The uptake of thiamine by isolated second-generation schizonts of Eimeria tenella and by host intestinal cells was found to consist of two components. One was passive and the other apparently an active process. The kinetic constants of the latter were compared in host (Km = 0·36 μm) and parasite (Km = 0·07 μm) and found to be significantly different. Both systems were competitively inhibited by amprolium but showed different affinities for the drug. (Host Ki = 326 μm; parasite Ki = 7·6 μm). Further differences were observed in schizonts of a drug-resistant line of E. tenella. These findings are discussed in terms of the inhibition of thiamine uptake being the basis of the anticoccidial activity of amprolium.

We investigated the diversity of Bartonella in Apodemus agrarius, an important rodent of peri-domestic habitats, which has spread into Europe in the past 1000 years. Spleen samples of 344 A. agrarius from Eastern Slovakia were screened for the presence of Bartonella spp. using 16S–23S rRNA internal transcribed spacer region and bacteria were detected in 9% of rodents. Based on sequencing of three housekeeping genes (gltA, rpoB and groEL) Bartonella genotypes were ascribed to the species typical for mice and voles: B. grahamii, B. taylorii and B. birtlesii. However, the study also confirmed presence of genotypes belonging to the B. clarridgeiae/B. rochalimae clade, and the B. elizabethae/B. tribocorum clade, which are not commonly found in woodland rodents. In addition, a potential recombination event between these two genotypes was noted, which highlights an important role of A. agrarius in shaping Bartonella diversity and evolution.

Toxocara canis larvae infecting dogs undergo tracheal migration following their ingestion in an experimental intermediate host. Development of the parasite then proceeds to the adult stage in the small intestine. Adult dogs are therefore capable of distributing fertile T. canis ova which, if ingested, could cause larva mirans in humans and other animals.

This work was financed by research grants from the University of Queensland and the National Health and Medical Research Council of Australia. The writer wishes to acknowledge the encouragement and criticism of Professor J. F. A. Sprent.

A new category of signalling molecules – transmitter gases – has appeared. Nitric oxide (NO) is generated by nitric oxide synthase (NOS) and diffuses as a short-lived transcellular messenger through the plasma membrane. NADPH-diaphorase (NADPH-d) is a marker enzyme for NO-producing neurons. In this study the pattern of NADPH-d stained neurons in Diphyllobothrium dendriticum is described and compared to the pattern of aminergic and peptidergic neuronal elements and to that of the musculature stained with TRITC-labelled phalloidin. NADPH-d staining was observed in neurons in the bilobed brain and along the 2 main nerve cords and in nerve fibres close to the body musculature and the musculature of the reproductive ducts, the walls of the testicular follicles and in sensory endings in the tegument. The NADPH-d staining reaction and the 5-HT or FMRFamide immunoreactivities occur in separate sets of neurons.

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.

Methodology is described for the laboratory maintenance of the life-cycle of Sanguinicola inermis, using common carp (Cyprinus carpio) as the definitive host and Lymnaea peregra as the intermediate host. Lymnaea auricularia was also infected but is not considered to be a suitable laboratory host. Tench (Tinca tinca) were susceptible to infection at high cercarial doses but subsequent infection rates were low. Goldfish (Carassius auratus) and Lymnaea stagnalis were refractory to laboratory infection.

The toxicity of the active oxygen species hydrogen peroxide, superoxide radical, hydroxyl radical and singlet oxygen to microfilariae (mf) has been studied in vitro, using active oxygen-generating systems and scavengers/inhibitors. Mf viability was monitored by uptake of the radiolabel, [3H]2-deoxy-D-glouse. Hydrogen peroxide and singlet oxygen, but not superoxide radical or hydroxyl radical, are toxic for mf. Hydrogen peroxide was toxic for mf within 2 h at concentrations as low as 5 ¼, an amount eosinophils have been shown to release in vitro (Weiss et al. 1986). Catalase and thiourea, but not inactivated catalase, superoxide dismutase (SOD), singlet oxygen scavengers, or hydroxyl radical scavengers, protected mf. Mf have relatively high levels of endogenous SOD but no measurable glutathione peroxidase and low levels of catalase when compared with other parasites (Callahan, Crouch & James, 1988). The low levels of hydrogen peroxide-scavenging enzymes correlate well with mf sensitivity to hydrogen peroxide and the protective effect of exogenous catalase.

While I was in Egypt conducting investigations into the etiology of dysentery with. Captain F. W. O'Connor at Alexandria in 1916 I was able to make some observations on the intestinal protozoa of three species of lizard. The occurrence of an Entamoeba and a Chilomastix (Tetramitus) in one of these, Agama stellio, was mentioned in our publication on the Human Intestinal Protozoa in the Near East, p. 147 (1917). The other lizards investigated were Chamaeleon vulgaris and Lacerta agilis. I will describe the observations on the protozoa under their respective hosts.

(1) Female Ixodes ricinus ticks which feed on a sheep infected with tick-borne fever are not capable of transmitting the infective agent to their progeny. Only the nymphs and females, therefore, can be infective to sheep. In the case of an infective female, the infection may have been acquired, (a) in the immediately preceding stage, or (b) in the larval stage, the infective agent remaining in the tissues of the tick during the nymphal engorgement and subsequent moult. This appears to occur irrespective of whether the host on which the nymph feeds is a susceptible or an insusceptible. animal.

(2) Ticks which have acquired infection are infective as soon after moulting to the next stage as they are able to attach to hosts. Unfed ticks were not found to be infective after a period of about 14 months.

(3) Infection is transmitted to the sheep in the course of the 2nd day after attachment of infective nymphs.

(4) The disease may be transmitted to sheep by inoculation of emulsions of infective ticks, if these have been allowed to commence engorgement. The use of saline for such emulsions appears to be inadvisable.

Phylogenetic relationships within the Capsalidae (Monogenea) were examined using large subunit ribosomal DNA sequences from 17 capsalid species (representing 7 genera, 5 subfamilies), 2 outgroup taxa (Monocotylidae) plus Udonella caligorum (Udonellidae). Trees were constructed using maximum likelihood, minimum evolution and maximum parsimony algorithms. An initial tree, generated from sequences 315 bases long, suggests that Capsalinae, Encotyllabinae, Entobdellinae and Trochopodinae are monophyletic, but that Benedeniinae is paraphyletic. Analyses indicate that Neobenedenia, currently in the Benedeniinae, should perhaps be placed in a separate subfamily. An additional analysis was made which omitted 3 capsalid taxa (for which only short sequences were available) and all outgroup taxa because of alignment difficulties. Sequence length increased to 693 bases and good branch support was achieved. The Benedeniinae was again paraphyletic. Higher-level classification of the Capsalidae, evolution of the Entobdellinae and issues of species identity in Neobenedenia are discussed.

Plasmodium vinckei infections, which usually kill mice within 7 days, can be terminated in the majority of cases by a single injection of chloroquine phosphate equivalent to 10 mg/100 g body weight. After such drug treatment the parasites disappear from the blood for 6–11 days, after which a recrudescence with a low peak occurs. Most animals overcome this second parasitaemia and the parasites finally disappear completely. After recovery, mice exhibit an immune response to a challenge infection and, after a short period of parasitaemia during which a low peak is reached, the animals recover completely. This immunity, once acquired, apparently persists for the lifetime of the host. The establishment of the immune state depends on the period of patent parasitaemia during the primary infection and the longer this period the greater is the chance of a solid immunity developing. Immunity may be exhibited by mice treated with chloroquine as early as the 3rd day of infection. Killed parasites have no immunizing effect. Mice splenectomized before infection are capable of an immune response, and mice splenectomized after radical cure are also able to overcome a challenge infection. In both these cases more than one injection of chloroquine is required. The immunity produced in mice is that of the true sterile type, equivalent to that produced against P. berghei in rats, and is species-specific, conferring no resistance to infection with P. berghei. P. vinckei infections in mice provide a useful immunological model for laboratory studies. The immune response is predictable and easy to induce, and this parasite is therefore superior to P. berghei for immunological studies. Attention is drawn to the fact that in P. vinckei the infection is brought under control by the immune response and not by the antimalarial drug; therefore this ought to be taken into consideration in the assessment of drug trials.

CBA/T6 strain mice infected with Plasmodium berghei ANKA develop cerebral symptoms and die, with mononuclear cell attachment to the cerebral microvascular endothelium, petechial haemorrhages and breakdown of the blood–brain barrier, some 6–7 days post-inoculation. The effects of dietary restriction on this process were examined. Mice were fed ab libitum (Group 1) or their food was restricted to produce body weight loss of 1·0–2·0 % (Group 2), 2·5–3·5 % (Group 3), 4·0–6·5 % (Group 4) or 7·0–9·5 % (Group 5) relative to Group 1. Dietary restriction reduced deaths caused by cerebral malaria from 100% in Group 1 to 47% (Group 2), 43% (Group 3), 10% (Group 4) and 53% (Group 5). Restriction of food intake had no effect on (1) the progression of parasitaemia in infected mice (2) changes in haematocrit, spleen weight, total lymph node cell number or (3) peritoneal exudate cell number in either malaria-infected or uninfected mice. P. berghei ANKA infection did not significantly affect the proportion of lymph node leucocytes that were Thy-1+T cells or CD8+T cells, but did lead to significant increases in the CD4+ and B cell populations. Dietary restriction alone increased the lymph node CD4+cell population but did not affect the increase in B cells in malaria-infected mice. P. berghei ANKA infection and dietary restriction together did not lead to increased CD4+cell numbers in lymph node leucocytes. The in vitro proliferative response of isolated lymph node cells to concanavalin A or phorbol myristate acetate plus ionomycin was measured and found to be identical in all treatment groups. Plasma levels of tumour necrosis factor (TNF) increased from undetectable in uninfected mice or P. berghei ANKA-infected mice on days 1–6 post-inoculation to 21 ± 2 ng/ml on day 7 when cerebral symptoms were at their height and death was imminent. This increase in plasma TNF was substantially inhibited in those mice subjected to regimes of dietary restriction. There was a good correlation (r2 = 0·73) between plasma TNF level and percentage mortality in the 5 treatment groups. The results indicate that dietary status is an important factor in the outcome of murine cerebral malaria and perhaps, by extrapolation, in the human disease.

The activation of the peptidergic neurosecretory system in Diphyllobothrium dendriticum was studied following cultivation of plerocercoids for short times in vitro and in vivo. In the plerocercoid the neurosecretory cells gave a very weak reaction with paraldehyde fuchsin (PAF). After cultivation for 1 h large numbers of neurosecretory cells filled with PAF-positive granules were evident. The significance of the activation of the neurosecretory system during the transfer of the worm from the cold-blooded fish host to the warm-blooded final host is discussed.

Giardiasis is a gastrointestinal disease that affects humans and other animals caused by parasitic protists of the genus Giardia. Giardia intestinalis (Syn. Giardia lamblia; Giardia duodenalis) infections can cause acute or chronic diarrhoea, dehydration, abdominal discomfort and weight loss. Metronidazole is the most widely used drug for treating giardiasis. Although effective, metronidazol has undesirable secondary effects. Plants used in traditional medicine as antidiarrhoeals or antiparasitics may represent alternative sources for new compounds to treat giardiasis. Heterotheca inuloides Cass. (Asteraceae/Compositae) plant is widely used in Mexican traditional medicine. The following secondary metabolites were isolated from H. inuloides flowers: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), 1R,4R-hydroxy-1,2,3,4-tetrahydrocadalen-15-oic acid (4), quercetin (5), quercetin-3,7,3′-trimethyl ether (6), quercetin-3,7,3′,4′-tetramethyl ether (7) and eriodictyol-7,4′-dimethyl ether (8). The activity of these compounds against Giardia intestinalis trophozoites was assessed in vitro as was the activity of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (9), 7-benzoxy-3,4-dihydrocadalene (10), 7-acetoxycadalene (11), 7-benzoxycadalene (12), quercetin pentaacetate (13) and 7-hydroxycalamenene (14). Among these, 7-hydroxy-3,4-dihydrocadalene (1) and 7-hydroxycalamenene (14) were the most active, whereas the remaining compounds showed moderate or no activity. The G. intestinalis trophozoites exposed to compound 1 showed marked changes in cellular architecture along with ultrastructural disorganization. The aim of this study was to evaluate the giardicidal activity of selected H. inuloides metabolites and some semisynthetic derivatives using an in vitro experimental model of giardiasis.

For the evaluation of the epidemiology of Theileria equi and Babesia caballi in a herd of 510 horses in SW Mongolia, several mathematical models of the transmission dynamics were constructed. Because the field data contain information on the presence of the parasite (determined by PCR) and the presence of antibodies (determined by IFAT), the models cater for maternal protection with antibodies, susceptible animals, infected animals and animals which have eliminated the parasite and also allow for age-dependent infection in susceptible animals. Maximum likelihood estimation procedures were used to estimate the model parameters and a Monte Carlo approach was applied to select the best fitting model. Overall, the results are in line with previous experimental work, and add evidence that the epidemiology of T. equi differs from that of Babesia spp. The presented modelling approach provides a useful tool for the investigation of some vector-borne diseases and the applied model selection procedure avoids asymptotical assumptions that may not be adequate for the analysis of epidemiological field data.

To address the molecular diversity and occurrence of pathogenic species of the genus Entamoeba spp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses on Entamoeba from wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) of Entamoeba spp. We identified Entamoeba spp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for six Entamoeba species/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli and Entamoeba polecki ST2). We recorded three Entamoeba species: E. coli (47%), E. dispar (16%), Entamoeba hartmanni (51%). Coproscopically, we could only distinguish the cysts of complex E. histolytica/dispar/moshkovskii/nuttalli and E. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences of E. dispar and E. coli from Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbour Entamoeba species similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence of Entamoeba species.

Parasitic infections in man and domestic animals exhibit two striking characteristics (a) their prevalence is high, but infections are unequally distributed among individuals within populations and (b) immunity is often slow to develop and appears, at best, only partially effective. Recent immunological and epidemiological studies suggest that effective immunity can develop, but that high prevalence within populations reflects the operation, not only of socio-economic and climatic factors, or husbandry practices, but also of powerful environmentally induced constraints upon the development of resistance. Immunogenetic studies suggest the operation of additional constraints which reflect individual genetic characteristics, and which influence the ability to develop and express effective immunity. A full understanding of all constraints is necessary before levels of population and individual resistance to infection can be increased; the need for such understanding has become more pressing with the prospect that anti-parasite vaccines may become available. Two aspects of environmentally induced constraints are considered, those arising from nutritional inadequacies and those resulting from exposure to infection in early life. Both are discussed primarily in terms of helminth parasites. Genetically determined constraints are discussed with reference to MHC-restricted recognition of malarial peptide vaccines and in terms of Class II molecule-directed control of T-cell function in Leishmania infections. Genetic influences are also considered from the standpoint of inflammatory cell function, in immunity against intestinal nematodes and in vaccine-induced immunity against Schistosoma. Finally, parasite-induced constraints, particularly those which down-regulate protective responses are discussed briefly.

1. Ixodes hexagonus is a three-host tick.

2. The length of the pre-oviposition period is fairly constant at 19° C. in a saturated atmosphere, varying between 12 and 14 days.

3. Oviposition takes place as in I. ricinus and, at room temperatures at 18–20° C., R.H. 100%, the number of eggs laid varies from 880 to 1449. Experiments on the effects of different temperatures and humidities on egg-laying are described.

4. Eggs hatch in 52–59 days under the conditions stated above.

5. The larvae gorge for 3–6 days, the majority becoming replete on the third and fourth days after attachment.

6. The metamorphosis of the larva is prolonged by cold conditions and is hastened during warm weather. In a saturated atmosphere at 15, 20, 22 and 25° C., the mean times for moulting are respectively 60, 33, 27 and 23 days.

7. The nymphs gorge for 5–7 days, the greater number dropping off between the sixth and seventh days.

8. At 18° C. and 100% R. H. moulting of the nymph takes place about 32 days after repletion.

9. After metamorphosis the adults rest for 8–12 days, and feeding lasts 8–16 days.

Larvae of the cattle tick Boophilus microplus will attach to thin (0·3–0·5 mm) slices of cattle skin and engorge on a partially denned medium at 35 °C. Forty-seven to 83% of the larvae had engorged by 8 days, and 51–71% of these moulted to nymphs. Tissue culture medium alone allowed little growth unless supplemented with dialysed, freeze dried bovine serum (7%, w/v). This medium could be further defined by substituting purified bovine serum albumin (Cohn fraction V) for the dialysed bovine serum. In one experiment, nymphs of Haemaphysalis longicornis engorged and later moulted to adults. Skin slices were used fresh or after freeze-drying and storing at − 25 °C. The possible uses of the culture method are discussed.

Perkinsus marinus is a protozoan responsible for dramatic mortality in the Eastern oyster, Crassostrea virginica, but not in the Pacific oyster, C. gigas. To understand the host–parasite relationship, we inoculated P. marinus trophozoïtes into the shell cavity of C. gigas and measured, over 2 months, (i) intensity of infection, (ii) protease inhibitory activities against P. marinus proteases and against bovine α-chymotrypsin, (iii) plasma haemagglutinin titre, (iv) plasma protein concentration, (v) plasma lysozyme activity and (vi) total haemocyte count. We observed that the highest protease inhibitory activities and haemagglutinin titres (3–10 days post-challenge) preceded parasite elimination (initiated 7 days post-challenge). In contrast, plasma protein concentration, lysozyme activity and total haemocyte count showed no significant modification following the challenge. It is hypothesized that the capacity of C. gigas to increase its protease inhibitors represents the key event in resistance to parasite infection by neutralizing the proteases secreted by P. marinus, thus preserving the oyster haemagglutinins from degradation. Such haemagglutinins will be ready to act as opsonins stimulating phagocytosis of parasites.