Peroxynitrite (ONOO-) is a cytotoxic anion, produced by interaction between nitric oxide and superoxide in vivo in some inflammatory cells. This study investigated its effects on Fasciola hepatica and Dicrocoelium dendriticum isolated from ovine livers and kept in bile at room temperature. Peroxynitrite was synthesized using a quenched flow reactor and assayed spectrophotometrically. It was applied at different concentrations (10-3.5 to 10-2.3 M) to the flukes kept in bile. The viability of the peroxynitrite-treated flukes was compared with a control group (n=5–7 per group). Control F. hepatica and D. dendriticum lived for 226±11 and 208±14 min, respectively. Life times were decreased by peroxynitrite at all concentrations used (P<0.001). At the highest concentration of peroxynitrite, F. hepatica and D. dendriticum lived only for 6.1±0.4 and 4.1±4.1±0.2 min, respectively. Correlation between peroxynitrite concentration and parasite viability was significant in the case of F. hepatica (r=-0.842; P=0.0035). A single application of peroxynitrite can decrease the life span of ovine liver flukes. A failure in the activation of hepatic macrophages in infected animals may lead to a decreased production of free radicals and, thus, peroxynitrite. Such a failure is likely to deprive the body of a defence tool against multicellular parasites.

Recombinant protein was produced from the cDNA library of Trichinella pseudospiralis, which seemed to form part of the excretory–secretory (ES) products. The library was constructed from cDNA of muscle larvae at 1 month post-infection, and immunoscreened with antibody against T. pseudospiralis ES products. A clone, designated Tp21-3, contained a cDNA transcript of 657 bp in length with a single open reading frame, which encoded 172 amino acids (19617 Da in the estimated molecular mass). The predicted amino acid sequence of clone Tp21-3 had a similarity of 76% to that of clone ORF 17.20 (GenBank under accession number U88239) from T. spiralis. The recombinant fusion proteins encoded by clone Tp21-3 were produced in an Escherichia coli expression system and affinity purified. On Western blotting analysis, Tp21-3 recombinant proteins migrated at 40 kDa and reacted to antibody against T. pseudospiralis ES products and T. pseudospiralis-infected sera. Sera were developed against Tp 21-3 recombinant proteins, which reacted to a single band migrating at 21 kDa in crude worm extract and ES products from T. pseudospiralis on Western blotting analysis, and reacted with stichocytes of T. pseudospiralis on immunohistochemical staining.

Abstract A total of 475 soil samples were collected from five public park playgrounds, 17 kindergarten sandpits and 124 housing estates in Resistencia, a medium-sized subtropical-region city in Argentina, and processed by the centrifugal flotation method. Eggs of Toxocara spp. were present in five (3.4%) of the 146 habitats surveyed and in six (1.3%) of the 475 samples examined. Twenty per cent of public parks, 5.9% of kindergarten sandpits and 2.4% of housing estates were contaminated with Toxocara eggs. Depending on the number of samples examined from the three types of habitat, contamination by Toxocara was 0.7% in public park playgrounds, 1.2% in kindergarten sandpits and 1.6% in the housing estates. High prevalences of Ancylostomidae eggs were also found especially in public park playgrounds with a value of 100%, compared with 19.4% found in housing estates and 11.8% in kindergarten samples. These results suggest that in Resistencia, human infections with Toxocara are likely to occur within the limits of housing estates more so than in public parks or open spaces.

The abomasal and small intestinal helminth fauna of three species of captive gazelles (Gazella dama mhorr, G. cuvieri and G. dorcas neglecta) kept in captivity in Almería (southeast Spain) have been studied, and the following species were identified: Nematodirus spathiger, N. filicollis, N. helvetianus, Camelostrongylus mentulatus, Trichostrongylus vitrinus, T. probolurus, T. colubriformis, Ostertagia ostertagi, O. harrisi, Teladorsagia (Ostertagia) circumcincta, and T. (Ostertagia) davtiani. Camelostrongylus mentulatus and N. spathiger were the most prevalent and abundant parasites. Ostertagia ostertagi, O. harrisi, N. helvetianus, and T. (Ostertagia) davtiani were identified for the first time in the genus Gazella. In addition, O. harrisi and Trichostrongylus probolurus are new records for Spain.

In this study, the excretory–secretory products (ESP) of the daughter sporocysts of Vesicocoelium solenophagum (Trematoda) and plasma proteins of its host, Sinonovacula constricta were characterized by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and gelatin-substrate gel analyses, and the relationships between them were analysed using immunoblotting. Proteinase activity was detected in the ESP from daughter sporocysts of V. solenophagum. Some polypeptides of the ESP were found to be recognized by antiserum, raised against plasma from non-infected S. constricta, suggesting that the ESP may mimic host molecules (molecular mimicry). In contrast, neither the obvious proteinase activity nor the binding to the antisera was observed for the soluble proteins of daughter sporocyst, indicating that the ESP may play a important role in the parasite–host relationship. Although the plasma of infected S. constricta contained polypeptides that were similar to the plasma of non-infected bivalves, increased quantities of proteins at >170 kDa, 15 kDa and decreased quantities at 60 kDa were observed in the plasma of infected bivalves. Immunoblotting analysis revealed that the plasma of infected bivalves had a faint reaction with both anti-non-infected plasma antisera and anti-sporocyst antisera. These results indicated that the structure and quantity of some polypeptides from the plasma of infected bivalves had changed because of the infection with V. solenophagum. The polypeptides between the plasma of bivalves from a non-epidemic area and that from an epidemic area were similar, but the former had more polypeptides of 170–220 kDa and much greater proteinase activity than the latter, suggesting that the increased polypeptides of 170–220 kDa and the high proteinase activity in plasma may be favourable for protecting the host from being invaded by the parasites.

Successful transmission of macroparasites is dependent on exposure of susceptible hosts to free-living infective stages. When these hosts are herbivores that feed mostly on a single food plant then natural selection should favour those infective larvae that selectively ascend this main food plant. Red grouse feed predominantly on heather, Calluna vulgaris, so we predict that the infective larvae (L3) of the caecal nematode Trichostrongylus tenuis selectively locate and ascend heather plants. To determine whether the presence of heather influences the horizontal dispersal of T. tenuis L3 across soil, the movement of L3 across trays of soil with and without heather was investigated in the laboratory. More T. tenuis L3 were recovered from soil when heather was present, implying that larval migration may be influenced by chemical cues produced by heather plants. This was investigated in a second experiment, in which the horizontal dispersal of T. tenuis larvae was examined in the presence of heather and grass vegetation. This trial was repeated with larvae of a second species, Haemonchus contortus, a nematode whose hosts feed on a wide range of grass and shrub species. Significantly more larvae of both nematode species were recovered in the region of the heather than the grass or controls. This implies that T. tenuis and H. contortus L3 exhibit selective migration towards heather, perhaps reflecting a general response to plant cues which may be stronger for heather than for grass.

To demonstrate the prevalence of Toxocara spp. and Toxascarisleonina eggs in parks in Murcia city, eastern Spain, a total 644 soil samples were examined from nine parks. More than 67% of parks and 1.24% of soil samples were contamined and the mean egg density per sample was 16.7±20 eggs per 100 g of soil. Over 97% of eggs identified were viable. Only one sample was positive for Toxascaris leonina. The present findings suggest that shady conditions are important for the occurrence and viability of Toxocara spp. and Toxascarisleonina eggs in soil as there were significantly more positive samples in shaded and moist areas compared with open and dry habitats.

The effects of a diet deficient in the B vitamins on infectivity, growth, and distribution of Echinostoma caproni in ICR mice were studied. The vitamin-deficient diet (experimental) was isocaloric to the control diet but lacked the B vitamins. Thirty-six female, 6- to 8-week-old ICR mice were each infected with 25 metacercarial cysts. From the day of infection to the day of necropsy, 18 mice were fed the experimental diet and the remaining mice received the control diet. Equal numbers of experimental and control mice were necropsied at 2, 3 and 4 weeks postinfection (p.i.). Mice on the experimental diet showed a significant loss in body weight between 2 and 4 weeks p.i. There was no significant difference in worm recovery at 2 to 4 weeks p.i. from mice on either diet. Worms from hosts on the experimental diet were more dispersed and located more posteriad in the small intestine than those from mice on the control diet. Worm dry weight was significantly less in hosts on the experimental diet at all weeks p.i. compared with that of hosts on the control diet. The body area of worms on the experimental diet was significantly less at 2 and 3 weeks p.i. than that of worms on the control diet. An isocaloric diet deficient in the B vitamins had a detrimental effect on the growth of E. caproni in ICR mice.

Outbred CD1 mice were administered doses of 1000 and 3000 Toxocara canis eggs and postmortem took place on days 7, 42 and 120 post-infection. Mice were killed by cervical dislocation and brains were sagitally bisected and fixed in 10% neutral buffered formalin prior to histological preparation and examination. The number of T. canis larvae were counted per brain and per section and the number of larvae cited for the first time per section were also recorded. These observations were compared by dose administered and by day of postmortem. The total number of larvae per brain and per section was higher for the 3000 dose compared to the 1000 dose. A different pattern emerged for the number of larvae observed in the brain over the three postmortem days depending upon the dose received. For the 1000 dose larval numbers increase from day 7 to day 120 whereas for the 3000 dose the opposite trend occurs. Larvae were assigned to one of five regions in the brain – the telencephalon, diencephalon, cerebellum, medulla, pons and brain stem and the olfactory bulb. Larvae did not show a random distribution in the brain. The majority of larvae were recorded from the telencephalon and the cerebellum. The percentage of sections with larvae in them is higher for the 3000 dose compared to the 1000 dose for all regions of the brain. For the majority of regions, the percentage of sections with larvae in them increases between day 7 and 42 and then decreases by day 120 and this is most pronounced for the cerebellum. For the telencephalon and diencephalon only, more larvae were detected on the right hand side of the brain compared to the left hand side. Statistical analysis revealed that dose and brain region are significant factors which influence the number of larvae observed in histological sections of the brain but day post-infection is not.

Steinernema longicaudum Shen & Wang is redescribed based on a comparative morphological study of specimens from the type isolate from China, and two other isolates recovered from Korea and the USA. For the first and second generation female, the location of the vulva, shape of the vulval lips, and shape and length of the tail were newly observed diagnostic characters. A more detailed description of the morphology of the male spicules and gubernaculum, and the arrangement of the genital papillae is included. A description, based on scanning electron microscopy observations, of the lateral field pattern of the third-stage infective juveniles is also provided. Additionally, restriction fragment length polymorphism profiles based on the internal transcribed spacer region, and cross-breeding tests supplement the description of this species.

Dactylogyrid species (Monogenea) communities were studied in roach, Rutilus rutilus, collected from two localities in the basin of Morava river, Czech Republic, during the period from April to November 1997 and March to September 1998 to determine the effect of water temperature on parasite abundance, species richness and diversity. Dactylogyrid species were found to co-occur on the gills of roach with up to six species found on the same host individual. Nine dactylogyrid species were identified with the abundance of each reaching a very low level. Niche size was considered to increase with species abundance even when water temperature was high. There was a strong effect of water temperature on abundance of the common dactylogyrid species (D. crucifer, D. nanus, D. rutili and D. suecicus) as well as of the rare species D. rarissimus. The temporary occurrence of the rare species was found without any temperature effect. Water temperature did not affect the relationship between abundance and niche size. Niche size increased with abundance, even when the water temperature was high, which suggests that negative interspecific interactions are not important within dactylogyrid communities.

Extraintestinal migration patterns of Pharyngostomum cordatum (Digenea: Neodiplostomidae) were studied in experimental rodents such as mice, rats, and hamsters. When metacercariae isolated from grass snakes were infected orally to rodents, they penetrated the intestinal wall at days 2–3 post-infection (p.i.) and were discovered mainly in the diaphragm, intercostal muscles, and vital organ such as the lungs at days 7–28 p.i., without morphological changes. Interestingly, from several rodents which died suddenly at days 2–9 p.i., small to considerable numbers of metacercariae were found, not only in the lungs, but also in the heart and brain. Within the tissues, worms were freely motile until day 7 p.i., but later they were surrounded by host cells, and finally tissue cysts were formed. When metacercariae harvested from the snakes and intercostal muscles of rodents were infected orally to cats, they developed into adult flukes in the small intestine. The results show that P. cordatum undergoes considerable extraintestinal migration including the vital organs of its rodent hosts.

Fifteen helminth species were collected from 47 wolves (Canis lupus ) which were surveyed from 1993 to 1999 in northwestern Spain. These included the trematode Alaria alata (2.1%); the cestodes Taenia hydatigena (44.7%), T. multiceps (29.8%), T. serialis (2.1%), Dipylidium caninum (6.4%) and Mesocestoides sp. aff. litteratus (4.2%); and the nematodes Pearsonema plica (7.4%), Trichuris vulpis (10.6%), Trichinella britovi (12.8%), Ancylostoma caninum (8.5%), Uncinaria stenocephala (51.1%), Toxocara canis (6.4%) Toxascaris leonina (4.2%), Angiostrongylus vasorum (2.1%) and Dirofilaria immitis (2.1%). Only two wolves were not infected. A single infection occurred in 28.9% of the cases, but the commonest infracommunity (31.1%) involved three species. The helminths Alaria alata, Taenia hydatigena, Mesocestoides sp. aff. litteratus, P. plica, Trichuris vulpis, and Ancylostoma caninum parasitizing C. lupus are reported for the first time in Spain. Taenia serialis and D. immitis are reported for the first time in wolves in Europe. Angiostrongylus vasorum represents a new host record for wolves. The helminth fauna of Spanish wolves is compared with that of other European wolf populations. Some epidemiological considerations of the helminth fauna of wolves in Spain and the health risk to humans are also discussed.

Studies on the seasonal population dynamics of Neoechinorhynchus qinghaiensis (Acanthocephala: Neoechinorhynchidae) in its fish host Gymnocypris przewalskii przewalskii in the Qinghai Lake, China, were carried out with samples taken in May 1991, August 1992, November 1992 and February 1993. Prevalences were higher than 44% in all seasons. The mean intensity of infection was above 124 worms per fish. The maximum intensity of worms recovered from a single fish was 1402 in the autumn of 1992. Differences in the mean abundance, mean intensity and prevalence are not statistically significant relative to season and this is likely to be related to the stable temperatures recorded at the bottom of Qinghai Lake. Over-dispersed distributions of N. qinghaiensis in the host population, due to heterogeneity and feeding habits, were observed in all seasons. The size composition of both sexes of N. qinghaiensis showed males to be less than 3.5 mm and females between 0.5 and 4.25 mm, with the main recruitment phase in the worm populations occurring in the autumn, extending through winter and spring with the lowest recruitment occurring in the summer. The maturation and copulation of worms were mainly focused in the summer season. The sex ratio of female to male was both high in winter (1.51:1) and spring (1.48:1). The higher proportion of females and the change in the worm sex ratio in winter can be attributed to the reduced longevity of male worms. As immature male worms exhibit a higher proportion of the worm population than females in all seasons, further studies are needed to determine if such a situation compensates for the shorter life span of males.

Cortisol production in fry of rainbow trout (367–456 mg body weight) infected with the ectoparasitic monogenean Gyrodactylus derjavini (mean intensities 4.7 and 4.9 parasites per fish) was studied at two temperature levels, 4–6°C and 11–12°C, respectively. Due to difficulties in obtaining an adequate amount of plasma from the small sized fish, the corticosteroid concentration was measured in the body fluid recovered (as supernatant) after sonication and centrifugation of whole fry. Infected fry at 11–12°C showed an elevated level of cortisol compared to uninfected fry. However, the cortisol concentration was lower than in fish exposed to handling stress. At 4°C, the cortisol level in infected fish compared to uninfected was insignificantly increased. The findings are discussed in relation to the role of monogeneans as inducers of secondary infections.

Analysis of mitotic and meiotic chromosome spreads of Aspidogaster conchicola (Trematoda: Aspidogastrea) reveals a diploid number of 10 for this species. Haploid sets containing 5 bivalents confirm the diploid number. The karyotype consists of one pair of comparatively long subtelocentric chromosomes and four pairs of shorter acrocentric elements. The results are discussed in comparison with existing data on chromosomes of aspidogastrean species and other related helminth groups.

Mitochondrial DNA polymorphism was characterized in a triploid form of Fasciola found in Japan in comparison with F. hepatica, F. gigantica and Korean Fasciola worm. Seventy worms of Fasciola from Japan, three of F. hepatica from Uruguay and Australia, two of F. gigantica from Thailand and one of Fasciola from Korea were used in the study. Mitochondrial DNA polymorphism was detected by restriction fragment length polymorphism (RFLP) using eight restriction enzymes, BamH I, Bgl II, Dra I, EcoR I, EcoR V, Hind III, Mfl I and Sca I. Three different types (types 1, 2 and 3) were detected from 76 Fasciola worms used in the study. Eight of 70 Japanese worms were categorized in type 2 (F. gigantica type), and the remaining 62 were in type 3 (F. hepatica type).

An examination of a sample of benthic invertebrates collected from the Upper San Marcos River in southwestern Texas, USA in September 1999 revealed that the nymph of the ephemeropteran Tricorythodes curvatus served as natural intermediate host of the nematode Rhabdochona kidderi texensis (Nematoda: Rhabdochonidae), an intestinal parasite mainly of the Rio Grande perch (Cichlasoma cyanoguttatum) in this locality; the prevalence of the parasite's third- and fourth-stage larvae in mayflies was 6.8% with the intensity of 1–2 larvae per nymph. Live R. kidderi texensis eggs collected from nematodes recovered from C. cyanoguttatum in Texas were transported to the Czech Republic, where they were used to experimentally infect nymphs of the palaearctic mayfly species Paraleptophlebia submarginata; the development of infective third- and fourth-stage larvae in this experimental intermediate host was completed after approximately 10 days at 19°C. Infected nymphs were fed to aquarium-reared fishes, four Cichlasoma nigrofasciatum and one Oreochromis niloticus, of which only three of the former became infected. The last (fourth) moult of a male nematode was observed in C. nigrofasciatum 23 days p.i. and adult males and gravid females with not fully mature (non-embryonated) eggs in uteri on days 40 and 51 p.i. The prepatent period of R. kidderi texensis is approximately two months.

Ten BALB/c mice were infected with the cestode Echinococcus granulosus. After the infection, serum was collected at different periods of time and monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) were determined. The level of MCP-1 increased from 290±12 pg ml-1 (mean±SD) at day 20 post infection (p.i.), to a maximum of 820±23 pg ml-1 on day 60 p.i., then decreased to 460±12.6 pg ml-1 on day 130 p.i. A second peak was observed at day 150 p.i. In addition, MIP-2 was detectable in serum as late as day 100 p.i. The highest level (100±11 pg ml-1) was observed on day 130 p.i., and decreased thereafter. Serum from noninfected animals (controls) contained no detectable levels of either MCP-1 or MIP-2. However, MCP-1 and MIP-2 appear to be implicated in E. granulosus infections, but their exact role during the disease is under determination.