, despite being closely related, there are significant differences between these two hosts with respect to their parasite communities. At our study site, prevalence of Balantidium, Trichuris and Watsonius was higher in P. papio. Papio papio harboured more parasites per host, and we found evidence of a positive association between Trichuris and Balantidium in P. troglodytes but not P. papio.

Individual infections of Egyptian and French Pseudosuccinea columella with five miracidia of Calicophoron daubneyi were carried out to determine whether this lymnaeid was capable of sustaining larval development of this parasite. On day 42 post-exposure (at 23 °C), infected snails were only noted in groups of individuals measuring 1 or 2 mm in height at miracidial exposure. Snail survival in the 2-mm groups was significantly higher than that noted in the 1-mm snails, whatever the geographic origin of snail population. In contrast, prevalence of C. daubneyi infection was significantly greater in the 1-mm groups (15-20% versus 3.4-4.0% in the 2-mm snails). Low values were noted for the mean shell growth of infected snails at their death (3.1-4.0 mm) and the mean number of cercariae (<9 in the 1-mm groups, <19 in the 2-mm snails). No significant differences between snail populations and snails groups were noted for these last two parameters. Most infected snails died after a single cercarial shedding wave. Both populations of P. columella showed an age resistance to C. daubneyi infection and only juveniles measuring 2 mm or less in shell height at exposure can ensure larval development of this digenean up to cercarial shedding., (© Y. Dar et al., published by EDP Sciences, 2015.)

Laboratory investigations on Galba truncatula experimentally infected with Calicophoron daubneyi were carried out to study the influence of natural light and sky nebulosity on cercarial emergence in snails subjected every week outdoors to a thermal shock (a mean of 12 °C for 3 h) during the patent period. The same study was also performed in G. truncatula naturally infected with Haplometra cylindracea according to the same protocol. Compared to infected controls always reared indoors at 20 °C, the number of cercariae emerging from snails subjected outdoors to a thermal shock was significantly greater, whatever parasite species. Natural light had an effect on snails releasing C. daubneyi cercariae after the thermal shock because their numbers were significantly higher between 601 and 1,200 lx and for the highest nebulosity values (7-8 octas). In contrast, the type of light used during thermal shock did not influence cercarial emergence of H. cylindracea because the numbers of cercariae per shedding snail noted under natural light or a 3,000-lx artificial light did not significantly differ from each other. Most snails releasing H. cylindracea cercariae were significantly more numerous for light levels higher than 3,000 lx. As for Fasciola hepatica cercariae, natural light had a significant influence on cercarial emergence of C. daubneyi, and this behavioural particularity for cercariae of both digeneans might be due to the fact that both species develop in the same ruminants and the same snail host.

The neuromuscular system of helminths is an important area for target identification and drug development. Many anthelmintics, namely ivermectin, levamisole, piperazine, pyrantel, praziquantel and organophosphates, produce paralysis of helminths by affecting their neuromuscular systems. The neuromuscular system of helminths is also an important area of research to identify some of the important differences between the neuromuscular physiology of helminths and mammals. The identification of differences would help in developing newer target-specific, safe and effective anthelmintics. The present study was carried out to investigate the effects of different adrenergic neurotransmitters (epinephrine, norepinephrine, dopamine, l-dopa) and their antagonists (propranolol and haloperidol) on the spontaneous muscular activity of isometrically mounted Paramphistomum cervi.

The prevalence and aetiology of natural paramphistomosis was investigated in cattle slaughtered in the Castilla y León region (Spain) over a 3 year-period. The overall prevalence of positive animals was 6.20%. The parasite burden per animal ranged from 8 to 8005 (median=144) and the ruminal atrium had the highest parasite burden whereas the ruminal dorsal sac the lowest. The prevalence and parasite burden increased with age while these parameters were lower in cattle under intensive management. Calicophoron daubneyi was the only Paramphistomidae species identified using morphoanatomical, histological and molecular methods in the studied animals., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Paramphistomiasis causes enteritis and anemia in livestocks and result in substantial production and economic losses. It is considered a neglected tropical disease, with no effective trematodicidal compound for treatment. Plumbagin (PB), a compound founds to be rich in the roots of Plumbago indica, is a naphthoquinone derivatives which can induce oxidative stress in parasites. In this study we have evaluated the anthelmintic activity of PB against adult Paramphistomum cervi by incubating the parasites in M-199 medium containing 0.1, 1.0, 10 and 100 μg/ml of the PB, and albendazole (ABZ) at the concentration of 100 μg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observed by scanning electron microscopy (SEM). After 12 h exposure with 100 μg/ml ABZ, flukes showed decreased contraction and motility. At 24 h incubation they showed only active movement of some part of the body. The PB-treated flukes at all concentrations showed rapid decrease of motility at 3 h incubation. In 0.1, 1.0 and 10 μg/ml of PB, the RM values were decreased sharply from 3 to 12 h, and then they were killed since 12 h in the incubation with 10 μg/ml of PB. The highest parasite mortality was found as early as 3h when they were incubated with 100 μg/ml of PB. The morphological changes on the tegumental surface were similar in both flukes treated with ABZ and PB, which sequentially comprised of swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesions were formed which exposed the basal lamina. The damage appeared more severe on the ventral than the dorsal surface, and earlier on the anterior part and lateral margins of middle third when compared to the posterior part of the parasites's bodies. The severity and rapidity of the damages were enhanced with increasing concentration of PB, which showed stronger activity than ABZ. Hence, PB has a potential to be an anthelmintic drug against adult P.cervi., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study explored various basic aspects of the epidemiology of paramphistomosis in Galicia, the main cattle producing region in Spain. In total, 589 cows from different farms located across the region were selected at random in the slaughterhouse for examination of the rumens and reticula for the presence of Paramphistomidae flukes. Paramphistomes were found in 111 of 589 necropsied cows (18.8%; 95% CI: 15.7-21.9%), with higher prevalences of infection in beef cows than in dairy cows (29.2% vs 13.9%). Although the number of flukes per animal was generally low (median=266 flukes), some cows harboured large parasite burdens (up to 11,895 flukes), which may have harmful effects on their health or productivity. Cows with higher parasite burdens also excreted greater numbers of fluke eggs in their faeces, which suggests that heavily parasitized mature cows play an important role in the transmission of paramphistomosis. This role may be particularly important in Galicia, where the roe deer, which is the only wild ruminant in the study area, was found not to be a reservoir for the infection. The use of morpho-anatomical and molecular techniques applied to a large number of fluke specimens provided reliable confirmation that Calicophoron daubneyi is the only species of the family Paramphistomidae that parasitizes cattle in Galicia. The environmental data from the farms of origin of the necropsied cows were used in Bayesian geostatistical models to predict the probability of infection by C. daubneyi throughout the region. The results revealed the role of environmental risk factors in explaining the geographical heterogeneity in the probability of infection in beef and dairy cattle. These explanatory factors were used to construct predictive maps showing the areas with the highest predicted risk of infection as well as the uncertainty associated with the predictions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The analysis of infection by Paramphistomidae trematodes was conducted in two agricultural regions with different knowledge on this parasitosis. Faecal and blood samples were collected from 374 cattle in Salto (NW Uruguay) where there is a lack of information about paramphistomosis. A total of 429 cattle from Galicia (NW Spain), an area with previous records of infection by gastric flukes, were sampled. Diagnostics of trematodosis was developed by using a copromicroscopic probe and an ELISA with excretory/secretory antigens collected from adult Calicophoron daubneyi (Paramphistomidae) specimens. Results were evaluated according intrinsic and extrinsic factors. In the Uruguay, the percentage of cattle passing Paramphistomidae-eggs by faeces was 7% (95% Confidence Interval 5, 10). A significantly higher prevalence of paramphistomosis in the Hereford × Angus cattle (OR=3.5) was recorded, as observed for the oldest ruminants (>3.5 years). An overall seroprevalence of 29% (25, 34) was obtained by ELISA, with the highest values in the Friesians (OR=3), the youngest bovines (<2.5 years) and dairy cattle (Friesians). Twenty-six percent (22, 30) of the cattle from Spain passed eggs by faeces, and cattle aged 2.5-7 years reached significant highest prevalences. By means of the ELISA, a percentage of 55 cattle (50, 59) had antibodies against the gastric fluke, and the highest seroprevalence was observed among the bovines under 6 years. It is concluded that paramphistomosis is on the increase in cattle from NW Spain, partly due to the absence of an effective treatment against the trematode. There is a need for reducing the risk of infection by Paramhistomidae spp. in cattle from Uruguay, especially by improving their management to avoid exposure to the gastric trematode. Further studies are in progress for identifying the species of Paramphistomidae affecting ruminants in Uruguay., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Experimental infections of Galba truncatula (two populations) with Fasciola hepatica or Paramphistomum daubneyi were carried out to study the effect of water temperature changes (3 h at a mean of 12 °C every week) on cercarial shedding during the patent period. The results were compared with those of control snails infected according to the same protocol and always maintained at 20 °C. Compared to controls, a significant increase in the number of cercariae-shedding snails, a significantly longer patent period and significantly greater cercarial production were noted in temperature-challenged snails, regardless of the type of digenean infection. In contrast, the number of incompletely formed metacercariae was significantly higher in temperature-challenged snails than in controls. Incompletely formed metacercariae of F. hepatica consisted of cysts whose colour remained whitish after shedding (25.4% for temperature-challenged snails) or whose dome was flattened after encystment (74.6%). Those of P. daubneyi were totally dark brown or blackish after formation. These incomplete metacercariae might originate from young differentiating cercariae within the snail body (F. hepatica) or from cercariae which died just after encystment (P. daubneyi). The use of regular temperature changes for snails infected with F. hepatica or P. daubneyi must be monitored carefully during collection of metacercariae to select completely formed cysts for infecting definitive hosts., (© D. Rondelaud et al., published by EDP Sciences, 2013.)

Populations of Biomphalaria straminea, Biomphalaria peregrina , Biomphalaria tenagophila, Biomphalaria orbignyi, and Biomphalaria oligoza from different Argentine localities were exposed to miracidia of Schistosoma mansoni EC strain, and Biomphalaria tenagophila, in addition to the SJ2 strain. Biomphalaria straminea and B. tenagophila displayed different susceptibility and compatibility (Frandsen's total cercariae production index class 0-II), whereas B. orbigny and B. oligoza were incompatible. Although B. peregrina and B. tenagophila were found naturally infected with the amphistome Zygocotyle lunata, all 5 species could be experimentally infected with Z. lunata. Exposure to Z. lunata infections with S. mansoni were obtained in natural populations of B. straminea and B. tenagophila with the EC strain (13.5-17.1% and 1.2%), respectively, and in B. tenagophila with the SJ2 strain (2.6%), 60 days postexposure [PE]), and in B. orbignyi and B. oligoza (31.1% and 26.7% 60 days PE, respectively, including single infections with S. mansoni and double infections with Z. lunata). The high susceptibility of B. orbignyi and B. oligoza is noteworthy, as these 2 species are considered resistant to S. mansoni .

The somatic extract of Zygocotyle lunata (Trematoda: Paramphistomidae) adults collected from experimentally infected mice was investigated using a proteomic approach to separate and identify tryptic peptides from the somatic extract of Z. lunata adult worms. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 36 proteins were accurately identified from the worms. The largest protein family consisted of metabolic enzymes. Structural, motor and receptor binding proteins and proteins related to oxygen transport were identified in the somatic extract of Z. lunata. This is the first study that attempts to identify the proteome of Z. lunata. However, more work is needed to improve our knowledge of trematodiasis in general and more specifically to have a better understanding about host-parasite relationships in infections with paramphistomes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Experimental infections of Galba truncatula with Paramphistomum daubneyi were carried out to determine at day 50 (at 24 degrees C) the numbers of sporocysts, which grew in infected snails via the count of first- and second-generation rediae. In snails individually exposed to one, two, three, four, or five miracidia, the numbers of first-generation rediae increased from the one-miracidium group to the five-miracidium snails (from a mean of 6.7 to 26.1), while second-generation rediae decreased in number (from 6.2 to 0.9, respectively). This scale of redial numbers was used to determine the number of sporocysts, which grew in naturally infected snails collected from sedimentary or acid soils between 1993 and 2006. In cercariae-containing snails, natural infections resulting from the development of one to five sporocysts were found in both samples of G. truncatula examined. The numbers of 3-, 4-, and 5-sporocyst infections were increasing over time since 1997, 2000, and 2003, respectively. The utility of such multiple-sporocyst infections is open to question, as the differentiation of second-generation rediae and that of procercariae were delayed and always limited. They might be interpreted as a consequence of a zoonosis, which has been spreading since 1990 in ruminants of central France.

Experimental infections of Galba truncatula with Paramphistomum daubneyi were carried out at 24 degrees C to study the dynamics of larval development in snails dissected at regular intervals and to determine if metacercarial production might be improved. When the shell height of snails (4, 5, 6 or 7 mm) at exposure increased (experiment A), the total number of metacercariae was significantly higher in the 6- and 7-mm snails than in the other two groups, and the differentiation period was shortened (the first cercariae encysted at day 35 post-exposure (p.e.) instead of day 40 in the 4- and 5-mm groups). When the number of miracidia (two, three or five) for each 6-mm high G. truncatula increased (experiment B), a significant decrease of snail survival at day 30 p.e., a significant augmentation of prevalence, and a significant increase of metacercarial production were noted. In the two- and three-miracidium groups, the number of metacercariae was close to that found in the 6-mm snails from experiment 1, whereas they showed slower growth from day 45 to day 65 in the five-miracidium group. In the two groups of lambs infected with metacercariae encysted at days 45 or 60 p.e., no difference in the numbers of adult worms was noted. In contrast, in the case of 35-day encysted larvae, the number of adult worms was clearly lower. Snail dissection allowed higher metacercarial production, a saving of 12-15 days at 24 degrees C to obtain these larvae, and a substantial decrease of their cost price for commercial production.

Helisoma trivolvis snails collected from a lake in Warren County, New Jersey, in June 2007 possessed the ectosymbiont Chaetogaster limnaei (Annelida). Some of these snails were also infected with larval stages of Zygocotyle lunata and Echinostoma trivolvis. Chaetogaster limnaei associated with the infected snails fed on the cercariae of both digeneans. Zygocotylae lunata cercariae were observed in the stomach of C. limnaei and whole cercariae were loosely attached to the ventral surface of the chaetogasters. Cercariae in the stomachs were digested within 48 hr and probably served as a source of nutrient for the annelids. Whole cercariae and 1 nonviable metacercaria of E. trivolvis were seen in the stomachs of the chaetogasters. The protective action of the chaetogasters on the transmission of the cercariae of E. trivolvis and Z. lunata to second intermediate hosts in the wild awaits further study.

The aims of the present study were to establish an adequate excystment method for metacercariae of Paramphistomum spp. and to compare the in vitro survival of paramphistomids in three different culture media. In a preliminary study, 31 different assays were performed solely with the purpose of obtaining the best percentage of metacercariae excystment. Once the best excystment method was selected, 21 further assays using 100 metacercariae/assay were carried out to determine the average percentage of excystment. For the in vitro culture of paramphistomids, the Hedon-Fleig, Rohrbacher, and RPMI-1640 culture media were compared by using 24-well immunoplates containing newly excysted flukes or 50-ml capacity tissue-culture flasks for adult paramphistomids. Results indicated an average of 69.0% excystment, the best culture medium to support the survival of immature and adult stages of the trematode being Hedon-Fleig and Rohrbacher, respectively. The best percentage of survival obtained was on day 4 (80%), with some immature and adult trematodes remaining alive until days 10 and 11, respectively.

The process of spermiogenesis in two paramphistomes, Sandonia sudanensis and Basidiodiscus ectorchis from the Nile fish Synodontis schall in Egypt was studied by transmission electron microscopy. Spermiogenesis is characterized by the outgrowth of the zone of differentiation, presenting two basal bodies separated by a microtubule organizing centre, each basal body developing into a flagellum. Proximodistal fusion of these flagella with a median cytoplasmic extension gives rise to the spermatozoon. The mature spermatozoon possesses two axonemes of the 9+'1' pattern typical of parasitic helminths. There are few ultrastructural studies on spermiogenesis in paramphistomes, which are considered the most primitive digenetic trematodes. The present study provides new and more detailed information on this process, including the presence of a lateral flange and external ornamentation of the cell membrane. The value of sperm ultrastructure as a taxonomic tool in phylogeny is also discussed.

The ultrastructural study of spermiogenesis and the spermatozoon of Paramphistomum microbothrium gave evidence of some characteristics of this digenean. The intercentriolar body located between two striated roots and two centrioles exhibits a symmetric plane. The two external bands of this intercentriolar body are made up of a line of granules. The striated roots are about 3.7-microm long. During spermiogenesis, a flagellar rotation of 90 degrees is described. The spermatozoon is characterized in its anterior part by the existence of external ornamentations and a lateral expansion exhibiting a "spine-like body." Such a spine-like body has never been described before in a digenean. The region II of the spermatozoon is asymmetrical and also characterized by the absence of mitochondrion. The posterior end of this spermatozoon exhibits a nucleus and a few microtubules.

Experimental infections of Galba truncatula with Fasciola hepatica, Fascioloides magna, or Paramphistomum daubneyi were carried out at 20 degrees C to determine if the use of 14-cm petri dishes for breeding lettuce-fed snails enhanced the characteristics of snail infections. Compared to infected snails raised in boxes up to day 30 post-exposure and later in individual 35-mm dishes, the survival of G. truncatula kept in 14-cm dishes and the shell height of cercariae-shedding snails during the first 45 days were higher, whatever the digenean species is. The consequence of such enhanced characteristics was a greater production of metacercariae in the case of F. hepatica (1.7 to 5.6 times higher) and P. daubneyi (2.3 times). In contrast, metacercariae of F. magna were few in number, whatever the method of snail breeding is, and this might be explained by a still incomplete adaptation between the parasite of Czech origin and the French population of G. truncatula. The use of these 14-cm dishes reduced the time necessary for snail maintenance and metacercaria collection, thus allowing a decrease in the cost price of these larvae for commercial production.

In an attempt to establish an ideal method for mass production of Calicophoron microbothrium metacercariae, a study was carried out to compare the shedding capacities of Bulinus tropicus naturally and experimentally infected with C. microbothrium. A total of 906 F1 B. tropicus between 4 and 5 weeks old were each experimentally infected with two C. microbothrium miracidia and monitored for 12 weeks. The infected snails were fed on dried lettuce and fish flakes and were kept in 1 l plastic aquaria housed in a snail room where temperature, light and humidity were controlled. Seventy-four percent of the experimentally infected snails died during the prepatent period and of the remaining, only 13.2% developed patent infection, while 12.5% were refractory. Snail growth rate was poor and the average shedding rate was 20 cercariae per snail per day. Compared to the experimentally infected snails, 2200 adult B. tropicus, collected from the field and naturally infected with C. microbothrium, yielded high numbers of metacercariae. Eighty-four percent of the snails died within 7 weeks of the study with peak mortality occurring from the 2nd to the 4th week of infection and coinciding with an overall decrease in the number of cercariae shed.

Four experiments on the metacercariae of Fasciola hepatica and Paramphistomum daubneyi were carried out under natural conditions in order to study their vertical location on submerged plants and to determine whether simultaneous cercarial shedding of both digenea causes changes in the distributions of the metacercariae. These experiments were performed in experimental boxes, each containing six tufts of rushes. Most metacercariae (73.0%) of F. hepatica were found along the walls of boxes, while 81.5% of P. daubneyi metacercariae were found on rush stems. In the case of snails infected by either of the two digenea, 80.1% of F. hepatica cercariae encysted on submerged parts of rushes and of the box walls near the water surface (to a depth of 1 cm), whereas 73.0% of P. daubneyi metacercariae were found in the lower sections (from -4 to -7 cm). If snails dually infected with P. daubneyi and F. hepatica were used, the vertical distributions of the metacercariae were significantly different from those found for snails infected by either of the two digenea. If snails having 42-day old infections with F. hepatica and other snails with 70-day old infections with P. daubneyi were simultaneously introduced into the boxes, the frequency of F. hepatica cysts was significantly lower in the section located under the water surface (29% only), while the frequencies of metacercariae in the lower sections (from -1 to -5 cm) were increased. Some significant changes were also observed for the metacercariae of P. daubneyi. The disturbance noted in the vertical distributions of F. hepatica and P. daubneyi metacercariae suggest that the encystment of F. hepatica cercariae can be disturbed by the simultaneous encystment of P. daubneyi cercariae, or conversely.

Biomphalaria peregrina was found to be naturally infected with cercariae of Zygocotyle lunata in a pond of the Zoological Garden of Buenos Aires. Mice and chicks were fed metacercariae and gravid adults recovered. Eggs recovered from mice feces were used for experimental infections. Laboratory-reared B. peregrina and 4 other Biomphaluria spp. were successfully infected with Z. lunata miracidia. Biomphaleria glabrata was refractory. Species of Helisoma, the only intermediate hosts of Z. lunata so far reported, have never been found in Argentina. Species of Biomphalaria may be intermediate hosts of Z lunata in the southern region of the Parana River Basin.

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.

As a test of the energy limitation hypothesis (ELH), we predicted that temperature would have a significant influence on the infectivity of metacercariae of the digenetic trematode Zygocotyle lunata. Snails infected with Z. lunata were collected from ponds near Crawfordsville, Indiana, isolated at room temperature, and examined for the release of cercariae. Newly encysted metacercariae were collected and incubated 1-30 days at 1 of 5 temperatures (0, 3, 25, 31, 37 C). Twenty-five cysts were fed to each of 5 or 10 mice per treatment group (temperature). At 17 days postinfection, mice were killed and worms were recovered; data were collected on levels of infection in each group and the total body area of each worm. No worms were found in mice fed cysts that had been held at 0 C or 37 C (after 30 days). There were no differences in prevalence, infectivity, or mean intensity among the 3, 25, and 31 C treatments. Infectivity of metacercariae incubated at 37 C for 1 day was significantly greater than in all other treatments, while infectivity of metacercariae in the 37 C/15-day treatment was significantly lower than in all others. Mean body area of worms at 37 C/15 days was significantly greater than at other temperatures, suggesting density-dependent increases in growth. These results, particularly those from the 37 C treatments, are consistent with the ELH; infectivity was lower at high temperatures or when incubated for more time at 1 temperature (fewer energy reserves). It is suggested that microhabitat conditions experienced by metacercariae of Z. lunata could contribute to longer larval life, thus influencing this parasite's temporal dispersal.

Dual infections of Lymnaea truncatula with Paramphistomum daubneyi and Fasciola hepatica were performed to determine whether temperature changes in snails (daily water change with spring water at 6 degrees-8 degrees C, which subsequently increased to room temperature at 20 degrees C) would influence snail infection and the production of cercariae by both trematodes. At day 30 post-exposure the surviving snails were individually placed in petri dishes to constitute two groups. Snails from the first group were maintained at a temperature of 20 degrees C, and the water in the petri dishes was changed daily. The protocol was identical for the second group of snails except that the water temperature was 6 degrees-8 degrees C when changed. The interval between exposure and the first shedding of cercariae in snails immersed in cold water for a short period was longer (67-69 days instead of 48-50 days in the 20 degrees C group). In both groups, snails infected only with F. hepatica or P. daubneyi or with both trematodes were detected. In snails infected only with F. hepatica the frequency of cercaria-shedding snails and the total number of metacercariae were significantly greater in the 20 degrees C group. Inversely, in snails infected only with P. daubneyi the frequency of cercaria-shedding snails and the number of metacercariae were significantly greater in the 6 degrees-8 degrees C group. In snails harboring both trematode larval forms, no significant difference in the frequencies of cercaria-shedding snails between the two groups was noted. Metacercariae of both trematodes were obtained from these snails. In the 20 degrees C group, F. hepatica metacercariae were more numerous, whereas in the 6 degrees-8 degrees C group the number of P. daubneyi metacercariae was greater. From these results it appears that greater activity of P. daubneyi cercariae occurs in snails subjected to daily temperature changes (from 6 degrees to 20 degrees C).

Natural infection of Lymnaea glabra and Lymnaea truncatula by Fasciola hepatica and/or Paramphistomum daubneyi was studied at two periods of risk (June-July and September-October) in 11 French farms known for their high prevalences of F. hepatica infection in ruminants. A total of 1,778 L. truncatula and 2,396 L. glabra measuring 6 mm or more in height were collected to determine the prevalence of natural infection with F. hepatica and P. daubneyi. The role of four factors, i.e. season, snail co-infection (F. hepatica-P. daubneyi), ruminant host and the existence of single or mixed intermediate host communities, was investigated. There were no differences in prevalences between the two risk periods. Co-infections in L. glabra were more frequent than expected. The prevalences of infection with F. hepatica or P. daubneyi in L. glabra were higher in farms rearing sheep than in farms rearing cattle. A similar finding was also noted for L. truncatula infected with P. daubneyi only. L. glabra was a much better intermediate host for F. hepatica and P. daubneyi when it was the only available snail, possibly indicating an adaptation of parasites to their less usual host in local conditions. The two trematodes preferably developed in L. truncatula rather than in L. glabra when both host species lived in the same places.

Preadult Lymnaea glabra measuring 4-6 mm in height were each exposed to 1 Paramphistomum daubneyi miracidium before being exposed to 1 miracidium of Fasciola hepatica. Total prevalence of infection in the snail groups from 3 different populations ranged from 33% to 39%. In each group, snails harboring larval forms of P. daubneyi, F. hepatica, or both, were noted. If the results from the 3 snail populations are pooled, the total prevalence of snail infection was 13.6% in snails harboring only F. hepatica larvae, 13% in those harboring only P. daubneyi larvae, and 10% in those with both trematodes. Cercarial shedding was obtained from snails harboring F. hepatica larval forms; in the case of snails infected with both trematodes, the mean number of metacercariae ranged from 13 to 21 for F. hepatica, and from 8 to 14 for P. daubneyi. No infected snails were found in snail groups exposed only to 1 trematode miracidia.

Objective: To determine the effect of treating naturally acquired gastrointestinal nematode and paramphistome infections on milk production in dairy cattle., Design: A field trial., Animals: One thousand two hundred and thirty nine dairy cows., Procedure: Cows were either not treated or treated with 4.5 mg/kg oxfendazole, 16.6 mg/kg oxyclozanide or 4.5 mg/kg oxfendazole and 16.6 mg/kg oxyclozanide in March, May and August., Results: A significant increase in milk production, averaging 0.4 L (SE 0.2) per day, was seen when dairy cows infected with gastrointestinal nematodes and paramphistomes were treated with oxfendazole or oxfendazole and oxyclozanide in March, May and August. Cows treated with oxyclozanide alone at these times produced no more milk than untreated cows. Faecal egg counts confirmed that oxyclozanide treatment reduced paramphistome populations and oxfendazole treatment reduced nematode populations in cows over the 7-month monitoring period., Conclusion: When dairy cows infested with gastrointestinal nematodes and paramphistomes were treated with oxfendazole alone or oxfendazole and oxyclozanide in March, May and August milk production increased.

Explanatum explanatum (Creplin, 1847) Fukui 1929, was found in three indigenous buffaloes in the west Ahwaz area, southwest Iran for the first time. The presence of this amphistome in bile ducts caused remarkable fibrosis of submucosa ad hyperplasia of the endothelial layer, such that villus-like structures and protuberances were formed in large bile ducts of the infected livers.

The tegument of juvenile Paramphistomum epiclitum and Fischoederius elongatus (Paramphistomidae: Digenea) resembles those of other digeneans. Seven types of papillae were observed, mostly on the oral and acetabular surfaces, and increase in number during migration. Also evident are two types of secretory body (T1 and T2) which are synthesized separately in tegumental cytons underlying the syncytium. Exocytosis of T2 bodies occurs at the apical membrane and appears to contribute to a fibrous glycocalyx. The tegumental syncytium lining the pharynx and acetabulum is thinner and has a higher capacity for vacuolation than the general tegument. These may represent important sites for osmoregulation. The absence of mitochondria from the tegument in migrating juveniles suggests limited involvement in energy demanding processes. Pigmentation of the subtegument is first evident in mature cercariae and is progressively eliminated during migration.

Outbred (CD-1 strain) mice were infected experimentally with metacercariae of Zygocotyle lunata obtained from naturally infected Helisoma trivolvis. Growth, survival, and fecundity of the parasite and gross pathology in the host were evaluated. Metacercariae from 5 snail sources produced 3 patterns of growth and survival: significantly smaller worms that survived less than 4 wk and did not mature, significantly larger worms that matured but survived less than 6 wk, and worms of intermediate size that matured and lived at least 19 wk. Parasite maturation (at 3-4 wk) and egg production (14 eggs.worm-1.hr-1) were similar among infections from different sources. Ceca of mice with infections at least 4 wk old were enlarged with localized mucosal alteration at the site of parasite attachment, but there was no evidence of splenomegaly. The differences in infection due to the source of metacercariae within 1 strain of final host were similar to differences reported among other species of final host. These results suggest that variation associated with the intermediate host may confound studies of specificity to the definitive host.

Various physico-chemical factors associated with the in vitro excystment of metacercariae of Zygocotyle lunata were studied. Metacercariae could excyst with or without the presence of trypsin or bile salts, although the rate of excystation was slower in the absence of both. Crude ox-bile extract promoted faster excystment than did more refined sodium taurocholate. Pretreatment of cysts with pepsin also resulted in faster excystment rates. Optimal excystation occurred at 40 degrees C and between pH 5.5 and 7.5. The broad pH and temperature ranges and the non-specific enzyme/bile salt requirements for excystation were correlated with the wide range of vertebrate hosts used by this trematode.

The occurrence of Paramphistomum cervi has been reported in Mexico, but its intermediate hosts have not been identified. Five species of snails of the genus Lymnaea, L. bulimoides, L. columella, L. cubensis, L. humilis and L. palustris, were collected in Mexico. Only three of the exposed species, L. palustris, L. cubensis and L. humilis, became infected with P. cervi. The first of these snail species was highly susceptible to infection and a higher number of the exposed snails shed cercariae. It is suggested that L, palustris may act as an important vector of P. cervi in Mexico.

Biochemical components, glycogen, protein, nucleic acids and lipid fractions were analysed every month from January to December 1986, in the liver and rumen amphistomes Gigantocotyle explanatum and Gastrothylax crumenifer, respectively. The results reveal a considerable seasonal variation. In the rumen amphistome the components reach their maximum level only once a year, whereas in the liver amphistome, more than one peak is observed in a year. In G. crumenifer the content of glycogen and nucleic acids increases before the onset of egg production while protein and lipids reach their maximum level during the egg production phase. The variations in biochemical components were associated with the reproductive cycles and gonad recrudescence of these parasites.

The formation of the egg mass in the ootype, containing the oocyte, vitelline cells, numerous spermatozoa and Mehlis' gland secretion marks the beginning of eggshell formation. Mehlis' gland cells pour their lipoprotein secretion into the ootype; this secretion forms a thin template around the egg mass and upon this template the shell globules are deposited. Eggshell formation begins in the proximal uterus and is completed in the middle uterus. The eggshells are devoid of phenols and phenolase indicating that hardening is not by quinone tanning. The eggshells contain keratin and are stabilized by disulphide linkages.

Survival of immature and mature stages of Paramphistomum cervi, collected from the intestine and the rumen of sheep has been studied in in vitro using Tyrode's, Hank's, Eagle's, Stephenson's saline and Hedon--Fleig media. Twenty-five ml quantities of carbon dioxide (CO2)-rich Hedon--Fleig solution at pH 6.7, at 37 degrees C supported the maximum survival of 5 worms of both immature and mature flukes for periods of 9 and 10 days, respectively. Alterations to the pH of the medium, the incubation temperature and the number of worms significantly affected the period of their in vitro survival. The tissues of immature and mature worms did not show any marked histological and histochemical change until 24 h of in vitro survival in Hedon--Fleig solution when the testes of adult worms started undergoing degeneration. There was a progressive depletion of stored lipids and carbohydrates in the parenchymatous tissue during the course of in vitro survival. After the second day of in vitro maintenance, the maturation of the worms' vitelline follicles was affected, and thereafter they started to degenerate. On Day 4, degeneration of some gastrodermal cells, and a ruptured body wall were observed in some worms. Though the degenerative changes in the tissues of worms appeared at 24 h and continued to increase during in vitro maintenance, the worms survived for 9-10 days.

Experiments have shown that developmental period of eggs of P. petrowi under humid conditions depends directly on temperature. At 30 C their development is over in 8, at 26-27 C in 14 and at 18.5 C in 40 days. Under natural conditions at daily average temperature of 17.5 C the hatching of miracidia occurred in 52 days. Intermediate host of this trematode in a zone of park deer breeding is a freshwater mollusc of the family Planorbidae, Anisus minusculus. The development of parthenites in the intermediate host at 11 to 22 C completes within 83 days.

Histological studies of the trematode Paramphistomum cervi collected throughout the year from the rumen of sheep, revealed the occurrence of gravid worms from April to August and immature worms from September to March. The maturation of the gonads and the accessory sex glands i.e., the prostate-, Mehli's- and vitelline-gland began during the period March-April and showed functional peaks during the period July-August, which coincided with the availability of the intermediate host, Indoplanorbis exustus in nature. Maturation as well as the incidence of infection of P. cervi have also been discussed in relation to various ecological factors, i.e., rainfall and temperature.

A total of 480 snails were collected from 3 habitats on the Mau Escarpment, Kenya, and were identified as Bulinus tropicus. Of the 351 snails examined alive in London, 75 were infected with Calicophoron microbothrium, 39 with C. microbothrium and Schistosoma bovis, 1 with S. bovis, 24 with other species of trematodes and 212 were uninfected. Examination of digestive glands of B. tropicus either uninfected or infected with both C. microbothrium and S. bovis demonstrated that it is possible to differentiate between parasite and host enzyme activity using glucose phosphate isomerase. However, malate dehydrogenase enables a much clearer differentiation between the enzyme activity of the schistosome and that of the amphistome. Laboratory snail infection experiments demonstrated that it is possible successfully to infect B. tropicus with S. bovis if the snails have previously been exposed to miracidia of C. microbothrium.

The effects of temperature, hydrogen ion concentration, salts, light, gravity and living organisms on the development, survival and behaviour of Paramphistomum microbothrium miracidia were studied. Isotonic water environment of pH 7-8 and temperature of 25 degrees C were found to be most favourable for miracidial development and hatching. Miracidia longevity was highest at 21 degrees C in an isotonic water environment of pH 6-8. Demineralized water reduced the longevity of miracidia. Miracidia exhibit a strong phototaxis and a weak geotaxis, the latter being manifested only in the absence of light, and they show a preference for a medium with a low Ca/Mg ratio. An unidentified fungus, probably of the group of Fungi Imperfecti, had a deleterious effect on egg development. Aquatic organisms such as molluscans, dipterans and amphibia affect the survival of the miracidia. The relationship between observed phenomena and dynamics of infection are discussed.