In spite of its limitations, the culture of Cryptosporidium parvum in monolayers of epithelial cells is a suitable model to study the interaction of this protozoan parasite with the host cell, to assay oocyst infectivity, and to screen drugs for anti-cryptosporidial activity. For unknown reasons, growth of Cryptosporidium in culture is limited in time and generally does not lead to the production of significant numbers of oocysts. In monolayers infected with high doses of oocysts, we observed that many cells remain uninfected, suggesting that some cells are less susceptible to the infection. Since C. parvum and the related species C. hominis lack many essential biosynthetic pathways, we tested whether the dependence of the parasite on host cell metabolites may favour the infection of cells in mitosis. The proportion of monolayer cells in stationary (G0/G1) phase and in mitosis (S/G2/M) was determined and the prevalence of infected cells in each subpopulation was quantified. Although C. parvum infects and develops in dividing and stationary cells, a significant preference for cells in S/G2/M phase was observed. Consistent with previous observations showing that C. parvum induces apoptosis in cell monolayers, infection was accompanied by a significant increase in the proportion of mitotic cells.

The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti-Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a ‘pre-existing’ nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a ‘pre-existing’ nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease.

The aim of this study was to elucidate the regulatory responses and the long-term effect of praziquantel treatment in chronically Schistosoma bovis-infected West African Dwarf goats. Forty-two goats were used and the design comprised a primary infection followed by treatment at week 13, challenge infection at week 36 and termination at week 52. Dependent variables included clinico-pathological data, worm numbers, faecal and tissue egg counts, and gross pathology of the liver. The results showed that primary infections remained suppressed for up to 52 weeks and, although challenge infections imposed on 36-week-old primary infections established fully, the impairment of their egg production capacity provided protection against clinico-pathological consequences measured by body weight and haemoglobin levels. The study also confirmed a high efficacy (97·7%) of praziquantel for treatment of S. bovis infection in goats and showed that anthelminthic removal of primary infections does not interfere with the ability of the goat to elicit a marked resistance to a subsequent challenge infection. Although treated goats had more fibrous scarring of livers than untreated goats, no negative effects of liver lesions were reflected in weight gains of treated goats. This study provides strong evidence for the beneficial effects of anthelminthic treatment of young domestic stock as an element of treatment and preventive programmes.

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed.

Infection with Trypanosoma cruzi causes acute myocarditis and chronic cardiomyopathy. Remarkable changes have been demonstrated in the structure and physiology of cardiomyocytes during infection by this parasite that may contribute to the cardiac dysfunction observed in Chagas' disease. We have investigated the expression of α-actinin, an actin-binding protein that plays a key role in the formation and maintenance of Z-lines, during the T. cruzi-cardiomyocyte interaction in vitro. Immunolocalization of α-actinin in control cardiomyocytes demonstrated a typical periodicity in the Z line of cardiac myofibrils, as well as its distribution at focal adhesion sites and along the cell–cell junctions. No significant changes were observed in the localization of α-actinin after 24 h of infection. In contrast, depletion of sarcomeric distribution of α-actinin occurred after 72 h in T. cruzi-infected cardiomyocytes, while no change occurred at focal adhesion contacts. Biochemical assays demonstrated a reduction of 46% and 32% in the expression of α-actinin after 24 h and 72 h of infection, respectively. Intracellular parasites were also stained with an anti-α-actinin antibody that recognized a protein of 78 kDa by Western blot. Taken together, our data demonstrate a degeneration of the myofibrils in cardiomyocytes induced by T. cruzi infection, rather than a disassembly of the I bands within sarcomeres.

Prolonged diapause is usually interpreted as an adaptation to unpredictable environmental conditions and resource availability. Many parasites usually face highly unpredictable environments, therefore prolonged diapause should be common among these organisms. Here we examine the occurrence and frequency of prolonged diapause in the ectoparasite Carnus hemapterus (Diptera: Cyclorrhapha, Acalyptratae). We found that the studied population is polymorphic with respect to diapause duration. Emergence of carnid flies after 2 and 3 wintering seasons was therefore detected in around 17% and 21% of the samples respectively. The number of flies with prolonged diapause ranked 0·88–50% with respect to the number of flies emerging during the first spring. Both the occurrence of prolonged diapause and the number of flies with a long life-cycle are related to the number of flies emerging during the first spring. The emergence pattern of flies with prolonged diapause was very similar to that observed for flies with a short cycle and occurred in synchrony with the occurrence of hosts. Prolonged diapause has been frequently reported in plant-feeding insects and in some host-parasitoid systems, but this is, to our knowledge, the second report ever on prolonged diapause in true parasites of animals. We discuss the reasons for the apparent rarity of prolonged diapause among these organisms.

Parasitism by the flea Synosternus cleopatrae does not affect the body mass of its principal rodent host, Gerbillus andersoni under natural infestation levels. We hypothesized that the lack of negative effects of flea parasitism on rodent body mass could be related either to the low level of natural infestation or to the differential susceptibility of rodent age cohorts to flea parasitism. We tested these hypotheses by measuring body mass change under flea parasitism in (a) adult rodents infested with fleas above the natural infestation level (the first hypothesis) and (b) juvenile rodents infested with fleas at natural infestation levels (the second hypothesis). Adult individuals parasitized by a number of fleas higher than in nature lost body mass at higher rates than non-parasitized control individuals. Parasitism significantly affected daily body mass change of juvenile gerbils. Juvenile rodents parasitized by fleas at the natural level of infestation lost body mass faster and gained body mass slower than control animals. We suggest that some regulating mechanisms may limit natural flea densities at a point at which the negative effect on hosts is below the accuracy of our measurements. However, natural flea densities are sufficiently high to harm the more susceptible, juvenile cohort.

Mature Fasciola hepatica of the triclabendazole-resistant Sligo isolate were incubated in vitro with 10 μg/ml of the sulphoxide metabolite of compound alpha [5-chloro-2-methylthio-6-(1-naphthyloxy)-H-benzimidazole]; the metabolite will be referred to as alpha.SO. Changes resulting from drug treatment were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tubulin immunocytochemistry (ICC). SEM revealed that disruption to the tegumental surface mainly took the form of swelling and blebbing. Extensive spine loss occurred on the ventral surface of the oral cone, and sloughing of the tegument was observed along the lateral margins of the fluke. Examination of sections from the anterior mid-body region at the TEM level revealed that treatment with alpha.SO led to swelling of the basal infolds and mitochondria within the tegumental syncytium; also, accumulations of secretory bodies beneath the apical plasma membrane. The tegumental cell bodies contained swollen mitochondria and cisternae of granular endoplasmic reticulum, but few Golgi complexes were observed. An increase in T2 secretory bodies was observed, whilst in the T1 tegumental cells, the T1 secretory bodies had decreased in number. Immunocytochemical (ICC) studies showed that incubation with alpha.SO, ABZ.SO and TCBZ.SO did not cause significant changes to the distribution of tubulin within the tegumental syncytium of the Sligo isolate. In contrast, alpha.SO, ABZ.SO and TCBZ.SO caused severe disruption to tubulin organization within the syncytial layer of the TCBZ-susceptible Cullompton isolate. The EM results confirm that compound alpha is a fasciolicide capable of disrupting the tegument of mature TCBZ-resistant F. hepatica; however, this was not accompanied by any change in tubulin immunoreactivity.

The variability inherent in different isolates of Fasciola hepatica has been evident from reports in the literature but to date there has been no systematic examination of the relationship between these differences and the fecundity of the parasite. In this study we have attempted to remedy this situation by comparing the relative efficiencies with which 2 well-characterized isolates of the liver fluke (Oberon and Fairhurst) progress through both their definitive and intermediate hosts. We did not observe a reduction in fitness in the Oberon isolate which has been reported to be triclabendazole-resistant, compared to the triclabendazole-susceptible Fairhurst isolate, but considerable inter- and intra-isolate variability at different life-cycle stages was recorded. Thus the Oberon isolate gave 4-fold the number of cercariae when 100 snails were each challenged with a single miracidium and was more successful in establishing productive infections in rats. Fairhurst metacercariae excysted at a higher rate than those from the Oberon isolate and Fairhurst flukes produced 4-fold more eggs. The extent of the intra- and inter-isolate variability revealed in this work will provide a basis for the development of models of population dynamics aimed at predicting the response of the liver fluke to changing environmental conditions such as the use of anthelmintics or climatic change.

Robinia aurata n. g., n. sp. is described from Liza aurata (Mugilidae), the golden grey mullet, from the Ebro Delta, Spanish Mediterranean. The new genus differs from all other hemiurid genera in the combined possession of muscular flanges and a vestigial ecsoma. Within the Bunocotylinae, which currently accommodates 2 genera, Bunocotyle and Saturnius, the new genus exhibits a unique combination of blind caeca, Juel's organ, post-ovarian bulk of the uterus in the hind-body, and tegumental papillae surrounding the oral and ventral sucker apertures. Furthermore, Robinia n. g. differs from both Bunocotyle and Saturnius in the nature of the muscular extensions around the oral sucker, with the shape of a muscular belt in the latter and numerous muscular papillae in the former. The phylogenetic hypothesis for the Bunocotylinae developed from sequence data analyses based on partial lsrDNA and complete ssrDNA combined (22 species) and V4 domain of the ssrRNA gene (37 species) supports the erection of the new genus and confirms its position within the Hemiuroidea. Both molecular analyses confirmed the monophyly of the Hemiuroidea, its division into 2 major clades and the polyphyly of the Derogenidae, as in previous studies, and suggest that the Gonocercinae (with 2 genera, Gonocerca and Hemipera), may require a distinct familial status. Finally, there was poor support for the distinct status of the Lecithasteridae and Hemiuridae, following previous suggestions based on different sequence data sets. A key to genera of the Bunocotylinae is presented.

Parasite life-cycles present intriguing model systems to study divergence in resource use and ecology between parasite taxa. In ecologically similar taxa, consistent selective forces may lead to convergence of life-history traits, but resource overlap and similarity of life-cycles may also promote divergence between the taxa in (1) use of host species or (2) specific niche within a host. We studied the life-history characteristics of 2 sympatric species of Diplostomum parasites, D. spathaceum and D. gasterostei, concentrating particularly on differences in intermediate host use and characteristics of the infective stages between the species. This group of trematodes is a notoriously difficult challenge for morphological taxonomy and therefore any information on the ecology of these species can also be helpful in resolving their taxonomy. We observed that these species indeed had diverged as they used mainly different snail and fish species as intermediate hosts and in controlled experiments infected different regions of the eye (lens and vitreous body) of a novel fish host. Interestingly, cercarial characteristics (activity and life-span) were similar between the species and the species were difficult to separate at the cercarial stage unless one observes their swimming behaviour. The release of cercaria from the snail hosts was higher in D. spathaceum, but when cercarial numbers were proportioned to the volume of the snail host, the production was higher in D. gasterostei suggesting differences in the rate of snail host exploitation between the parasite species. These results corroborate the prediction that closely related parasite taxa which are competing for the same resources should have diverged in their life-history characteristics and host use.

Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus datasetNucleotide sequence data described in this paper have been deposited in the EMBL database under the Accession number AM177177.. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34–40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.

Protozoan cysts and helminth eggs preserved in a coprolite from the Early Cretaceous Bernissart Iguanodon shaft in Belgium demonstrate that representatives of 3 phyla parasitized dinosaurs by that period. These fossil parasite stages are described and their possible effect on dinosaurs discussed. These findings represent the earliest fossil records of protozoan and helminth parasites of terrestrial vertebrates.

The study of host-parasite relationships usually requires reliable estimates of parasite intensity, which is often estimated from parasite propagule concentration in faeces. However, parasite excretion in faeces may be subject to variation due to endogenous or exogenous factors that must be identified to obtain reliable results. We analysed the effect of the hour of sample collection on propagule counts of 2 intestinal parasites infecting the red-legged partridge: the capillarid nematode Aonchoteca caudinflata and coccidia of the genus Eimeria (Protozoa). Also, we test whether there are differences in propagule counts between caecal and intestinal faeces. Individual faecal samples from infected birds were collected daily at 4 different hours during several days. The hour of the day exerted a very strong effect on propagule counts, excretion of both types of parasites showing a clear and constant increase from dawn to dusk. Also, capillarid eggs were more abundant in intestinal than in caecal faeces, whereas the inverse pattern was found for coccidian oocysts. Standardization of the hour of sample collection or statistical control of this variable is recommendable to prevent bias. Similarly, in bird species with long caeca, consistent collection of one type of faeces may avoid significant errors in parasite burden estimates.