Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

A phylogenetic study of the cecal amphistome Zygocotyle lunata (Trematoda: Zygocotylidae), with notes on the molecular systematics of Paramphistomoidea.

Zygocotyle lunata inhabits the caecum of birds and mammals from the American continent. This amphistome parasite is easily maintained in the laboratory and serves as a model organism in life-cycle studies, but it has seldom been studied using molecular data. Neither the position of Z. lunata in the superfamily Paramphistomoidea nor the monophyly of the Zygocotylidae has been evaluated with molecular phylogenetic methods. In the present study, adult specimens of Z. lunata obtained experimentally in mice from Brazil were submitted to molecular studies. Partial sequences of nuclear (1261 bp of 28S and 418 bp of 5.8S-ITS-2) and mitochondrial (1410 bp of cytochrome c oxidase 1, cox1) markers were compared with published data. In the most well-resolved phylogeny, based on 28S sequences, Z. lunata clustered in a well-supported clade with Wardius zibethicus, the only other species currently included in the Zygocotylidae, thus confirming the validity of this family. Divergence of 28S sequences between these species was 2.2%, which falls in the range of intergeneric variation (0.9-5.6%) observed in the other two monophyletic groups in the 28S tree, i.e., representatives of Gastrodicidae and Neotropical cladorchiids (Cladorchiidae). Analysis of ITS-2 and two parts of the cox1 gene placed Z. lunata within poorly resolved clades or large polytomies composed of several paramphistomoid families, without clarifying higher-level phylogenetic relationships. The cox1 of a Brazilian isolate of Z. lunata is 99.6% similar to a Canadian isolate, confirming the pan-American distribution of the species. Finally, our phylogenetic reconstructions of Paramphistomoidea revealed a complex scenario in the taxonomic composition of some amphistome families, which highlights a need for further integrative studies that will likely result in rearrangements of traditional morphology-based classifications.

Pseudosuccinea columella: age resistance to Calicophoron daubneyi infection in two snail populations.

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.

The production of mammalian trematode infective stages by the snail Galba truncatula.

Several experiments on the breeding of trematode-infected Galba truncatula for obtaining and packaging Fasciola hepatica and Paramphistomum daubneyi metacercariae were carried out to determine the more convenient methods to use for commercial production of these infective stages. Compared to the breeding of infected snails in aquaterraria, the use of 14-cm Petri dishes allowed a greater prevalence of snail infection and a higher number of metacercariae. The production of these larvae was still 2.3-3.4 times greater if infected snails were dissected during the patent period. The aspiration of these metacercariae at the extremity of a Pasteur pipette significantly shortens the time necessary for their transfer from Petri dishes to Eppendorf tubes. Using 14-cm Petri dishes, snail dissection and metacercarial aspiration for their transfer strongly reduce the cost price for metacercarial production of the trematodes Fasciola hepatica and Paramphistomum daubneyi.

Influence of Calicophoron microbothrium amphistomosis on the biochemical and blood cell counts of cattle.

Sixteen Tuli steers aged 1 year were subdivided into four equal groups (I-IV) and infected with Calicophoron microbothrium metacercariae. Group I received a low dose (LD) of 5000 metacercariae, group II a medium dose (MD) of 15,000 metacercariae, group III a high dose (HD) of 25,000 metacercariae while group IV was the non-infected control (C) group. The experimental animals were monitored daily for clinical signs while ethylene diamine tetraacetic acid (EDTA) blood and serum samples were collected every 7 days until day 28 post-infection, when sample collection was terminated. Samples were processed for full blood count, eosinophils and blood biochemical values for calcium, magnesium, phosphorus, total protein and albumin. Moderate to severe diarrhoea developed in the MD and HD groups at day 21 post-infection. The diarrhoea coincided with a significant decrease (P < 0.05) in total plasma protein, calcium and phosphorus levels, particularly in the MD group. Similarly, a significant decrease (P < 0.05) in the packed cell volume (PCV), the haemoglobin (Hb) and red blood cell (RBC) levels occurred in the MD and HD groups from day 21 post-infection, while a significant increase (P < 0.05) in the circulating eosinophils occurred between 7 and 21 days post-infection in the LD and the HD groups.

Efficacy of oxyclozanide against the rumen fluke Calicophoron daubneyi in experimentally infected goats.

Paramphistome infections are very common in ruminants and may induce clinical signs, but little is known about effective treatments. In this study, the efficacy of oxyclozanide against Calicophoron daubneyi was studied in goats and its activity tested against immature stages (10 days post-infection) at a dose of 22.5mg/kg bodyweight (BW) and against adult stages using two doses (15 and 22.5mg/kg BW). There was a reduction (82%) in the number of immature worms (compared to controls) but the result was not statistically significant. When tested against adult stages, however, oxyclozanide reduced the worm burdens by 95.6% and 95.9% at doses of 15 and 22.5mg/kg BW, respectively, with no significant difference between the two doses. The experiment demonstrated that oxyclozanide is highly effective in reducing the number of adult paramphistomes in goats.

The redial and cercarial production of a digenean in the snail host is lower when no cercarial shedding occurs.

Single- and double-miracidium exposures of Galba truncatula with Fasciola hepatica (two groups) or with Paramphistomum daubneyi (two groups) were carried out under laboratory conditions to compare parasite production in cercaria-shedding snails (CS snails) with that found in snails without emission (NCS snails). Free rediae and cercariae were thus counted in snails from both categories after their dissection at regular intervals (at 24 degrees C). In the four groups, the numbers of free rediae and free cercariae found at day 75 post-exposure (F. hepatica) or at day 85 (P. daubneyi) were significantly greater in CS snails than in NCS ones. The number of cercariae in NCS subgroups did not show any significant variation from day 45 p.e. to day 75 (F. hepatica, the two groups) or from day 55 to day 85 (P. daubneyi, single-miracidium infections), while it significantly decreased with increasing time of infection in the double-miracidium infections with P. daubneyi. In NCS snails, the presence of too numerous free cercariae within the snail's body (the volume of the body allows development only of a given number of rediae) might rapidly block out redial development and intraredial differentiation of other cercariae. The numerical diminution of P. daubneyi cercariae in the NCS snails (double-miracidium group) might probably be due to the lysis of new cercariae just formed, probably in reason of a lack of nutrients available for these larvae within the snail.

[Gastrodiscoidosis is a dangerous zoonosis].

The paper gives data on the helminthiasis--gastrodiscoidosis, a zoonotic disease caused by the trematode Gastrodiscoides hominis (Lewis et McConnall, 1876) parasitizing in the animal and human intestine in the endemic foci of both Russia and foreign countries. It also presents information on the morphology of the helminth, the biological cycle of development of the parasite and its habitat and spread, as well as its induced abnormalities and on the method of diagnosing of the disease.

Paramphistomum daubneyi: the number of sporocysts developing in experimentally and naturally infected Galba truncatula.

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.

Paramphistomum daubneyi: production dynamics and infectivity of metacercariae originating from snails dissected at regular intervals.

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.

Parasite development and visceral pathology in Galba truncatula co-infected with Fasciola hepatica and Paramphistomum daubneyi.

Histological investigations in Galba truncatula naturally or experimentally co-infected with Fasciola hepatica and Paramphistomum daubneyi were carried out to study parasite development and the responses of the digestive gland and kidney of snails, as larval forms of these digeneans often use these two sites for their growth within the snail's body. The number of live rediae per snail ranged from 2.4 to 4.2 for the dominating parasite (it developed in the digestive gland) and was less than 2.0 for the other species. When the dominating species was F. hepatica, most snails harboured cercariae-containing rediae; if this parasite was P. daubneyi, procercariae-containing rediae with or without free procercariae were observed in most snails. In contrast, most rediae of the other species were immature. The pathology caused by the dominating species in the digestive gland was greater than that recorded in the kidney, where the other parasite was generally located. The most frequent tissue lesions in the digestive gland were generalized epithelial necrosis and epithelial reconstitution. In the kidney, multifocal epithelial necrosis was frequently observed, particularly when P. daubneyi was the dominating species. The frequencies of lesions in the digestive gland agreed with percentages reported by our team in other snails mono-infected with F. hepatica or P. daubneyi. In contrast, multifocal necrosis in the kidney was clearly greater in the present study and this finding might be explained by assuming that a sufficient number of free larvae within the snail would be necessary for the development of epithelial necrosis in the whole kidney.

Prevalence and intensity of infections in the lymnaeid snail Omphiscola glabra experimentally infected with Fasciola hepatica, Fascioloides magna and Paramphistomum daubneyi.

Single and double infections of juvenile Omphiscola glabra (Gastropoda: Lymnaeidae) with Paramphistomum daubneyi and/or Fasciola hepatica were carried out to determine the redial burden and cercarial production in snails dissected at day 60 or at day 75 post-exposure (p.e.) in the laboratory at 20 degrees C. The results were compared with those obtained with single-miracidium infections by Fascioloides magna. Compared to F. hepatica, low values were noted at day 75 p.e. for the prevalence of snail infections with P. daubneyi (4.6-8.3% instead of 23.6-25.9%), the total number of free rediae (10.7-17.9 per snail instead of 26.3-34.7), and that of free cercariae (112.8-136.9 per snail instead of 177.8-248.5). Despite a greater number of free rediae at day 75 p.e. (36.2-45.6 per snail), the prevalences of snail infections with F. magna and cercarial production were similar to those noted for F. hepatica. The results concerning F. hepatica and P. daubneyi might partly be explained by a progressive adaptation of O. glabra to sustain the larval development of these digeneans over the years, as this snail is a natural intermediate host of F. hepatica and P. daubneyi in central France since 1995. Compared with the high number of fully-grown rediae of F. magna in O. glabra, cercarial production seemed limited and this might be explained by the presence of high numbers of rediae which reduced the avaibility of nutrients for cercarial differentiation within the snail.

Seasonal pattern of bovine amphistomosis in traditionally reared cattle in the Kafue and Zambezi catchment areas of Zambia.

Seasonality of bovine amphistomosis in the Southern province of Zambia was established after examining 268 faecal samples from cattle presented for slaughter at Turnpike slaughter slab, Mazabuka. Amphistomosis was found present throughout the year but the highest abundance rate was found during the post-rainy season (47.8%) and the lowest during the cold dry season (24.8%). In the rainy and post-rainy seasons, higher mean egg counts and cattle found positive were recorded than in any other season. The distribution of amphistome eggs was significantly different (p < 0.001) among the four seasons, with the rainy season having higher median egg counts than others. There were no significant differences in abundance rates between sexes or between ages of cattle. A similar seasonality to that of fasciolosis exists and may help in strategic management of Fasciola and amphistomes.

Observations on mass production of Calicophoron microbothrium metacercariae from experimentally and naturally infected Bulinus tropicus.

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.

Distribution, habitats and role as intermediate host of the freshwater snail, Bulinus forskalii, in South Africa.

This paper focuses on the geographical distribution and habitats of Bulinus forskalii, the snail intermediate host of the conical fluke of equids, Gastrodiscus aegyptiacus as reflected by the 1209 samples in the database of the National Freshwater Snail Collection of South Africa. The 362 different loci on record represent an extensive distribution in KwaZulu-Natal Province, the Limpopo Province, the coastal areas of the Eastern Cape Province and the south-eastern part of the North West Province. Although it was recorded from all types of water-body represented in the database, the highest percentages of samples were recovered from dams (30.4%) and brooks (28.2%). The majority of samples came from perennial habitats (59.1%), 60.7% from habitats with standing water, 54.0% from habitats with clear water and 71.8% from habitats of which the water was described as fresh. The majority of samples (39.5%) were collected in habitats of which the substratum was recorded as muddy. The highest percentage of samples, by far (81.5%), was collected in habitats that fell within the mean yearly temperature interval ranging from 15-20 degrees C. An integrated decision tree constructed from the data in the database indicated that temperature and type of water-body played a decisive role in determining the presence of B. forskalii in a given area. The results of experimental exposure to miracidia of a local strain of both Schistosoma haematobium and Schistosoma mattheei in the laboratory indicated that a local strain of B. forskalii was incompatible with both these strains of parasite. Research to clarify the role of B. forskalii in the transmission of both Calicophoron microbothrium and G. aegyptiacus in South Africa, is recommended.

Fasciola hepatica and Paramphistomum daubneyi: vertical distribution of metacercariae on plants under natural conditions.

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.

Use of remote sensing and geographical information systems to identify environmental features that influence the distribution of paramphistomosis in sheep from the southern Italian Apennines.

A geographic information system (GIS) was constructed using remote sensing (RS) and landscape feature data together with Calicophoron daubneyi positive survey records from 197 georeferenced ovine farms with animals pasturing in a 3971 km(2) area of the southern Italian Apennines. The objective was to study the spatial distribution of this rumen fluke, identify environmental features that influence its distribution, and develop a preliminary risk assessment model. The GIS for the study area was constructed utilizing the following environmental variables: normalized difference vegetation index (NDVI), land cover, elevation, slope, aspect, and total length of rivers. These variables were then calculated for "buffer zones" consisting of the areas included in a circle of 3 km diameter centered on 197 farms. The environmental data obtained from GIS and RS and from data taken by the veterinarians on the field (stocking rate and presence of streams, springs and brooks on pasture) were analyzed by univariate (Spearman and ANOVA) and multivariate (discriminant) statistical analyses using the farm coprological status (positive/negative) as the dependent variable. Sheep on 32 of the 197 (16.2%) farms, were positive for C. daubneyi, with an average intensity of 52 epg. A multivariate stepwise discriminant analysis model was developed that included moors and heathland, sclerophyllous and coniferous forest vegetation, autumn-winter NDVI and presence of streams, springs and brooks on pasture. The variables entered in the model were also correlated with C. daubneyi positive farms in the univariate tests and are consistent with the environmental requirements of C. daubneyi and its snail intermediate host.

Variability in the prevalence of infection and cercarial production in Galba truncatula raised on a high-quality diet.

Two experiments using seven populations of Galba truncatula were carried out to analyse the effect of food quality (cos lettuce only, or cos lettuce+Tetraphyll) on the characteristics of infections: (1) in a single population of G. truncatula infected by one of three digenea (first experiment), and (2) in seven populations of G. truncatula differing in their susceptibility to Fasciola hepatica miracidia (second experiment). In most groups, food quality did not have a significant effect on the survival of snails. The prevalence of infection in five populations was significantly higher in snails raised on lettuce+Tetraphyll (first and second experiments), whereas it was close to those noted in lettuce only-reared groups in the last two populations (second experiment). Despite the higher growth of cercariae-shedding snails when raised on the mixed diet, no significant differences were noted. Significant effects of parasite species (first experiment) and of snail population (second experiment) on the life-spans of cercariae-shedding snails were noted, whereas food quality did not influence this parameter. Except for a single snail population, cercarial production in groups raised on lettuce+Tetraphyll was significantly higher than that in groups on lettuce. The variability noted in the prevalence of snail infections and in the intensity of cercarial shedding might be explained by differences in the susceptibility of snail populations to F. hepatica infections, and/or by the fact that Tetraphyll would not have the same appetency for all populations of G. truncatula.

Fasciola hepatica and Paramphistomum daubneyi: changes in prevalences of natural infections in cattle and in Lymnaea truncatula from central France over the past 12 years.

A retrospective study was carried out over a 10- to 12-year period to analyse the changes in prevalences of natural fasciolosis and paramphistomosis among cattle and snails in central France, and to determine the causes which had induced these changes. The prevalences of natural fasciolosis in cattle increased from 1990 to 1993 (13.6% to 25.2%) and diminished afterwards up to 1999 (at 12.6%). Those of natural paramphistomosis showed a progressive increase between 1990 and 1999 (from 5.2 to 44.7%). The prevalences of natural infections and the numbers of free rediae counted in the snails (Lymnaea truncatula) infected with F. hepatica did not show any significant variations over time. By contrast, the prevalences of natural paramphistomosis in snails significantly increased from 1989 to 1996 and remained afterwards in the same range of values (3.7-5.3%), while the number of free rediae significantly increased up to 2000 (from a mean of 6.5 to 13.8 rediae per infected snail, respectively). Three hypotheses may explain the increase of paramphistomosis in cattle and snails: a better quality of diagnosis for the detection of P. daubneyi eggs in veterinary analysis laboratories, the use of specific molecules in the treatment of cattle fasciolosis since 1993, and the lack of an effective treatment up to now against cattle paramphistomosis. Since the objective of most farmers in central France is to obtain the highest antiparasitic efficiency with a single treatment of cattle per year, it is reasonable to assume that the prevalence of bovine paramphistomosis will continue to increase in the future.