Varying degrees of naturally occurring immunoglobulin (Ig) M antibodies to Toxoplasma gondii were observed in almost all of adult Japanese populations negative for IgG antibodies as determined by an enzyme-linked immunosorbent assay (ELISA). The antibody activity was reduced by absorption of sera with Toxoplasma soluble antigen or by 2-mercapto-ethanol treatment of the IgM fraction after sucrose density gradient centrifugation. Umbilical cord sera had almost no detectable IgM antibodies, and the ELISA value in infants increased with age until 1 year. Most paired sera collected from adults at a 1-year interval showed constant IgM antibody levels, suggesting retention of natural IgM antibodies throughout their lives without changing ELISA values of ≥ 0·1/year. Polar staining was observed in immunofluorescent studies for 14 serum samples with natural IgM antibody levels of ≥ 0·5, while samples obtained from a pregnant woman with natural IgM antibody levels of ≥ 0·9 reacted to the whole organism as was observed in positive sera of acute patients. Western blotting analysis revealed that natural IgM antibodies were directed to multiple antigen molecules. The band pattern differed slightly from one serum to another, but almost consistent patterns were observed in sera sequentially obtained from an adult over 4 years.

Giardia infections were detected in 14 out of 32 weaned, 1 to 2-month-old pigs, used in experiments with coccidia. Giardia trophozoites were present in the small intestine from the duodenum to the ileum with maximum numbers in the cranial part of the upper jejunum. They were localized mainly on the surface of intestinal crypts. No histological changes accompanied the infection and the infected animals were asymptomatic. Giardia trophozoites obtained from the intestines of necropsied pigs were isolated in axenic culture in a bile-supplemented TYI-S-33 medium. Morphological features of the trophozoites revealed by both light and scanning electron microscopy proved that the organism belongs to the Giardia intestinalis group.

In an area of holoendemic malaria in Northern Tanzania, Anopheles gambiae s.l. females were age-graded by Polovodova's method and dissected for sporozoites. Age-specific sporozoite rates implied that mosquitoes acquired new infections at all ages. The extrinsic period lasted just over 3 gonotrophic cycles (9–11 days). Very high sporozoite rates in the oldest females implied the absence or rarity of genetic refractoriness to infection. A method is described for estimating the proportion of bloodmeals which result in mosquito infection. This method makes relatively few assumptions about mosquito behaviour, and could be useful for evaluating transmission-blocking interventions. Overall, it is estimated that about 21% of meals are infectious. This is much higher than previous estimates derived either from experimental mosquito feeding studies or from similar age-grading data collected from the same area in 1962. Various alternative explanations are considered, and it is concluded that there has been a 2.5-fold increase in human infectiousness in the last 25 years. This is partly attributable to suppression of human infectiousness by widespread chloroquine usage during the 1960s, followed by removal of this effect by drug resistance. It is argued that chloroquine would be expected to select for increased infectivity in the parasite, and this may also have contributed to the observed increase.

The asymmetric distribution of phospholipids in the erythrocyte membrane during the intracellular development of the human malaria parasite Plasmodium falciparum was studied. Infected cells of high parasitaemia were treated with phospholipase A2 or sphingomyelinase C, followed by isolation of the host red cell membrane using the Affigel (731) bead method. Additionally, phosphatidylserine on the surface of infected cells was probed using a phosphatidylserine-sensitive prothrombinase assay. Trophozoite-infected cells showed an increase in phosphatidylethanolamine and phosphatidylserine and a decrease in phosphatidylcholine in the outer leaflet. In addition to the changes already present in trophozoite-infected cells, schizont-infected cells showed a decrease in sphingomyelin as well as a further increase in phosphatidylserine in the outer leaflet. The results are discussed with respect to possible mechanisms and consequences of these changes.

DNA–DNA hybridization techniques have been found to be very useful in the classification and identification of Leishmania parasites. We report here the cloning of a mini-circle from Leishmania strain UR6 in a plasmid and subcloning of the mini-circle fragments in M13 mp9. Clone MLURk32, containing a 560 bp fragment of mini-circle, has been found to have unique specificity. Application of this specific probe in identifying different Leishmania isolates reveals that the probe reacted only with strains of post-kala-azar dermal leishmaniasis but not with strains or isolates of visceral leishmaniasis.

The midgut epithelium of Glossina morsitans centralis, G. austeni, G. pallidipes, G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes, G. tachinoides and G. brevipalpis from ILRAD-bred colonies was examined, by electron microscopy, for the presence and distribution of Rickettsia-like organisms (RLOs). RLOs were present in the midgut epithelial cells of all non-teneral tsetse. In G. m. centralis, G. pallidipes and, to a much lesser extent, G. brevipalpis, RLOs were numerous and were present in all the specimens examined. RLOs were present in fewer numbers in the epithelial cells of tenerals of these three tsetse species. In contrast, RLOs occurred in very much lower numbers within the midgut cells of nonteneral G. austeni, G. p. palpalis, G. p. gambiensis, G. f. fuscipes and G. tachinoides; were not seen in every specimen, and were rarely observed in the midgut cells of teneral tsetse. The RLOs were typical rod-shaped bacteria with an inner and outer membrane, which occurred free within the host cell cytoplasm and appeared to cause no obvious pathology. The micro-organisms divided by binary fission and at least two distinct morphological forms plus a range of intermediate forms were seen in the midgut cells. A comparison of the presence and numbers of RLOs within the midgut cells and the midgut infection rates of both Trypanosoma congolense and T. b. brucei, both between Glossina species and also within the same stock of tsetse, clearly indicates that the ability of trypanosomes to establish and develop to mature infections is unlikely to be correlated solely with the presence of RLOs within the tsetse midgut.

Maintaining the puparial stage of successive generations of a population of tsetse 3 °C lower than normal reduced the numbers of rickettsia-like organisms (RLO) carried by emerging flies. The susceptibility of these flies to midgut infection with Trypanosoma congolense was also significantly reduced compared with control flies held at normal temperature. These results support the view that the relationship between RLO and susceptibility is quantitative – teneral flies with heavier RLO infections being more susceptible to trypanosome infection.

This study examines the effects of host age and sex on the frequency distribution of Brugia malayi infections in the human host. Microfilarial (mf) counts for a large data base on the epidemiology of brugian filariasis in Shertallai, Kerala, South India are analysed. Frequency distributions of microfilarial counts partitioned by age are successfully described by zero-truncated negative binomial distributions, fitted by maximum likelihood. This analysis provides estimates of the proportion of mf-positive individuals who are identified as negative due to sampling errors, allowing the construction of corrected mf age–prevalence curves, which indicate that the observed prevalence may under-estimate the true figures by between 18 and 47%. There is no evidence from these results for a decrease in the degree of over-dispersion of parasite frequency distributions with host age, such as might be produced by the acquired immunity to infection. This departure from the pattern in bancroftian filariasis (where there is evidence of such decreases in over-dispersion; Das et al. 1990) is discussed in terms of the long history of filariasis control (and consequently low infection prevalence) in Shertallai.

Pseudodiplorchis americanus accumulates its entire reproductive output in utero during hibernation of its host, Scaphiopus couchii. Infective larvae are released only during the toads' brief entry into water for breeding. In one host population studied prior to transmission, in S.E. Arizona, adult worms contained 0–326 infective stages; 19% of 178 worms produced 51% of the total larvae. Reproductive output was positively correlated with body length which was considered to reflect worm age; first-, second- and third-year worms produced means of 5, 43 and 96 larvae, respectively. Within second- and third-year cohorts, there was a density-dependent reduction in offspring numbers. Under controlled laboratory conditions, the mean rate of offspring accumulation during the first 6 months of development was significantly reduced only in burdens in excess of those found naturally (36–68 worms). In older parasites, a density-dependent reduction in the mean accumulation rate occurred in burdens of 20–32 worms, where 88% of parasites produced offspring at less than half the maximum rate. The asymmetry in individual reproductive output increased with competition and there were significant reductions in the median rates of offspring accumulation at smaller burdens (17–25 and 5–14 worms, in experimental and natural infections, respectively) than evident from decreases in the mean. Thus, individual variation is a more sensitive measure of competition than is the mean.

Sixteen strains of trypanosomes isolated from the blood of 10 freshwater fish species (all but one from South Bohemia) were characterized by enzyme analysis using dise electrophoresis in 5% polyacrylamide gel. Six enzymes were selected: ADH, LDH, MDH, ME, G6PDH and GPI. The most variable were ADH and LDH, whereas MDH, G6PDH and GPI showed the same pattern in all strains analysed. Cluster analysis indicated a very high degree of similarity among strains, even those isolated from phylogenetically distant hosts. Moreover, no correlation between enzyme pattern and host species was found.

Bloodstream forms of Trypanosoma congolense (2 clones: ILNat3·1 and IL3000, and 4 stocks: IL2079, IL2466, IL3266 and CP-81) were continuously cultivated in vitro at 34–36 °C in the absence of feeder cell layers, using HMI-93 medium which was modified from Iscove's modified Dulbecco's MEM (Flow Laboratories, Irvine, Scotland). The modification was done by supplementing the medium with 0·05 mM bathocuproine sulphonate, 1·5 mM L-cysteine, 0·5 mM hypoxanthine, 0·12 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0·16 mM thymidine, 20% (v/v) heat-inactivated young goat serum and 5% (v/v) Serum Plus™ (Hazleton Biologics, Lenaxa, KS, USA). Trypanosomes obtained from two different sources were used to initiate primary cultures: (1) metacyclic forms which were produced in vitro at 27 °C, and (2) bloodstream forms obtained from Balb/c mice which had been infected with the bloodstream forms transformed in vitro from the metacyclic forms. Metacyclic forms placed in 25 cm2 T-type (T-25) flasks rapidly attached to the bottom of the flasks and transformed to bloodstream forms during the initial 24 h and continued to proliferate. The bloodstream forms isolated from the infected mouse blood by means of diethylaminoethyl cellulose (DE52) column chromatography also continued to proliferate in the flasks. Cultures were maintained by replacing the medium every 24 h. Every 4–5 days, the attached bloodstream forms were resuspended in fresh medium by gentle pipetting and then were subcultured. The method was further simplified by initiating primary cultures directly with 10 μl of the tail blood of infected mice in 24-well culture plates and then by subcultivating either in wells or in T-25 flasks. The shortest population doubling time, 9 h, was achieved by seeding subcultures with 106 bloodstream forms/ml. The bloodstream forms propagated in this system were morphologically similar to those seen in infected mouse blood, they were covered with a surface coat as examined by electron microscopy and they were infective to mice.

During 1988 and 1989, the mesenteric veins of 901 cattle were examined for the presence of schistosomes at the Kandy slaughterhouse (Sri Lanka). The overall prevalence of infection was 31·2%. Animals younger than 2 years were less infected (21·3%) than those older than 5 years (47·9%). Based on the number of paired worms counted, three intensities of infection were recognized: low (1–20 pairs), moderate (21–100 pairs) and heavy (> 100 pairs). Intensities increased with the age of the animals but remained low (average 10 worm pairs). The worm burden increased by approximately 20% for each step in age group. The number of miracidia/100 g faeces was measured in 85 animals of all age groups and intensities of infection; 77% of the samples contained less than 100 miracidia. Miracidia counts decreased with age; moderately and heavily infected animals in each age group had the highest and the lowest counts, respectively. This may be due to a host immune response. The results raise questions on the sensitivity of faecal egg counts as a diagnostic method for visceral schistosomiasis in cattle.

The present study shows an in vivo role for interleukin-2 in the formation of hepatic granulomatous inflammation in murine Schistosoma japonicum infection. Mice which had been administered an inhibitor to interleukin-2 function during acute infection were noted to have reduced cell-mediated immune responses to ConA and S. japonicum soluble egg antigen (SEA). Lymphoid tissue from these treated mice also have reduced numbers of cells which could be activated by ConA to produce interleukin-2 mRNA as shown by in situ hybridization studies. Mice with less activated lymphocytes had, on the average, a 70% reduction in the granulomatous inflammation surrounding eggs deposited in hepatic tissues of acutely infected mice. The data demonstrate that IL-2 is a key factor in the generation of granulomatous inflammation in S. japonicum infection and suggest that a potential anti-pathology vaccine could be generated based on limiting the presence of IL-2 generated during acute infection.

Muscle cells from adult male Schistosoma mansoni have been isolated using a combination of papain digestions and mechanical dissociation procedures. The muscle fibres isolated in this way lacked nuclei but they did contract and relax in response to high [K+], a response which was blocked in the presence of Co2+. From this we conclude that the isolation procedure yields viable muscle fibres useful for physiological studies. Patch-clamp recordings taken from the isolated fibres show a variety of discrete ionic conductances. In inside-out patches one prominent channel was a Ca2+-activated K+ channel with a conductance of 195 pS and a selectivity greater than 10:1 for K+ over Na+, Cs+ or NH4+. Percentage open time was dependent on [Ca2+] at the intracellular face. With [Ca2+] at 1 μM or greater percentage open time was > 95%; at 0.1 μM it was < 2%. No voltage sensitivity could be detected in the voltage range from –50 to –10 mV membrane potential. Ba2+ (10 mM), but neither tetraethylammonium nor 3,4-diaminopyridine blocked the channel from the intracellular face. This Ca2+-activated K+ channel in the muscle membrane of this acoelomate animal is similar in most respects to the maxi-K+ channels which have been described in a variety of cells from more highly evolved animals.

It has been shown that in intact cercariae of Schistosoma mansoni in water, both head and tail had an identical, aerobic energy metabolism. As long as the environment was water, glucose was mainly degraded to carbon dioxide by both head and tail whether or not these two were still connected to each other. Transfer of intact cercariae into a simple salt medium supplemented with glucose resulted in a very rapid transition towards a more anaerobic energy metabolism: the production of lactate and pyruvate increased, whereas the production of carbon dioxide remained more or less constant. A concomitant rise in temperature to 37°C was not essential for this biochemical transition, but made it more pronounced. Experiments on isolated cercarial bodies and tails in a transforming medium demonstrated that the tails oxidized glucose to carbon dioxide, whereas bodies produced mainly pyruvate and lactate. The results showed that the metabolic transition towards a more anaerobic energy metabolism occurred only in the head and not in the tail of the cercariae. Loss of the tail was shown not to be a pre-requisite for this transition, nor did it by itself trigger a metabolic switch in the resulting cercarial body.

The activities of glucose-6-phosphatase (G6Pase), frucrose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) were determined in homogenates of adult Schistosoma mansoni worms and compared with the activities in homogenates of rat liver and rat skeletal muscle, tissues with a high and a low gluconeogenic capacity, respectively. All four gluconeogenic enzymes were present in S. mansoni. The enzymes were less active than in rat liver, but the activities of G6Pase, PEPCK and PC were at least an order of magnitude higher than in rat skeletal muscle whereas FBPase was approximately equally active in S. mansoni and in rat muscle. Experiments with 14C-labelled substrates or [14C]NaHCO3 failed to demonstrate the actual occurrence of gluconeogenesis in S. mansoni. Some possible other functions of the gluconeogenic enzymes were investigated. Experiments with inhibitors of PEPCK gave no indications that this enzyme was involved in the degradation of glucose. This was confirmed by 13C-NMR experiments which indicated that lactate was formed from phosphoenolpyruvate via the actions of pyruvate kinase and lactate dehydrogenase, and that PEPCK did not participate in the formation of lactate. Substrate cycling between fructose-6-phosphate and fructose-1,6-bisphosphate was demonstrated to occur in adult S. mansoni. This shows that FBPase participates in the glucose metabolism of this parasite.

The relationship between faecal examination for egg output and worm burden of Opisthorchis viverrini in man of 181 autopsy cases from Northeast Thailand is described. Diagnosis of the parasite infection by stool examination for the presence of eggs was less sensitive than the worm recovery technique. Using Stoll's dilution and formalin–ether technique, no eggs were detected in the faeces of 20 cases harbouring low worm burdens (< 20 worms). The quantitative faecal egg count by Stoll's dilution technique showed a strikingly close positive correlation with the number of worms recovered (r = 0·96, P < 0·001) indicating a strong linear association between eggs per gram of faeces (epg) and worm burden. The number of epg per worm was inversely correlated to the worm burden (P < 0·001), suggesting that density-dependent constraints on fecundity could operate to restrict egg output in heavy infections. The accuracy of egg counts for estimating worm burden and its relevance to parasite epidemiological research are discussed.

Three techniques for estimating the intensity of Opisthorchis viverrini infection in individuals from a Northeast That community are compared. Egg counts were determined using a quantitative formalin/ethyl acetate technique, worm burdens were estimated by expulsion chemotherapy and antibody levels were measured by ELISA. Log-transformed worm and egg counts were closely correlated (r = 0·80), suggesting that both measurements provide good assessments of relative intensity of infection. However, no Opisthorchis worms were recovered from 34 people with high egg counts; probably due to problems with the expulsion technique in some individuals. Examination of egg production per fluke indicated that each fluke contributed an average of 180 eggs per gram (epg) of faeces and fecundity was negatively associated with total worm burden. Serum IgG levels correlated significantly with Opisthorchis egg count (r = 0·61) at two independent assessments. Although significant associations were observed between antibody levels and echinostome infection, analysis suggested that these reflected independent associations between these two variables and Opisthorchis infection and age. We conclude that all three measurements are useful for epidemiological studies.

Chub and eels were experimentally infected via intermediate hosts harbouring cystacanths, with Pomphorhynchus laevis alone, or Acanthocephalus anguillae alone, or simultaneously with mixtures of both species in varying proportions, and sampled at 7, 56 or 112 days post-infection. Examination of chub revealed that both species showed low establishment and growth rates, differing markedly from British field data, where chub is apparently one of the most important hosts, and preventing further meaningful experiments. Both species showed higher establishment rates in eels, but A. anguillae both grew faster and matured to a greater extent than P. laevis, reflecting field data. No evidence of any interactions, competitive or otherwise, was found in the mixed infections in eels at low or high infection levels. The results were interpretated with regard to British field data and current theories of the role of interspecific competition in structuring fish helminth communities.

Female sex pheromone reception was inhibited in a bioassay by treating males of the beet cyst nematode, Heterodera schachtii, with the sulphydryl reagent, mersalyl acid. This effect was blocked by the sulphydryl group protectant, dithiothreitol, indicating the occurrence of sulphydryl groups in the receptor molecules and a role for sulphydryl groups in pheromone reception.