The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

Metacestodes of Hymenolepis diminuta cause a perturbance of vitellogenesis in the intermediate host Tenebrio molitor. The reduction in host reproductive output associated with infection may be due to this pathophysiology. Many of these events are regulated by host juvenile hormone (JH). A comparison of the titre of JH and its rate of degradation in female control and parasitized 15-day-old insects has been made. Haemolymph from female beetles contained 1·27 pMol JH equivalents/100 µl. No significant difference was associated with infection. However, topical application of a JH analogue, methoprene, at the time of infecion or 8 days post-infection reduced the significant accumulation of vitellogenin usually found in the haemolymph of females 12 days or more post-infection. These findings indicate that parasite-induced alteration of host vitellogenesis is not mediated via alteration in JH titres, although observations made after hormone supplementation suggest some form of interaction between the parasite and the host endocrine system.

The size and form (length, width, and length: width ratio) of eggs of Diphyllobothrium dendriticum, D. ditremum and D. laturn vary considerably among individual worms within each species. The size of eggs varies with host species and a decrease in egg size with increasing intensity of infestation is indicated. The egg size of D. laturn increases during the first 10–12 days of egg production. For single worm infections in golden hamsters the mean egg length and width of D. ditremum are significantly smaller than the corresponding means of D. dendriticum and D. latum, while D. latum has significantly wider eggs than D. dendriticum. As taxonomic characteristics, egg size and form may contribute to species delimitation at the population level. For identification at the individual level the best possible accuracy is about 80%. This accuracy is considerably reduced when variation in host species and intensities of infestations are introduced. Scanning electron microscope studies did not reveal any differences among eggs of the three species.

To determine whether rabbits may serve as reservoir hosts for Lyme disease spirochaetes in Europe, we compared their competence as hosts for Borrelia afzelii, one of the most prevalent European spirochaetal variants, with that of the Mongolian jird. To infect rabbits or jirds, at least 3 nymphal or adult Ixodes ricinus ticks infected with spirochaetes fed to repletion on each animal. Whereas jirds readily acquired tick-borne Lyme disease spirochaetes and subsequently infected vector ticks, rabbits exposed to tick-borne spirochaetes rarely became infectious to ticks. Only the rabbit that was infectious to ticks developed an antibody response. To the extent that I. ricinus ticks feed on European rabbits, these mammals may be zooprophylactic by diverting vector ticks from more suitable reservoir competent hosts.

Infection of small macropodids with the larval stage of Echinococcus granulosus can cause fatalities as well as significant pulmonary impairment and other adverse sequelae. The brush-tailed rock-wallaby (Petrogale penicillata) is a small macropodid listed as vulnerable on the IUCN's Red List of Threatened Species. This study used radiographic techniques to determine the prevalence and severity of pulmonary hydatid infection and growth rates of hydatid cysts in a wild population of this macropodid. The overall prevalence was 15·3% (9/59 animals) with 20·0% (8/40 animals) of adults infected. During the study period, the death of at least 1 infected animal was directly attributed to pulmonary hydatidosis. Rapid cyst growth occurred in some animals (up to 43% increase in cyst volume in 3 months). Cyst volume reduced lung capacity by up to 17%. Secondary pulmonary changes were uncommon but, in 1 animal, resulted in reduction in lung capacity by approximately 50%. Infection was associated with a higher blood urea concentration, but no significant differences in other blood variables were detected. These results indicate that hydatid infection may be a significant risk to threatened populations of small macropodids and should be addressed in conservation management plans for these animals.

Gyrodactylid parasites were observed on non-native populations of North-American freshwater catfishes, Ameiurus nebulosus and Ameiurus melas (Siluriformes: Ictaluridae), at several sites in the Elbe River basin, Czech Republic, Europe. Using a combination of morphological and genetic analyses, the parasites infecting A. nebulosus were determined to be Gyrodactylus nebulosus, a North American parasite co-introduced to Europe along with its Ameiurus fish hosts. Subtle morphometrical differences, as well as seasonal variations, were observed among parasites collected from A. nebulosus and A. melas. The host-related variation was further supported through genetic analysis of the partial 18S rDNA, ITS1-5.8S-ITS2 and COI, showing 0.2, 3.0 and 4.8% divergence, respectively. Consistent genetic differences indicated there were two distinct genotypes. Subtle morphological differences associated with the shape of sickle toe, anchor root and ventral bar membrane, according to host species, also supported the description of a new cryptic species, Gyrodactylus melas n. sp., infecting A. melas. Multivariate morphometrical analysis of haptoral hard parts showed significant differences between the anchor lengths of G. nebulosus and G. melas n. sp. However, the measurements of the haptoral hard structures partially overlapped between species, limiting the usage of these parameters for species delineation.

Irreversible pathological lesions were noted in the organs of mice infected with 1 of 3 rodent malaria species: Plasmodium chabaudi chabaudi, P. vinckei petteri and P. yoelii nigeriensis at different times during the course of the primary parasitaemia and long after microscopical clearance of the parasites. Moreover, similar lesions were also obtained when parasite levels were kept below 1%by subcurative drug treatment. The frequency and severity of the lesions correlated with the duration of the infection. Accumulation of tissue damage during chronic low-grade malaria infections has implications for the design of control measures.

Five chromosomes and 17 isoenzyme loci were analysed in 4 allopatric populations of Leishmania (Viannia) peruviana, and molecular distances calculated with 2 estimators, Chromosomal Size Difference Index and Jaccard Distance. Chromosome and isoenzyme data were in overall concordance: 13/30 isolates clustered similarly on the dendrograms constructed from the different estimators, and a significant correlation (P<0·001) was observed between the molecular distances calculated from the two sets of characters. This indicates an evolutionary association between chromosomal size polymorphism and isoenzymes. Chromosomes have a faster molecular clock than isoenzymes; twice as many genotypes were identified by chromosome analysis and significant size differences (for a total of up to 500 kb for 5 chromosomes together) were observed within a given zymodeme. Chromosomes most likely represent better indicators of genetic drift than isoenzymes, as suggested by the higher correlation between both estimators of chromosomal size-polymorphism and eco-geography. Some chromosomes might present an adaptive response to environmental variation.

The in vitro leishmanicidal activity of a series of imidazole-containing phthalazine derivatives 1–4 was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, and their cytotoxicity on J774·2 macrophage cells was also measured. All compounds tested showed selectivity indexes higher than that of the reference drug glucantime for the three Leishmania species, and the less bulky monoalkylamino substituted derivatives 2 and 4 were clearly more effective than their bisalkylamino substituted counterparts 1 and 3. Both infection rate measures and ultrastructural alterations studies confirmed that 2 and 4 were highly leishmanicidal and induced extensive parasite cell damage. Modifications to the excretion products of parasites treated with 2 and 4 were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds 2 and 4 were potent inhibitors of iron superoxide dismutase enzyme (Fe-SOD) in the three species considered, whereas their impact on human CuZn-SOD was low. Molecular modelling suggests that 2 and 4 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the antioxidant features of the enzyme.

A considerable proportion of mice lose acquired immunity to Plasmodium berghei during the first pregnancy. Immune parous mice, however, have a better immune status than virgin mice, the risk of loss of immunity during a subsequent pregnancy is greatly reduced, the capacity to clear parasites is enhanced, and the maintenance of immunity is less dependent on certain splenic functions. The establishment of improved immunity is dependent on the presence of proliferating parasites during the second half of pregnancy when immunosup pression results in recrudescence. Immune reactivity is also improved after a (chemothera peutically controlled) recrudescent infection provoked by immunosuppressive treatment of immune mice with corticoids or anti-T cell serum. This mimics the situation encountered during pregnancy. Hence, improved immunity after pregnancy is a consequence of a reconfrontation of a suppressed and/or convalescent immune system with proliferating parasites.

Prototransversotrema steeri gen.nov., sp.nov. is found under the scales of the mullet, Aldrichetta forsteri, a euryhaline fish.

It is distinguished from Transversotrema Witenberg, the only other genus of the family Transversotrematidae by the possession of an oral sucker as well as a pharynx. This necessitates alteration of the family diagnosis.

Knowledge of the life-histories of Transversotrema spp. is summarized. The so-called ‘metacercariae’ found under the scales of freshwater and euryhaline fish are regarded as adults. When they have no eggs or only one egg they have probably developed in a fish which is not the normal host.

P. steeri escapes from under the scales of the fish shortly after the latter is caught. It is suggested that this habit may account for the small number of species known in the family.

A cell-sorting method is described for the analysis and separation of red blood cells in Plasmodium berghei-infected mouse blood based on their DNA content. This method involves a selective uptake of the bis-benzimidazole dye 33258 Hoechst, a DNA-binding dye, by red blood cells containing parasites. Infected blood is incubated at 37 °C with the dye then washed at 4 °C to remove unbound dye. Uninfected cells are then non-fluorescent at the characteristic wavelengths for 33258 Hoechst excitation and emission, whereas parasitized cells display fluorescence intensities in approximately direct proportion to the number of parasite nuclei (i.e. amount of parasite DNA) within the cell and can be sorted accordingly. Providing cells were incubated in a complex nutrient medium during dye uptake at 37°C, the sorted parasite cells produced lethal P. berghei infections when injected into BALB/c mice. The dyelabelling technique is simple and sufficient red blood cells at various stages of infection can be collected for biochemical or immunochemical studies by cell sorting.

Pulsed-field gel electrophoresis of intact chromosomes from 2 isolates of each of the 2 most pathogenic species of large Babesia parasites that infect dogs, i.e. Babesia canis (European species) and B. rossi (South African species), revealed 5 chromosomes in their haploid genome. The size of chromosomes 1–5 was found to be different in the 2 species, ranging from 0.8 to 6.0 Mbp. The genome size was estimated to be approximately 14.5 Mbp for B. canis and 16 Mbp for B. rossi, respectively. Within each species, the size of chromosomes 1–3 of B. canis and 1–2 of B. rossi was conserved between the 2 isolates, whereas the size of chromosomes 4–5 of B. canis and 3–5 of B. rossi was variable. Chromosomes 1–5 hybridized with a 28-mer telomeric oligonucleotide probe derived from Plasmodium berghei. When NotI-digested chromosomes of the 4 isolates were hybridized with the telomeric probe a maximum of 10 fragments was revealed. Moreover, hybridization of this telomeric probe to a Southern blot of genomic DNA from the 4 isolates, digested with a series of restriction enzymes, revealed a species-specific restriction map. Hybridization of intact or NotI-digested chromosomes of both species with 2 sets of 3 cDNA-antigen probes derived from each species, revealed no cross-hybridization between these B. canis and B. rossi genes.

The cestodes Echinococcus granulosus, Taenia multiceps, T. pisiformis, T. hydatigena, Hymenolepis diminuta, Moniezia expansa and Anoplocephala perfoliata all produced substances that stimulated thymidine incorporation by whole blood lymphocyte cultures. This mitogenic activity was demonstrated in metacestode cyst fluids, live protoscoleces and scoleces, parasite culture supernatants, and extracts of adult parasites. T. multiceps metacestode cyst fluid mitogen adhered to, but would not pass through, cellulose dialysis tubing. This adherence was reduced or prevented by D-glucose and by proteins. The mitogen was weakly anionic. With Sephadex G75 gel filtration, its elution volume was greatly decreased when the elution buffer contained 0·5 M D-glucose. Mitogenic activity was protease resistant and could be separated by gel filtration from all the cyst fluid proteins.

Mice of the inbred P strain fail to develop significant resistance to challenge Schistosoma mansoni infection at 6 weeks after either low-grade primary infection or vaccination with attenuated homologous parasites, in contrast to other strains such as C57B1/6, and thus provide a model for comparison of potential immune resistance mechanisms in low versus high responder animals. In this study, the antigen-specific cellular responses found to correlate with resistance in these strains were delayed cutaneous hypersensitivity, production of macrophage activating lymphokine and macrophage larvicidal activity, all of which were greater in infected or vaccinated C57B1/6 mice than in similarly immunized but non-resistant P mice. Humoral responses correlating with resistance were IgM reactivity to schistosomula surface antigens in both infected and vaccinated animals, as well as both IgM and IgG reactivity to soluble schistosome antigens in infected mice. Immune responses that showed no relationship with resistance included IgG reactivity to larval surface antigens and immediate hypersensitivity to soluble worm antigens. In infected mice, neither granuloma size nor extent of hepatic fibrosis correlated positively with resistance to challenge infection. Thus, similarities exist between patterns of resistance and immune response at this early time after immunization with either viable or attenuated parasites. These observations suggest that common immune effector mechanisms could be involved, with activated macrophages playing a central role in resistance.

Experiments upon the duration of chronic infections of Plasmodium relictum in canaries prove that parasites may persist in the peripheral blood, as ordinary schizogonic stages, up to 8 years and 3 months after infection and that the blood of such birds is infective for clean birds for at least the same period.

The fine structure and histochemistry of the envelopes surrounding the mature oncosphere of Hymenolepis diminuta have been investigated by transmission electron microscopy and light microscope observations of JB-4 embedded material. The fate of these envelopes during the hatching sequence in vitro has been followed by phase-contrast and scanning electron microscopy. Hatching is considered to comprise 4 stages. Stage 1 involves the mechanical breakage and removal of the shell and the outer cytoplasmic layer of the inner envelope; Stage 2 the activation of the oncosphere and the swelling of the gelatinous layer of the inner envelope; and Stage 3 the digestion and rupture of the embryophore. This is accomplished both by secretions from the penetration gland and by the action of external digestive enzymes, together with hook activity. Trypsin is more effective than amylase in digesting the embryophore. Stage 4 involves the enzymatic weakening of the gelatinous layer which helps the oncosphere to tear itself free with its hooks. Amylase is more effective than trypsin in attacking the gelatinous layer. On emergence from the gelatinous layer, the oncosphere is still enveloped by the ‘oncospheral membrane’, although this covering is soon lost. Once activated, the oncosphere is capable of completing the hatching sequence by itself, without the addition of enzymes. This process is a lengthy one, however, taking up to 2 h, and the expenditure of its glandular and energy reserves makes successful gut penetration by the oncosphere unlikely.

An experiment has been conducted to examine the effects of discontinuous infection with H. contortus in sheep on the development of resistance to infection. When sheep were given four doses of 3000 larvae at fortnightly intervals and these sensitizing infections were removed on day 56, there was no evidence of resistance at that time nor of any residual effect of the sensitizing infections on the response to challenge at day 140. When, however, the sensitizing infections were permitted to persist until day 132, there was strong resistance to the establishment of a challenge infection whether or not the sensitizing worm burden was removed prior to challenge, and, in the case of superimposed challenge, resistance was coupled with rejection of most of the sensitizing population. When a single infection with 3000 larvae was followed 22 days later with a further single dose of 3000 larvae, there was no evidence of resistance whether or not the first infection was removed with anthelmintic. If the interval between two doses of 3000 larvae was extended to 76 days there was resistance to the establishment of the second dose if this was superimposed on the first, but not if the first infection was removed 8 days prior to the second dose.

It is suggested that the manifestations of resistance appear in a sequence of (i) resistance only to a superimposed infection, followed by (ii) resistance to a challenge infection in the presence or absence of a previously existing worm burden, coupled in the former case with rejection of much of the existing population. This sequence could be related crudely to a product of larval dose and time. Although larval dose and time were to some extent confounded in the design of the experiment strong resistance was associated with prolonged uninterrupted infection.

The aim of this paper was to test the hypothesis that when the 2 species of Acanthocephalan Pomphorhynchus laevis and Acanthocephalus clavula are found concurrently within the intestine of brown trout under field conditions, they have the potential to interact negatively. Evidence has shown that Acanthocephala are more likely to exhibit negative interactions with their own and other species, under both field and experimental conditions. Furthermore, the likelihood of these interactions is increased in Ireland because of the absence of certain definitive hosts and the fact that concurrent infections by two or more species of Acanthocephala are more commonly observed in fish. Data collected from wild and stocked brown trout and from 2 lakes provided an opportunity to compare the 2 potentially interacting helminth species in their fundamental and realized niche and several pieces of convincing evidence are provided here to support the hypothesis. A significant negative association between the numbers of each species found in individual fish was reported and this was consistent for both wild and stocked trout. Furthermore, an analysis of the proportions of low, moderate and high intensity infections in single and concurrent infections revealed a significant reduction in increasing intensities in concurrent infections compared to single infections. Finally, strikingly different patterns of niche inhabitation were observed, particularly for P. laevis in the presence of A. clavula in wild trout. Results from the niche width analysis also support the observations on average position in single and concurrent infections. The niche width of P. laevis when it co-occurred with A. clavula decreased markedly in high intensity infections compared to low intensity infections.

The proton-translocating inorganic pyrophosphatases (H+-PPases) are primary electrogenic H+ pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H+-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61·7 kDa and an isoelectric point of 5·0. Several motifs characteristic of plant vacuolar H+-PPases (V–H+-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V–H+-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K+-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V–H+-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.