Three isolates of Babesia divergens have been cultured continuously for 6 months in rat erythrocytes using the candle jar technique (Trager & Jensen, 1976). One isolate was already rat-adapted, the other two became adapted to rats through continuous culture in rat erythrocytes. Parasites were cultured in rat erythrocytes in RPMI medium supplemented with 20% foetal calf serum. The highest parasitaemia obtained was 35% and multiparasitization of red blood cells was often observed. Cultures of B. divergens remained infective to splenectomized rats. Cultures with high parasitaemias contained a large number of extracellular merozoites. When separated from the red blood cells, these extracellular merozoites retained their infectivity.

A monoclonal antibody (OW-IGO) raised against purified excysted oocysts of Cryptosporidium parvum reacted in an immunofluorescence assay with the oocyst wall. The corresponding antigen was localized by immunoelectron microscopy in fibrillous material present in the parasitophorous vacuole of developing macrogametes and in the wall of both single and double layered sporulating oocysts. Gold particles were also detected over electron-lucent vesicles of the macrogametes by immunoelectron microscopy. On Western blotting of C. parvum oocyst extracts, major bands at 250 and 40 kDa and several minor components were recognized by Mab OW-IGO. Almost complete abolition of Western blot reactivity occurred after periodate oxidation of oocyst antigen, suggesting that monoclonal antibody OW-IGO reacts with a carbohydrate epitope. Taken together, our results suggest that a fibrillous glycoproteinic material is released in the parasitophorous vacuole from electron-lucent vesicles during gametogenesis, and later condensed in the oocyst wall.

Transfer of Eimeria apionodes from wood mice directly into untreated laboratory mice was unsuccessful but transfer into corticosteroid-treated animals produced an oocyst output, about 1000 times less than that observed from wood mice after a similar inoculum. Repeated passage through corticosteroid-treated laboratory mice resulted in a line adapted to survival in untreated animals. This line was compared with the parent strain maintained in wood mice and some features of the oocyst output patterns, notably the pre-patent period, appeared to be controlled by the host species. The oocyst production of each population was higher in the host species to which it was adapted than in the other host species (P > 0·001). Once adapted to laboratory mice, the line produced insignificantly different levels of oocysts in corticosteroid-treated and untreated animals (P > 0·05).

Reversal of chloroquine (CQ) resistance by verapamil, a Ca2+ antagonist, has been shown in CQ-resistant human and rodent malaria parasites. Here, we report ultrastructural changes associated with this phenomenon in CQ-resistant Plasmodium chabaudi (AS strain) after infected mice were administered CQ and verapamil. At parasitaemias of 5–7%, CQ at 6 mg/kg caused little morphological effect on CQ-resistant parasites. In contrast, co-administration of CQ and verapamil at 50 mg/kg induced swelling of food vacuoles with clumped pigment at 2.5 h. Morphological changes other than food vacuole enlargement occurred at 21 and 45 h: disappearance of endoplasmic reticulum, formation of myelin structures, focal cytoplasmic vacuolization and coarse clumping of electron-dense material in nuclei. These structural changes appeared to be very similar to those observed in CQ-sensitive P. chabaudi in mice injected with CQ alone or CQ plus verapamil. On the other hand, verapamil at 50 mg/kg alone did not induce any effect on both CQ-sensitive and CQ-resistant P. chabaudi. These results suggest that swelling of the food vacuoles is an initial event associated with reversal of CQ-resistance by verapamil.

In the rodent malarial parasite Plasmodium berghei sexual parasites are produced in a single major wave with maximal numbers between day 7 and day 16. Irrespective of their time of appearance during infection these sexual parasites are equally fertile in vitro. In contrast, in vivo infectivity to the mosquito is maximal at day 3–5 when gametocyte numbers are only 9% of the peak levels seen between days 7 and 16. Up to 96% of natural potential infectivity of gametocytes for the mosquito is therefore suppressed. The suppression is humoral, reversible and correlates with the appearance of an effective host response to the initial rapid increase in asexual parasitaemia. These data are consistent with published evidence which indicates that a reduction in parasitaemia may cause an increase in infectivity of gametocytes to the mosquito vector. Therefore the impact of strategies aiming to control asexual parasites is re-examined. Inefficient strategies might be predicted to increase and not suppress malaria transmission.

Tubulin biosynthesis was rapidly induced during transformation of the mammalian (amastigote) stage of the kinetoplastid parasite Leishmania donovani to flagellated promastigotes. However, transcription of β-tubulin genes occurred constitutively, as judged by nascent RNA synthesis in isolated nuclei and Northern blotting of steady-state mRNA. Two mRNA species of 2.2 and 2.4 kb were shared by the two cell-types, while a third 2.6 kb species, constituting about 20% of the total, was present in large amounts in promastigotes. RNase protection experiments demonstrated sequence micro-heterogeneity in the 5′-untranslated region, the pattern of which was identical in promastigotes and amastigotes. By primer extension assays, heterogeneity in the 5′-terminal cap structure of amastigote β-tubulin mRNA and differential pausing of reverse transcriptase within the mini-exon leader region were detected. These differences correlated with enhanced translational efficiency of tubulin mRNA from promastigotes in a rabbit reticulocyte lysate system. The results indicate that translational control plays a major role in tubulin induction during L. donovani differentiation.

A number of authors have demonstrated that the schistosomicidal compound, Praziquantel (Pzq), depends for its action upon the immune status of the host (Sabah et al. 1985; Brindley & Sher, 1987; Doenhoff et al. 1987). We have attempted to define the synergistic interaction between immuno- and chemotherapy further, using the murine irradiated vaccine model of schistosomiasis mansoni. In vaccinated mice, resistance operates in the skin but not the lungs; drug targeted towards lung-stage worms exacerbates lung-phase immunity, however, as depicted by the increased number and size of inflammatory reactions in the pulmonary tissues. Parasites are often found trapped within such foci. In the present investigation, light and ultrastructural studies have been utilized to examine the nature and extent of damage inflicted upon lung-stage larvae recovered from day 6 Pzq-treated vaccinated mice. Such studies have revealed that damage involves muscle disorganization, internal disruption and occasionally, loss of the tegument; in the latter case, cells are often seen attached to the denuded lung worms. To identify the crucial cellular effector cell(s) involved in the synergy between immuno- and chemotherapy, cell depletion studies have been performed in vivo. It would appear from these experiments that eosinophils or lymphocytes rather than neutrophils or macrophages are important effector cells in this synergy. Histological studies argue in favour of eosinophils being the key effector cells.

T cell-deprived mice acutely infected with S. mansoni suffer microvesicular hepatocyte damage which is not seen in infected, immunological intact animals. A cationic fraction (CEF6) of the PBS-soluble portion of S. mansoni eggs (SEA) induces antibodies which, on passive transfer, prevent hepatocyte damage. CEF6 contains 2 antigens, ω1 and α1, and has also been shown to be a useful serodiagnostic reagent. This paper describes the purification and characterization of the 2 antigens present in CEF6. ω1 is a monomeric glycoprotein with a pI > 9·0 and a molecular weight of 31 kDa. α1 consists of two immunologically cross-reactive dimers, 41 and 36 kDa in non-reducing conditions, each of which consists of one unique and one common glycoprotein subcomponent. In ELISA with mouse and human infection sera ω1 is shown to be S. mansoni specific and is better able to distinguish S. mansoni infections from other schistosome infections than are unfractionated SEA, CEF6 or α1. Passive transfer of monospecific anti-ω1 sera into S. mansoni infected, T cell-deprived mice completely prevented the occurrence of microvesicular hepatocyte damage in these animals. Monospecific anti-α1 serum had no hepatoprotective capacity.

Culture characteristics of two species of piscine trypanosomes were compared. Trypanosoma catostomi from Catostomus commersoni was isolated in hypo-osmotic blood agar media but not in hyper-osmotic media. However, T. phaleri from Amia calva was isolated in both media. Only T. catostomi was stimulated to divide by diluting infected blood with water. The maximum number of T. phaleri and percentage of trypomastigotes were not affected by NaCl concentration. The maximum number of T. catostomi was inversely related to osmotic pressure and the percentage of trypomastigotes was directly related to osmotic pressure. This was confirmed by using equi-osmotic concentrations of NaCl, NaBr, Na acetate, KCl, choline-Cl or sucrose. Supplementation with urea had no effect on the number or morphology of trypanosomes. The survival of T. catostomi following cryopreservation was enhanced by prior incubation in hyper-osmotic media. Differential culture characteristics of these species may reflect adaptation to different host–vector systems.

The accumulation of the trypanocide isometamidium chloride (Samorin®, RMB Animal Health Ltd, UK) by a range of clones of Trypanosoma congolense with varying sensitivity to the drug, was measured by methods based on the fluorescence of isometamidium. Fluorescence microscopy and flow cytometry showed a reduction in drug accumulation by resistant clones. Fluorescence spectrophotometry demonstrated an inverse correlation between the intensity of cell-associated fluorescence and the level of resistance of the clones expressed in vivo. The addition of the metabolic inhibitor SHAM/glycerol to the incubation medium resulted in a reduction of this apparent difference in drug accumulation between the clones; those clones which were sensitive to isometamidium showed a reduction in fluorescence while a percentage increase in fluorescence was observed as clones became more resistant to the trypanocide. These observations may be of value for the in vitro detection of resistant T. congolense populations and may also be used to estimate the mean level of resistance in a given sample. The results also imply that decreased accumulation of isometamidium by drug-resistant clones of the parasite may be responsible for the reduction in sensitivity.

Larvae of the monogeneans Pseudodiplorchis americanus and Neodiplorchis scaphiopodis develop within egg capsules retained in the uterus of the parent parasite. This study reveals adaptations for the storage and nutrition of infective stages which have no known precedent amongst other platyhelminths. The vitelline system is greatly reduced and appears to contribute little to embryo nutrition. An electron-dense ‘shell’ which encloses the newly formed embryos is subsequently replaced by a thin flexible sac composed of concentric layers of membranes which are derived from the uterus wall. Further membranes are added to this multi-laminate structure as the encapsulated embryos pass along the uterus, and this enables the sac to expand continuously as the larva inside it grows. The capsule lining extends into fine cytoplasmic processes which ‘plumb in’ to the larval tegument between ciliated cells. These connexions are packed with glycogen and appear to perform a placenta-like function. The unique adaptations correlate with the demands of the life-cycle. Large numbers of oncomiracidia (up to 300) can be packed, in membranous sacs, within the storage capacity of the uterus; direct transfer of nutrients from parent to offspring enables resources to be supplied over an extended period; and infective larvae can be maintained in readiness for an unpredictable opportunity for transmission.

The rostellar distal cytoplasm of Echinococcus granulosus protoscoleces is characterized by extensive basal membrane infoldings, prominent hemidesmosomes and is subtended by a lamina reticularis with microfibrils of approximately 10 nm diameter that occasionally show a 55 nm banding periodicity. The rostellar hooks, in 2 rows, each have a blade, guard and handle region and possess a central amorphous pulp, a middle microfibrillar medulla with microfibrils of approximately 4 nm diameter, and a complex outer cortex in all but the proximal region of the guard and the base of the handle. In these regions additional material, of similar electron density to the medulla, but lacking the fibrillar substructure, occurs and gives the areas a lobed appearance. Energy-dispersive X-ray microanalysis of whole hooks demonstrated the presence of sulphur and trace quantities of phosphorus. X-ray near-edge absorption spectra resembled those of cystine, feather and hair and showed the sulphur to be predominantly in the form of disulphide linkages. X-ray diffraction patterns of whole hook preparations revealed 2 diffuse rings with equatorial spacings of 7·99 Å and 15·22 Å, thus differing from vertebrate keratins.

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PH1-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

A study to compare effects of mass, targeted and selective chemotherapy with levamisole (Ketrax) as an action for the control of Ascaris lumbricoides was carried out in three communities in rural Oyo State, Nigeria. Selective treatment was applied in one village by treating the most heavily infected 20% of the inhabitants, targeted treatment in the second village involved children aged 2–15 years, while mass treatment was offered to all inhabitants excluding infants under 1 year and pregnant women in the third village. Recommended doses of levamisole were given in the villages, as described above, at 3-monthly intervals during a period of 1 year. Prevalence and intensity (epg) of A. lumbricoides infection were determined immediately before and 3 months after the period of intervention using a modified Kato-Katz technique. In the selective treatment village, no significant differences were found between the pre- and post-treatment egg counts (mean(±S.D.) epg 6776±10791 versus 4259±10909 respectively) of A. lumbricoides in the total population. In the targeted treatment village, significant differences were recorded in pre- and post-treatment egg counts for the total population (9057 ± 15 797 versus 2579±6529) among the children alone (10935±20094 versus 992±3175) and among the untreated adults (7742±9782 versus 4561±8798). In the mass treatment village, significant differences in pre- and post-treatment egg count values were also recorded (11 907±17220 versus 1489±5165). The intensity of Trichuris trichiura and hookworm infections among the villagers before and after intervention were not observed to have changed significantly regardless of selective, targeted or mass treatment.

The white colour of the cyst wall of the metacercaria of Microphallus sp., even in the presence of dihydroxy phenol and phenoloxidase, is due to proteins generated through a possible quinone methide interaction. The quinone methide isomerase converts the phenoloxidase mediated quinone into β-hydroxy catechol. Formation of catechol from quinone by cyst extracts was observed spectrophotometrically and chromatographically. This enzyme is involved in detoxification of excess quinone and β-sclerotization of the cyst wall.

Proteases from infective larval (L3) and adult stages of Nippostrongylus brasiliensis were investigated with a combination of techniques involving gelatin degradation and cleavage of fluorogenic substrates. Analysis of L3 excretory–secretory (ES) products revealed enzymes of Mr 51, 58, 79, ~ 150 and ~ 250 kDa. Inhibition profiles indicate that the major 51 kDa protease is a metallo-enzyme. Significantly, little activity was present in larval somatic extracts, suggesting the synthesis of zymogens or precursor forms prior to secretion. Adult ES contained a distinct enzyme, of 50 kDa, and a number of other proteases were detected in somatic extracts of this stage, ranging from 51 to > 300 kDa. The largest of these adult somatic enzymes is also a putative metallo-protease. While nearly all enzymes from both L3 and adult are heat labile, incubation at 100 °C generated a previously unobserved activity at 20 kDa. Furthermore, a protease of similar size may be found in uninfected rat intestinal tissue, suggesting specific uptake of a host-associated enzyme by the parasite in the form of an inactive, heat-labile complex.

A genomic library of Meloidogyne incognita Race 1 has been prepared in the bacteriophage λgt10 and screened for specific DNA sequences by hybridization with radio-isotope labelled total genomic DNA from a number of Meloidogyne species. One clone isolated (MR1#15), although not totally species specific, clearly showed preferential hybridization to M. incognita. Following subcloning and sequencing of the 255 bp insert, four stretches of the sequence corresponding to oligonucleotides of approximately equal length (~60 bp) were synthesized and examined for specificity. One of them, MR1#15.2, showed the necessary specificity to be used as a diagnostic tool.