Genetic studies have demonstrated profound differences between the ‘porcine’ genotype of Cryptosporidium parvum, versus ‘human’ and ‘bovine’ genotypes. The study analysed infectivity and pathogenicity of the ‘porcine’ genotype (CPP-13 isolate) of C. parvum, and compared the results with published data on the ‘bovine’ genotype (CPB-0 isolate). This was investigated in calves and piglets from commercial herds. Piglets were mildly affected by the CPP-13 isolate, contrary to piglets infected with the CPB-0 isolate, which caused diarrhoea of a mean duration of 3·5 days. CPP-13 produced no or very mild clinical signs in piglets despite the excretion of high numbers of oocysts. Concomitant infection with rotavirus, however, caused a dramatic aggravation of the clinical signs, and 5 of 6 experimentally infected piglets died. CPP-13 appeared to be adapted to porcine hosts as illustrated by the lack of infectivity to 1 experimentally inoculated calf, and the absence of clinical signs, the long pre-patent period (15 days), and the excretion of very low numbers of oocysts following experimental infection of another calf. Thus, in accordance with other molecular studies, our results support the genetic evidence for the existence of a new species of Cryptosporidium adapted to pigs.

Cystic echinococcosis, caused by Echinococcus granulosus, is an emerging disease in many parts of the world and, in particular, in eastern Europe and the former Soviet Union. This paper examines the abundance of infection of E. granulosus in the definitive host in southern Kazakhstan. Observed data are fitted to a mathematical model in order to decide if the parasite population is partly regulated by definitive host immunity and to define parameters in the model. Such data would be useful to develop simulation models for the control of this disease. Maximum likelihood techniques were used to define the parameters and their confidence limits in the model and the negative binomial distribution was used to define the error variance in the observed data. The results indicated that there were 2 distinct populations of dogs in rural Kazakhstan which had significantly different exposures to E. granulosus. Farm dogs, which are closely associated with livestock husbandry, particularly sheep rearing, had a relatively high mean abundance of 631 parasites per dog and a prevalence rate of approximately 23%. The best fit to the model indicated that there was significant herd immunity in the dog at this infection pressure. In contrast, village dogs which were more likely to be kept as pets had a much lower mean abundance of parasites of only 27 parasites per dog and a lower prevalence of 5·8%. With this village population of dogs, the best fit indicated negligible herd immunity.

The mechanism by which nitric oxide (NO)-dependent cytotoxicity acts against Toxoplasma gondii tachyzoites is poorly understood. An NO donor, sodium nitroprusside (SNP), was used to induce death in T. gondii tachyzoites in vitro as a model for investigating (i) whether NO is capable of inducing apoptosis-like death in tachyzoites and (ii) whether a calcium signal transduction pathway is involved. Exposure to 2 mM SNP resulted in a pattern of tachyzoite death that shares many features with metazoan apoptosis and it may involve a calcium signal transduction pathway. Motility and cell survival in these parasites showed a gradual decline with increasing levels of SNP. Features common to metazoan apoptosis are observed after exposure to 2 mM SNP. Ethylene glycol bis-(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA), Verapamil and bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid/acetoxymethyl ester (BAPTA/AM) partially increased the cell survival concomitant with decreased [Ca2+]i in cells exposed to SNP. An NO scavenger (N-acetylcysteine), the analogue of SNP (devoid of NO), inhibited the rate of apoptosis after SNP treatment compared with SNP treatment without scavenger, but alone did not induce apoptosis. Taken together, the results indicate that SNP is capable of inducing apoptosis in T. gondii tachyzoites via a calcium signal transduction pathway.

A new histozoic species of myxosporean was found to infect the gill filaments, gut, kidney and liver of the freshwater teleost Leporinus friderici, collected from the estuarine region of the Amazon, near the city of Belém, Brazil. The plasmodia show asynchronous development, at any one time composed of mature spores and all sporogonic stages. The ellipsoidal spore body, measuring 10·4 μm long and 5·7 μm wide, consists of 2 equal shell valves adhering together along the straight suture line. Each valve has a caudal process measuring 23·3 μm in length. There are 2 symmetric polar capsules, without intercapsular appendix, measuring 5·0 μm×2·1 μm, and each has a polar filament with 7–8 coils. In general, ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. Some ultrastructural aspects such as cellular alterations of the pericyte in the different organs infected and characterization of the sporoplasmosomes during the sporoplasm maturation are described. This parasite was studied under light and electron microscope and compared with others species of the genus Henneguya, considering also host specificity. From our observations we propose the creation of a new species, Henneguya friderici n. sp.

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS–PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.

We compared parasite communities in fish taken from a polluted lake (L. Vatia) and two control lakes before (1986) and after (1995) nine years of markedly reduced chemical and nutrient loading from a pulpmill in central Finland. Discriminant analyses of the 1995 data, using a function based on the 1986 data, showed that the parasite communities in the fish from the two control lakes had changed relatively little, whereas those from L. Vatia had converged on those from the mesotrophic control lake, indicating substantial recovery from the effects of pollution. Only a few species of parasites provided evidence for recovery. These were anodontid glochidia, which had increased markedly in perch, Rhipidocotyle fennica in roach and R. campanula in both fish species. This suggests that the recovery of the polluted lake involved increased populations of anadontid clams in shallow waters. On the other side decrease of Dermocystidium percae on perch fins and Ichthyophthirius multifiliis on roach indicate increased immune responses in the fish, reflecting better water quality. Other parts of the system have apparently not yet recovered.

The aims of this study were to characterize human American tegumentary leishmaniasis, which includes cutaneous, mucocutaneous and mucosal leishmaniasis, in Northwest Argentina, to determine the prevalence of double infection with Trypanosoma cruzi and to identify the species of Leishmania in this area. Most of the 330 leishmaniasis patients presented cutaneous ulcers (96·1%), 2·4% mucocutaneous and 1·5% the mucosal form (‘espundia’). The aetiological agents, determined by isoenzyme electrophoresis, were identified as Leishmania (Viannia) braziliensis in 16 out of 20 isolates and in the remaining 4 as Leishmania (Leishmania) amazonensis, the first ever-documented in Argentina. Sera analysed by ELISA and IFA using complex antigen from both T. cruzi and L. braziliensis showed a very high percentage of positives (66·3–78·2%). When antigens for specific diagnosis of Chagas' disease were used, 40·9% of the leishmaniasis patients were also found to be infected by T. cruzi. These results indicate that the strong immune response against T. cruzi gave no protection to Leishmania, in spite of the serological cross-reaction between these parasites. In addition, we showed that more than 40% of the patients would be misdiagnosed as chagasic if complex antigens, as epimastigotes or soluble fraction from epimastigotes, were used in IFA or ELISA. This is of paramount importance not only because patients' treatment would be associated to misdiagnosis but the fact that in many countries in Central and South America, a positive test for Chagas' disease means a rejection for those seeking employment.

As has been shown previously, immunologically intact mice with patent Schistosoma mansoni infections has a significantly lower mean platelet number than intact uninfected mice (P<0·0001). However, platelet numbers in T-cell deprived mice with patent infections were not significantly different from those in uninfected T-cell deprived mice. Also, platelet counts in both the infected and uninfected T-cell deprived groups were not significantly different from those in intact uninfected mice. The S. mansoni-induced thrombocytopaenia in mice is thus seemingly immune dependent. Immunologically intact mice with chronic 12-week-old S. mansoni infections has IgG antibodies that were reactive in an ELISA-type assay wit whole fixed platelets of both mouse and human origin. In Western immunoblots the IgG antibodies from chronically-infected mice reacted in particular against mouse and human platelet antigens of 90, 37 and 30 kDa. Antisera raised from 2 rabbits, immunized respectively with mouse and human platelet antigens, cross-reacted with antigens of the larval, adult worm and egg stages of S. mansoni. These results support the hypothesis that an anti-platelet antibody response may be the cause of the thrombocytopaenia observed in mice with patent schistosome infections.

An ultrastructural study of microfilariae of Wuchereria bancrofti was performed after in vitro treatment with diethylcarbamazine. One of the first morphological alterations produced by treatment with 5, 10 or 50 μg/ml of DEC was the loss of microfilarial sheaths. Drastic effects of DEC were only detected when microfilariae were exsheathed. Microfilariae treated with 5, 10 or 50 μg/ml DEC showed severely affected organelles, formation of several vacuoles mainly in the hypodermis, and cytolysis. Some microfilariae showed extreme cellular disorganization with abundance of electron-dense degenerating organelles, numerous large vacuoles, and nuclear condensation. Lamellar bodies probably related to an assembly of endoplasmic reticulum membranes were observed in some damaged microfilariae. Thus, in vitro treatment with concentrations of DEC similar to therapeutic conditions had direct effect by causing a loss of the microfilarial sheath of W. bancrofti with subsequent damage of organelles and apoptosis.

Schistosomiasis is initiated when cercarial larvae invade human skin. Contrary to long-held assumptions, most cercariae of Schistosoma mansoni do not shed their propulsive tails as they penetrate. Scanning electron microscopy studies and infection experiments with entire human skin and differentiated, stratum corneum-like, human keratinocyte cultures, have shown that most cercarial tails enter the skin along with their bodies. We propose that this behaviour is an adaptive trait linked with concomitant immunity.

Aqueous extracts of Schistosoma mansoni eggs have been shown to have fibrinolytic activity inhibitable by a serine protease inhibitor. Fibrinolytic activity was not present in extracts of either adult worms or cercariae. A 27 kDa enzyme that was proteolytically active on fibrinogen in zymography and that degraded fibrinogen in a pattern similar to that of plasmin, is presumed to be responsible for the schistosome egg fibrinolytic activity. Anti-human fibrinogen antisera were shown to have antibodies that cross-reacted with mouse fibrinogen in Western immunoblots. Electroblotted sera from S. mansoni-infected and control uninfected mice displayed different antigenic profiles when probed with the cross-reactive anti-human fibrinogen antibodies, suggesting an alteration in mouse host fibrinogen metabolism as a result of the parasitic infection. We discuss the possibility that modulation of fibrinogen metabolism is a factor in a recently discovered anti-atherogenic effect exerted by schistosomes.

Intestinal helminths of fish are of increasing interest as potential bioindicators for heavy metal contamination in aquatic habitats. Among these parasites cestodes and acanthocephalans in particular have an enormous heavy metal accumulation capacity exceeding that of established free living sentinels. Metal concentrations several thousand times higher in acanthocephalans than in host tissues were described from field and laboratory studies. Whereas larval stages inside their intermediate hosts are not able to take up high quantities of metals, young worms begin to take up metals immediately after infection of the final host. After four to five weeks of exposure, the parasites reach a steady-state concentration orders of magnitude higher than the ambient water level. Thus, acanthocephalans are not only very effective in taking up metals, but they can also respond very rapidly to changes in environmental exposure. The mechanism which enable acanthocephalans to take up metals from the intestinal lumen of the host appears to be based on the presence of bile acids, which form organo-metallic complexes that are easily absorbed by the worms due to their lipophilicity. Investigations of the environmental conditions affecting metal uptake have shown that the parasites are more consistent and reliable indicators for metal pollution than the host tissues as metal levels of the latter are much more dependent on the water chemistry. Thus, after some years of research on the uptake of metals by acanthocephalans and on the factors affecting metal accumulation in intestinal parasites it should be asked if acanthocephalans meet the criteria commonly accepted for sentinels. If parasites can be considered as promising sentinels, we need reasons for the establishment of ‘new’ indicators. Therefore, this review summarises the present knowledge about parasites as bioindicators and compares the accumulation properties of parasites and established free living indicators. Finally, this review presents possible answers to the question why it could be advantageous to have new and even more sensitive indicators for environmental monitoring purposes.

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

Enzyme-linked immunosorbent assays (ELISAs) were developed in order to detect coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes (FIC) in rats experimentally infected with Trichinella spiralis. The usefulness of these assays was compared to that of a conventional ELISA for detection of serum antibodies (Abs) to muscle larvae excretory–secretory products (ML–ESP). The ELISA for CAgs was the first parameter to give a positive result but the detection was limited only to day 2 p.i. CAbs against ML–ESP and adult worm excretory–secretory products (AW–ESP) was first positive on day 4 p.i. Anti-ML–ESP remained positive until day 12 p.i. while CAbs against AW–ESP remained positive throughout the study period. Specific IgE and IgA were found. FIC were detected between days 2 and 8 p.i. Serum Abs began to appear on day 10 p.i. Therefore, the ELISA for CAbs was a suitable assay for the detection of the enteral and early phases of the infection.

Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (<400 g) during investigations of T. cruzi.

This paper describes two approaches to evaluate the use of fish macroparasites as bioindicators of heavy metal pollution at selected river stretches in Austria. Firstly changes in the diversity and richness of endoparasites of the cyprinid barbel, Barbus barbus (L.), were tested in relation to heavy metal contents in the aquatic system. Secondly, the bioaccumulation potential of cadmium, lead and zinc was assessed in the acanthocephalan, Pomphorhynchus laevis (Müller, 1776), and compared with that in the muscle, liver and intestine of its barbel host. The present results indicated that in order to validate the role of parasite community patterns related to heavy metal pollution, more investigations on food web dynamics, interelationships between parasites and the presence/absence of intermediate hosts will be essential. Heavy metal concentrations differed significantly between the organs of barbel and P. laevis (P=0·001) with levels up to 2860 fold in the parasite. The high level of heavy metal accumulation in P. laevis compared with that in its barbel host, suggests that despite variability in the parasite infrapopulation, host mobility and feeding behaviour, P. laevis is a most sensitive indicator of heavy metals in aquatic ecosystems.

A morphological study has been carried out to determine the effect of the active sulphoxide metabolite of the benzimidazole anthelmintic, albendazole (ABZ-SO) on the adult liver fluke, Fasciola hepatica. Whole flukes were treated with ABZ-SO for 12 and 24 h at a concentration of 10 μg/ml. The changes in response to drug treatment were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tubulin immunocytochemistry (ICC). No surface changes were apparent following 12 h ABZ-SO treatment, but localized blebbing was observed after 24 h, which became more extensive towards the posterior region of both surfaces. TEM of sections from the posterior midbody region revealed that ABZ-SO caused the accumulation of secretory bodies in the tegumental cells and in their cytoplasmic connections and, after 24 h, just above the basal plasma membrane. Localized blebbing of the apical membrane also occurred. The morphology of the Golgi complexes within the tegumental cells began to change after 12 h treatment with ABZ-SO and, by 24 h, few complexes were observed. A distinct increase in tubulin immunoreactivity occurred after 12 h treatment, but this decreased after 24 h. The results obtained are consistent with those expected for microtubule inhibition. They are discussed in relation to the action of established microtubule inhibitors, as well as the benzimidazole derivative, triclabendazole.

Host metabolic changes have been observed to affect Schistosoma mansoni egg production, worm survival and morphology. We examined worms recovered from streptozotocin-induced diabetic mice by morphometric and morphological analysis through brightfield and confocal laser scanning microscopy. Tegument thickness was slightly smaller and changes in the reproductive organs were observed in 23–30% and consecutively 88–100% of the worms. The testicular lobes had a large diminution of cells in one or more of the lobes, which was associated with a lack of spermatozoids in the seminal vesicle. Ovaries were atrophied, manifested by a complete or large reduction in oocytes but other parts of the reproductive system like the vitelline glands were mainly unaffected. Streptozotocin (STZ) instead of hyperglycaemia caused the degeneration since worms from mice injected with a non-diabetogenic dose, or with nicotinamide to prevent diabetes showed the same alterations. The drug did not affect worm survival or pairing. We conclude that STZ, an alkylating agent that provokes chromosome and DNA damage, changes the morphology of ovaries and testicular lobes in S. mansoni worms in vivo. This is the first report of STZ action in helminths and we suggest that STZ affects oogenesis and spermatogenesis and might cause sterilization of schistosomes.

In this paper we describe the effect of poly-L-lysines of different molecular weight on the schistosomula. In the control sample, the schistosomula of Schistosoma mansoni take up fluorescent Texas Red conjugated to bovine serum albumin (TxR–BSA) into the gut. Following slight damage by 24·0 kDa poly-L-lysine, a high proportion of schistosomula take up fluorescent TxR–BSA into the excretory system. Subsequently, the dye diffused into the bodies of the schistosomula. We suspected that this diffusion involved the process of endocytosis so we investigated this with the use of endocytosis inhibitor, Latrunculin A. Addition of the endocytosis inhibitor Latrunculin A following poly-L-lysine treatment inhibited gut uptake of TxR–BSA as well as the diffusion of excretory-ingested TxR–BSA molecules.

Viable eggs produced weekly per infective stage was used as a measure of the reproductive success of Echinostoma friedi during the first 12 weeks of infection in hamsters. The weekly reproductive success was not constant during the experiment in relation to the egg output and the proportion of viable eggs produced. The egg release started during week 2 post-inoculation, attaining a maximum during week 3. A decline in egg output was observed from week 9. Viable eggs were only produced from week 3 post-inoculation and a maximum was attained at week 4 of the experiment. A decline in egg viability was observed from week 9. Considering together the egg output and the egg viability, the maximum weekly reproductive success was obtained during week 4 post-inoculation. The changes in the weekly reproductive success were not reflected in variations in worm numbers and body sizes during the course of the infection. The humoral immune response of golden hamsters during the infection with E. friedi was determined. Increases of IgG levels against somatic and excretory/secretory products of E. friedi were detected coinciding with the reduction in the reproductive success.