Polymerase chain reaction (PCR) combined with non-radioactive DNA hybridization was applied for the detection and characterization of a 150 bp tandem repeat of Onchocerca volvulus. DNA of worms from western Uganda was amplified and then probed with a digoxygenin-labelled oligonucleotide, specific for the forest form of O. volvulus and compared to samples from various African countries. Hybridization was only observed with PCR products from the forest in Liberia, south-eastern Ghana, Benin and southern Cameroon, but not with worms from Uganda or the savannah in Burkina Faso and northern Ghana. A nested PCR using primers derived form the forest form-specific DNA sequence confirmed these results. Morphometric studies revealed length differences between the microfilariae of Ugandan O. volvulus to those of West Africa, especially to those of the savannah in Burkina Faso. It is concluded that the forest/savannah classification of O. volvulus from West Africa is not suitable for Simulium weasel-transmitted O. volvulus from Uganda.

The population dynamics of outbred laboratory mice in indoor enclosures in the absence and presence of a naturally transmitted direct life-cycle nematode Heligmosomoides polygyrus Dujardin 1845 were reported previously. This manuscript presents further information on the age and sex structure of the populations, results of experiments designed to estimate the density-dependent effect of the parasite on host survival and reproduction, and a mathematical model of both uninfected and infected mouse populations. In the uninfected mouse population, survival of female mice was age- and density-independent, survival of male mice was age-dependent and density-independent, and recruitment was density-dependent. Independent experiments revealed that the parasite had no density-dependent effect on mouse reproduction, but had density-dependent effects on both acute and chronic survival of mice. An age-structured Leslie matrix model captured the exponential growth and plateau of the uninfected mouse population. Modification of the model to incorporate the effects of the parasite provided a good fit to the data from the infected populations, supporting the hypothesis that density-dependent effects of the parasite on host survival could lead to regulation of host abundance.

This study reports on the prevalence and severity of infections caused by the parasitic dinoflagellate, Hematodinium in juvenile edible crabs (Cancer pagurus) found in 2 intertidal survey sites (Mumbles Head and Oxwich Bay) in the Bristol Channel, UK. Crabs were assessed for the presence and severity of Hematodinium infections by the histological examination of infected tissues. Such infections were found to exhibit a seasonal trend in the 2 study areas with high numbers of animals (ca. 30%) infected in the spring to summer but with low severity. Conversely, in November only ca. 10% of crabs were infected but these animals had large numbers of parasites in their haemolymph and other tissues. At this time, the carapace and underlying tissues of infected crabs had the chalky, pinkish-orange appearance that is characteristic of this disease. Hematodinium-infected crabs ranged in size from 12 to 74 mm carapace width. Overall, it is concluded that the high prevalence of infection of juvenile crabs in this area may have implications for the sustainability of the edible crab fishery in the Bristol Channel.

The free-living nematode Caenorhabditis elegans is the simplest animal model organism to work with. Substantial knowledge and tools have accumulated over 50 years of C. elegans research. The use of C. elegans relating to parasitic nematodes from a basic biology standpoint or an applied perspective has increased in recent years. The wealth of information gained on the model organism, the use of the powerful approaches and technologies that have advanced C. elegans research to parasitic nematodes and the enormous success of the omics fields have contributed to bridge the divide between C. elegans and parasite nematode researchers. We review key fields, such as genomics, drug discovery and genetics, where C. elegans and nematode parasite research have convened. We advocate the use of C. elegans as a model to study helminth metabolism, a neglected area ready to advance. How emerging technologies being used in C. elegans can pave the way for parasitic nematode research is discussed.

We surveyed nine populations of the three-spined stickleback infected by the diphyllobothriidean cestode Schistocephalus solidus from south-central Alaska for two apparent forms of tolerance to infection in females capable of producing egg clutches notwithstanding large parasite burdens. Seven populations exhibited fecundity reduction, whereas two populations showed fecundity compensation. Our data suggest that fecundity reduction, a side effect resulting from nutrient theft, occurs in two phases of host response influenced by the parasite : host body mass (BM) ratio. The first is significantly reduced ovum mass without significant reduction in clutch size, and the second one involves significant reductions in both ovum mass and clutch size. Thus, ovum mass of host females who are functionally being starved through nutrient theft seems to be more readily influenced by parasitism and, therefore, decreased before clutch size is reduced. This inference is consistent with expectations based on the biology of and effect of feeding ration on reproduction in stickleback females. Fecundity compensation appears to be uncommon among populations of three-spined stickleback in Alaska and rare among populations throughout the northern hemisphere. Fecundity reduction seems to be common, at least among stickleback populations in Alaska.

Factors which may determine trematode–snail interactions were assessed in the present study. Compatibility was examined using a bacterial clearance assay to detect the modulatory effects of both compatible and incompatible trematode infections on the activity of haemocytes from Lymnaea stagnalis, during the early stages of infection. Exposure to and injection with Trichobilharzia ocellata, a compatible trematode, or the incompatible Schistosoma mansoni, resulted in modulation of haemocyte activity. However, T. ocellata activated haemocytes 1·5 h post-infection (p.i.) and then suppressed activity 24–72 h p.i. whereas with S. mansoni no suppression, only activation of haemocytes was observed throughout the test period (1·5–72 h p.i.). In previous studies, modulation of the haemocyte clearance activity by T. ocellata was found to be mediated by 2 E–S fractions, an activating fraction and a suppressing one. Investigations to assess whether the lack of suppression of haemocyte activity, observed in the S. mansoni–L. stagnalis incompatible trematode–snail interaction studied, was due to either the absence or ineffectiveness of the suppressing E–S fraction, were performed on a second incompatible combination, T. ocellata–Planorbis corneus. Using this combination it was revealed that only the activating E–S fraction had modulatory effects on P. corneus haemocytes, indicating that the suppressing E–S fraction, which actively interferes with the clearance activity of haemocytes from L. stagnalis, appears to act in a host-specific manner. In conclusion, the suppressing E–S fraction determines, at least in part, compatibility in the trematode–snail association studied. This is also probably likely in other trematode–snail combinations.

The development of molecular diagnostic methods, particularly those utilizing PCR for the detection of parasitic protozoa will contribute greatly to the identification and control of these pathogens, by increasing speed of diagnosis, specificity and sensitivity, reproducibility and ease of interpretation. PCR methods are not without their problems however, and there is a need for laboratory procedures to be refined before PCR-based assays are accepted as the tools of choice for the routine detection of protozoan parasites. The application of PCR detection to various parasites is discussed.

To examine levels of heat shock proteins (HSPs) in host responses to helminth infection, rats were infected with Trichinella spiralis. The kinetics of HSP25, HSP60, HSP70 and HSP90 production in the liver and muscle of infected rats was compared with that of non-infected controls. HSPs were detected using electrophoretic analysis of fixed amounts of proteins (0·02 mg) and then blotting and incubation of membranes with polyclonal anti-HSP25 antibody, or monoclonal antibody against HSP60, or HSP70 or HSP90. Quantitation of blotted separated polypeptides reactive with the specific anti-HSP antibodies was achieved using an image analyser. Enhancement of HSP25 production was observed in the liver of infected rats, whereas muscle from the same rats exhibited enhanced production of HSP25 and HSP60 one day after infection only. These data indicate that HSPs levels can be used successfully to measure stress injury brought about by helminth infection in organs and tissues of the host.

Interactions among different parasite species within hosts can be important factors shaping the evolution of parasite and host populations. Within snail hosts, antagonistic interactions among trematode species, such as competition and predation, can influence parasite abundance and diversity. In the present study we examined the strength of antagonistic interactions between 2 marine trematodes (Maritrema novaezealandensis and Philophthalmus sp.) in naturally infected Zeacumantus subcarinatus snails. We found approximately the same number of snails harbouring both species as would be expected by chance given the prevalence of each. However, snails infected with only M. novaezealandensis and snails with M. novaezealandensis and Philophthalmus sp. co-occurring were smaller than snails harbouring only Philophthalmus sp. In addition, the number of Philophthalmus sp. rediae was not affected by the presence of M. novaezealandensis sporocysts and the within-host clonal diversity of M. novaezealandensis was not influenced by the presence of Philophthalmus sp. Our results suggest that antagonistic interactions may not be a major force influencing the evolution of these trematodes and that characteristics such as host size and parasite infection longevity are shaping their abundance and population dynamics.

Recently, a way was opened with the development of many mathematical methods to model and analyze genome-scale metabolic networks. Among them, methods based on graph models enable to us quickly perform large-scale analyses on large metabolic networks. However, it could be difficult for parasitologists to select the graph model and methods adapted to their biological questions. In this review, after briefly addressing the problem of the metabolic network reconstruction, we propose an overview of the graph-based approaches used in whole metabolic network analyses. Applications highlight the usefulness of this kind of approach in the field of parasitology, especially by suggesting metabolic targets for new drugs. Their development still represents a major challenge to fight against the numerous diseases caused by parasites.

Toxoplasma gondii stimulates a potent pro-inflammatory response and neutrophils are involved in early infection. Galectin-3 (Gal-3) is an endogenous modulator of inflammatory processes and anti-infective agents, but its interaction with neutrophils in T. gondii infection is still unclear. Here, we evaluated the role of Gal-3 in peritoneal inflammation, reactive oxygen species (ROS) production by neutrophils and survival, after in vivo T. gondii infection with virulent RH strain, using Gal-3 deficient and wild type mice. Animals were inoculated with thioglycollate or tachyzoites, and peritoneal cells were harvested for analysis of the influx of leukocytes. Neutrophils were isolated from peritoneal exudates from infected mice and stimulated with phorbol myristate acetate (PMA) to evaluate ROS production by luminol-dependent chemiluminescence assay. Our results showed that: (1) Gal-3 upregulates peritoneal inflammation, with enhanced recruitment of neutrophils and lymphocytes after thioglycollate stimulation, but does not influence the enhanced neutrophil influx after early T. gondii infection; (2) Gal-3 upregulates ROS generation by inflammatory peritoneal neutrophils from infected mice, but downregulates its production in non-infected mice and (3) Gal-3 does not influence the survival of mice after infection with the virulent T. gondii strain. In conclusion, Gal-3 is essential for ROS generation by neutrophils in the initial acute phase of T. gondii infection and this phenomenon may constitute an attempt to control parasite growth during in vivo infection with the T. gondii virulent strain.

P. falciparum malaria severely affects pregnant women and children. Despite immunity through lifelong exposure to malaria, pregnant women become susceptible to infections causing anaemia, abortions and low birth weight. They experience massive accumulation of infected erythrocytes (IEs) in the placenta. Adhesion of IEs to host endothelial receptors is mediated by members of a large diverse protein family called P. falciparum erythrocyte membrane protein 1 (PfEMP1). Pregnancy malaria is generally associated with the emergence of a distinct subset of parasites expressing a unique PfEMP1 that binds to the host-receptor chondroitin sulfate A (CSA). Resistance to pregnancy malaria is associated with the acquisition of antibodies that block IEs binding to placental CSA. The absence (or rare occurrence) of CSA-binding parasites in malaria patients (children, men and non-pregnant women) suggests that these parasites become virulent only during pregnancy. The molecular mechanisms used by P. falciparum to achieve the timely expression of the Pf-CSA ligand in pregnant women remain puzzling. In this review we will discuss two hypothetical mechanisms by which CSA-binding parasites may arise during pregnancy. The first, a selection process by the placenta of a distinct sub-population of P. falciparum expressing a particular PfEMP1. The second, an induction mechanism that facilitates the expression of a particular PfEMP1 protein by specific host factor(s) present only during pregnancy.

Surgical treatment of human hydatidsosis involves the use of various scolicidal agents to kill infective Echinococcus granulosus protoscoleces that may disseminate into the peritoneal cavity during surgery and potentially re-infect the patient. Currently, no scolicidal agent is completely effective in killing intracystic protoscoleces in humans. Cyclosporin A (CsA) has previously been found to be lethal for E. granulosus protoscoleces in vitro. In this study, we further assessed the effectiveness of CsA as a scolicidal agent by testing the toxic effect of CsA at higher doses over various time-periods. Experiments were performed on activated and unactivated protoscoleces cultured in nutrient medium or sheep hydatid cyst fluid. All activated protoscoleces were killed following culture in 100 μg/ml of CsA for 3 days and 50 or 20 μg/ml for 5 days. The lethal effect of CsA on unactivated protoscoleces varied but reached 100% over 15 days in culture with 100 or 50 μg/ml of CsA. Pulse treatment of protoscoleces with 50, 20 or 10 μg/ml of CsA for 5 min or 72 h killed all parasites by day 10 and day 5 respectively. Untreated protoscoleces remained greater than 95% viable for the duration of experiments. Changes in protoscolex ultrastructure induced by treatment with 10 μg/ml of CsA over 10 days in in vitro culture was assessed by TEM. Protoscolex alterations observed in treated parasites included an increase in cellular vacuolization, swelling of mitochondria, rounding of cells, damage to the tegument, decrease in glycogen, a breakdown of the extracellular matrix and an increase in lipid globules. The untreated protoscoleces, by comparison, had few changes during the 10-day culture period with the exception of large amounts of extracellular glycogen observed in the protoscoleces at culture days 7 and 10. From these results, CsA is clearly an effective scolicidal agent in vitro that may have potential application as a new therapeutic agent in the treatment of human hydatid disease.

PCR amplification of 16S rDNA was used to determine the diversity of bacteria associated with 3 strains of sheep scab mite, Psoroptes ovis. Eight species of bacteria were identified by phylogenetic analysis of the PCR product sequences. Seven of these species are previously unreported in association with sheep scab mites. Five species were matched to Serratia marcesens, Proponibacteium acnes, Phyllobacterium rubiacearum, Pantoea agglomerans, Curacaobacter baltica, whereas the remaining 3 sequences matched unclassified sequences belonging to the gamma proteobacteria, pseudomonads and streptococci. Bacterial diversity of the in vivo cultured strain was very low and did not match the diversity of the 2 wild collected isolates. The diversity of the bacteria in relation to the disease of sheep scab and the possible importance of these bacteria in the diet of the mites are discussed.

The family Hippoboscidae is a less known group of blood-sucking flies. Deer ked are particularly important for animal health; they may act as potential vectors of disease to ungulates, and may transmit pathogens to animals and humans. The aim of this study was to investigate the presence of Trypanosoma (Megatrypanum) DNA in deer keds using molecular methods. Results prove the presence of Megatrypanum trypanosome DNA in the studied winged adult deer keds and this is the first detection of this pathogen in Lipoptena fortisetosa. In addition, this paper evidences the occurrence of L. fortisetosa in two new locations: one in the Białowieża Primeval Forest, and another in the Strzałowo Forest Inspectorate (Piska Forest), both in north-eastern Poland.

Due to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

Glycogen reserves of whole worms and their body wall, intestines, ovarian and testicular tubules of the avian intestinal nematode, Ascaridia galli, were assayed and on dry-weight basis found to be 14–20 %, except in the testicular tissue which contained only 7 % glycogen.

Segments of whole male worms or testicular tubules were found to produce more lactic acid than segments of whole female worms or ovarian tubules. The body wall and intestine of the worms had also appreciable glycolytic activity. Whereas in the segments of whole worms, male and female alike, glycolysis was more active under anaerobic conditions, no differences in glycolytic rates were seen between aerobiosis and anaerobiosis when isolated tissues were used. Exogenously added glucose did not stimulate glycolysis either aerobically or anaerobically to any extent greater than observed in the absence of glucose.

Homogenates of whole worms or their different anatomical parts were assayed for enzymic activities associated with the Embden-Meyerhof pathway of glucose assimilation. The crude extracts prior to differential centrifugation, were found to contain glycogen phosphorylase, aldokinases, pyruvate kinase, phosphatases acting on glucose 6-phosphate and hexose diphosphate and 6-phospho-gluconate dehydrogenase. The 105000 g particulate-free supernate was found to have significant activities of the following enzymes: fructokinase, phosphoglucomutase, phosphoglucoseisomerase, phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, lactic dehydrogenase and glucose-6-phosphate dehydrogenase. This fraction was devoid of NADH or NADPH oxidase activities in the absence of added substrates.

Although there was some indication of a negative correlation between low glycogen reserves and high glycolytic activity in the testicular tubules, in general, there was no relationship between glycogen reserve and glycolytic activity on the one hand or between the rate of glycolysis and the specific activities of some of the key glycolytic enzymes in either whole worm or in tissues other than the testicular tubules.

The 105000 g supernate was fractionated with ammonium sulphate. The fraction precipitating between 25 and 80 % saturation of the salt was recovered, dialysed and chromatographed on DEAE cellulose column. By a step-wise elution schedule using increasing molarity of NaCl in tris-HCl buffer, pH 7·4, three main protein fractions were obtained representing respectively enolase, aldolase and glucose-6-phosphate dehydrogenase.

The recovery of enzyme activity after chromatography on DEAE cellulose was higher than the amount applied to the column suggesting that during the fractionation some naturally occurring inhibitors were removed. About 10- to 20-fold purification of the enzymes was achieved by anion-exchange chromatography.

Some properties of the purified enzymes were studied with respect to the affects of enzyme and substrate concentrations, temperature of preincubation and action of divalent cations, some anions, metal chelating agents and SH reagents. The Km values of enolase, aldolase and glucose-6-phosphate dehydrogenase of A. galli were 5·9 × 10−4M, 4·5 × 10−3M and 2·4 × 10−3M respectively. Glucose-6-phosphate dehydrogenase was found to be very sensitive to both heat and cold losing activity rapidly even at 43 °C or by freezing and thawing.

The SH groups of aldolase were readily blocked by pCMB and presumably by o–phenanthroline. No requirement of any divalent cations was shown by this enzyme which was, however, inhibited by borate ions.

Enolase of A. galli showed a requirement of Mg2+ for full activation. Phosphate, fluoride, EDTA, o–phenanthroline, αα-bipyridyl inhibited the enzyme. Veronal was found to inactivate the enzyme.

Glucose-6-phosphate dehydrogenase of A. galli was also found to be sensitive to SH reagents and metal chelating agents. The enzyme was activated by Co2+, Mn2+ and Mg2+.

The evidence presented indicates that segments of whole worms of A. galli or its anatomical parts are equipped with the enzymatic machinery required to mediate anaerobic breakdown of glucose and to derive energy by this mechanism.

The authors are grateful to Dr R. K. Kaushik for help in identifying the worms, to Mr P. A. George for help in the statistical analysis of the data and to Messrs A. C. Kol and S. K. Bose for skilful technical assistance.

An examination of 2 strains of Eimeria acervulina var. mivati (since 1973 E. mivati has been regarded as a variant of E. acervulina) showed that previous confusion over the taxonomic status of E. mivati arose because the investigations were done using laboratory cultures of E. mivati which were contaminated with E. acervulina.

Electrophoretic analyses of enzymes, host specificity and cross-immunity tests have revealed that:

(1) The 1971 Houghton strain of E. acervulina var. mivati was a mixture of 2 parasites. (a) Passage of this strain in embryonating eggs resulted in a selection against that parasite previously characterized as E. acervulina. (b) The parasite which did reproduce in eggs did not immunize chickens against subsequent challenge with E. acervulina. This parasite is most likely E. mivati. (c) E. mivati recovered from eggs did, however, immunize chickens against challenge with a new field strain which was morphologically identical to E. mivati and characterized by the same electrophoretic forms of 2 enzymes.

(2) A strain of E. acervulina var. mivati from the USA was also a mixture of E. acervulina and E. mivati.

Cell cloning techniques and schizodeme analysis were used to detect mixtures of subpopulations in Leishmania parasites, isolated from humans and a reservoir host. Clones were obtained by plating promastigotes, at limited dilution, on solid medium. The resultant colonies were analysed by the restriction profile of kinetoplast DNA minicircles (schizodeme analysis). The efficiency of plating was around 40–60%, and subpopulations of different schizodemes, in stocks of Leishmania isolated from 2 cases of human cutaneous leishmaniasis were detected. The presence of different schizodemes in one isolate from a sylvatic animal suggested the possibility of a mixed natural infection.

It was noticed that some stock cultures of Laemophloeus minutus Olivier and L. ferrugineus Stephen were showing an abnormally high mortality. The numbers of living larvae appeared to decrease, many pupae died inside their cocoons and most of the few adults which emerged died within a few weeks. As adult Laemophloeus live for about 6 months at the conditions under which the cultures were kept (25° C. and 70% r.h.) it was apparent that something was killing the beetles at all stages in development. Microscopic examination of dead larvae and adults which had been chopped up and mounted in Berlese's fluid (Swan's formula) revealed huge numbers of the lemon-shaped spores (sporocysts) or pseudonavicellae of a sporozoan parasite. Living larvae from infected cultures were dissected in saline, and smears were fixed in Bouin's fluid (aqueous) and stained with Heidenhain's haematoxylin. Other larvae were fixed in Bouin's fluid, sectioned, stained with Ehrlich's or Heidenhain's haematoxylin and eosin, cleared in xylol and mounted in Canada balsam. These preparations revealed various stages of a schizogregarine which appeared to be Mattesia dispora which was described by Naville (1930) from Ephestia kühniella Zell. (Lepidoptera, Phycitidae) and recorded by Musgrave & Mackinnon (1938) from Plodia interpunctella Hbn. (Lepidoptera, Phycitidae). Careful examination of sections and measurement of various stages confirmed this identification. All stages were found, with the possible exception of young gamonts formed by macronuclear schizogony.