To investigate the quantitative distribution of Plasmodium falciparum gametocytes into the vector bloodmeal, Anopheles arabiensis mosquitoes were fed on 3 volunteers naturally infected with gametocytes. The content of each mosquito midgut was smeared on a microscope slide and Giemsa stained. The distribution of gametocytes ingested by mosquitoes followed a negative binomial distribution, with apparently constant overdispersion (parameter k±S.E. = 3·105±0·392) for the 3 series. This aggregation of gametocytes in some midguts probably facilitates the conjunction of gametes and fertilization. This suggests that P. falciparum gametocytes in the peripheral blood flow of infected man do not follow an independent, homogeneous pattern but show a significant aggregation.

The oocysts of Plasmodium falciparum that form on the midgut wall of anopheline vectors represent the meiotic products of the sexual cycle of the parasite and are useful for genetic analyses. Recognizing the limitations of current markers for the analysis of genetic variation, we describe a protocol for the DNA isolation and PCR amplification of microsatellite loci from individual oocysts. We present a simple method for the storage and isolation of individual Plasmodium oocysts from wild-caught mosquitoes and demonstrate that it is possible to extract sufficient oocyst DNA for approximately 50 amplifications. Genotyping was successful for 10 microsatellite loci and, using Genescan technology, we found extensive microsatellite polymorphism in an analysis of a dozen oocysts from each of 2 Anopheles gambiae female specimens collected within 6 months at the same location in northern Tanzania. All of the loci exhibited at least 2 alleles, with a maximum of 5. Homozygous and heterozygous oocysts could easily be discerned, but most of the oocysts were homozygous. The procedure has great potential for shedding light on genetic mechanisms operating during meiosis in mosquito vectors and the influence this may have on the genetic structure of natural populations of P. falciparum.

The differentiation of the two sexes in the gametocytogenesis of Plasmodium falciparum was investigated using a plaque assay and antibodies specific for various stages and sexes of gametocytes. Immunofluorescence assays on plaques of cultured parasites grown in monolayers of erythrocytes revealed that the merozoites released from a single sexually-committed schizont became either all male or all female gametocytes. Thus, the commitment of this species to differentiate into one sex or the other is likely to occur prior to the nuclear division of the sexually-committed schizont. The characteristic female-biased gametocyte sex ratio observed for many Plasmodium species is manifested in P. falciparum by a greater percentage of schizonts that produce female gametocytes (67–71%) than those that yield males. From the plaque assay, it was determined that the number of gametocytes produced per sexually-committed schizont was similar for both sexes, indicating that allocation of parasite resources was equal for each sex of gametocyte. The timing of sexual differentiation and features of the gametocyte sex ratio is discussed in relation to previous observations on P. falciparum and related malaria parasites.

The elongation factor-1 alpha (EF-1α) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEα) that codes for a major part of the coding region of the EF-1α protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5′ and 3′ ends of the EF-1α gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5′ end of B. hominis C EF-1α were identified. A comparative study on the deduced amino acid sequences of BEα of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the speciesThe nucleotide sequence data reported in this paper are available in the GenBank database with the accession numbers as shown in Table 1.. A phylogenetic reconstruction analysis with other eukaryotic EF-1α sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.

Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

The in situ polymerase chain reaction (PCR) results revealed that mouse type A and type C retroviral sequences were transmitted horizontally from the host to schistosomes. The signals to these retroviral sequences were observed in the nuclei of the mesenchymal and reproductive cells of 8-week Schistosoma japonicum. These signals were also detected in the nuclei of the mesenchymal and reproductive cells and in the cytoplasm of the tegumental tubercles of 24-week S. mansoni. Furthermore, mouse type A retroviral sequence was detected in the DNA extracted from the cercariae of both species. However, mouse type C retroviral sequence and mouse type 2 Alu sequence (B2) were difficult to detect in the cercarial DNA of either species. These findings may indicate that some host sequences are propagated in the schistosome progeny, that is to say, not only horizontal but also vertical transfer of the host gene may occur in schistosomes.

Using the filamentous actin marker, FITC-conjugated phalloidin, the major muscle systems of adult male and female schistosomes have been examined. The body wall musculature comprises an outer sheath of circular fibres, within which there is a compact layer of short, spindle-shaped longitudinal fibres and a lattice-like arrangement of inner diagonal fibres. Within the oral sucker and acetabulum 3 fibre types, circular, radial and longitudinal can be distinguished. The wall of the oesophagus is lined by a grid-like array of circular and longitudinal fibres, whereas the walls of the intestinal caeca contain only comparably broad circular fibres. Within the female reproductive system, only circular fibres are present in the oviduct, vitelline duct and uterus. In contrast, the wall of the ootype displays closely arranged circular and longitudinal muscle fibres. Antisera to previously identified myoactive compounds (serotonin [5-hydroxytryptamine, 5-HT], neuropeptide F [Moniezia expansa] and GYIRFamide [Bdelloura candida, Dugesia tigrina]) were used as neuronal markers in a preliminary study of the spatial inter-relationships of specific nerve fibres and various muscle systems. Serotoninergic fibres innervate both suckers and also constitute a subtegumental nerve net. In males they provide innervation to the dorso-ventral muscle fibres of the gynaecophoric canal, and in females they innervate the circular and longitudinal muscle fibres of the ootype. Neuropeptide F and the FMRFamide-related peptide, GYIRFamide are both localized within nerve plexuses associated with the dorso-ventral fibres of the gynaecophoric canal, and are evident in the innervation of the ventral and oral sucker.

This paper presents a first attempt at modelling the possible cost and effectiveness of reaching non-enrolled children through school-based programmes using empirical data from Egypt. A sex/school-attendance/age-structured population dynamic model was used to predict trends in infection and early disease. Four treatment delivery strategies were compared: school-based (coverage of 85%) and school-aged targeted (coverage of 25, 50 and 85%). The school-aged targeted strategies also included the school-based programme. For each alternative strategy, the maximum unit cost was calculated to obtain a cost-effectiveness ratio equal or smaller to the one obtained with the school-based programme (unit cost of US$ 0·60). The analysis showed that, for S. mansoni in Lower Egypt, a programme where only 85% of children attending school were treated would still prevent 77% of the early disease cases prevented with a programme where 85% of all school-age children were treated. However, using the school-aged targeted strategy, from US$ 0·06 to US$ 1·03 extra unit costs could be spent to reach non-enrolled children and still be more cost-effective. Treating non-enrolled children is an important consideration in maximizing the effectiveness of treatment programmes while maintaining a cost-effectiveness comparable to school-based delivery.

In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive λ bacteriophage clones containing F. hepatica cDNA have been isolated. Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elementsThe nucleotide sequence data reported in this paper are available in the GenBank database with the accession number AF153056.. All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions. Antisera raised against a β-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens. Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument. The wall of the uterus showed strong reactivity in the adult. Rats immunized with the β- galactosidase fusion protein showed enhanced resistance to challenge infections. The role of these antigens in the host response to infection by F. hepatica is discussed.

Lungworms of the genus Dictyocaulus from cattle, roe deer, and moose in Sweden were subjected to morphological and molecular analyses. The objectives of the study were to investigate whether mixed or monospecific Dictyocaulus infections occur in Swedish cattle and whether wild cervids may act as reservoirs. The morphological characters examined were thickness and shape of the buccal capsule wall (BCW) and total spicular length (TSL). Morphometry was also done on the total body length, and BCW thickness and length. In the molecular identification, we used a PCR-linked hybridization assay to probe worm DNA with species-specific oligonucleotide probes to the second internal transcribed spacer (ITS2). The results showed that the BCW shape was the most reliable morphological character for identification. Significant differences were observed in this character, but an overlap occurred between lungworms from each of the host species. With the hybridization assay, all lungworms from cattle were identified as D. viviparus, whereas those from roe deer represented a novel Dictyocaulus species demonstrating that each host had a monospecific lungworm infection. In moose, 61 (78·2%) worms belonged to the new species and 17 (21·8%) were D. eckerti. This study shows the usefulness of hybridization assay as an epidemiological tool for the specific identification of lungworms of cattle and wild cervids.

This study describes morphological changes during capsule formation of Trichinella spiralis with emphasis on nuclei within the capsule. The nuclei of the pre-existing muscle cell may become hypertrophied or degrade soon after the entrance of newborn larvae. Cytoplasm associated with these hypertrophied nuclei was basophilic in H&E staining and composed of mitochondria and rough endoplasmic reticulum. Satellite cells underwent cell division and joined to the nurse cell. These nuclei may also become hypertrophied or degrade. Cytoplasm associated with nuclei of satellite cell origin was eosinophilic in H&E staining and composed of mitochondria, polysomes, and smooth and rough endoplasmic reticulum. Eosinophilic cytoplasm (of satellite cell origin) became prevalent in the capsule as capsule formation proceeded. Infiltrated cells were observed within the capsule. Thus this study showed diversity of nuclei in the capsule of Trichinella.

Previous studies have indicated that between 60 and 80% of a population of entomopathogenic nematodes do not infect their insect hosts at any one period in time. Two hypotheses explain this behaviour: the first that there is a subpopulation of non-infectious nematodes and the second that the non-infectious group is created by inhibitory cues derived from infected insects. Through an experimental approach with the Galleria mellonella–Steinernema feltiae system we show that both mechanisms operate together. When conditions for infection were optimized, the sum of individual infection behaviours was similar to the number infecting as a population, implying observed infection rates are driven by intrinsic mechanisms. In addition, there was evidence that an infected host released a chemical cue into the environment which inhibited subsequent levels of infection. This degree of inhibition was independent of the number of infecting nematodes. Both these mechanisms are dynamic, so the observed proportion of infectious nematodes depended heavily on the time of exposure. The implications of these findings for both the design of laboratory trials and the use of entomopathogenic nematodes in biological control are discussed.

Five groups of ten 7-month-old Romney lambs were challenged twice-weekly (2 × 3000) with either Ostertagia circumcincta (Groups 1–3) or Trichostrongylus colubriformis (Groups 4 and 5) infective-stage larvae (L3) following administration of controlled release capsules (CRC) containing either albendazole (ABZ–CRC) (Groups 2 and 5) or ivermectin (IVM–CRC) (Group 3). Larval challenge comprised a mixture of equal numbers of drug-susceptible and -resistant L3 (OR/TR[ratio ]OS/TS) during the 14-week period of drug release. Positive faecal egg counts (FEC) were recorded in each group during this period. Eggs recovered from faeces were used in an egg hatch assay (EHA) utilizing different concentrations of ABZ. O. circumcincta eggs from Groups 2 and 3 had significantly higher LC50 values than Group 1; eggs of T. colubriformis from the ABZ–CRC treated Group 5 had a higher LC50 than control Group 4. Following the exhaustion of drug release, larval challenge was changed to consist of only susceptible parasites and EHA performed weekly to determine whether LC50 declined, which would indicate dilution or replacement of drench survivors. In those animals challenged with O. circumcincta, LC50 values were still significantly higher in Groups 2 and 3 at week 19 than in the previously untreated Group 1. For T. colubriformis, LC50 values declined steadily following exhaustion of drug release and were not significantly different between Groups 4 and 5 by week 20. This decline in LC50, and corresponding rise in FEC, was largely the result of 3 animals which dominated with high FEC. The remaining 6 animals showed little decline in LC50 or rise in FEC. The results demonstrate the ability of persistent drugs to screen for resistant parasites and establish that the period of selective advantage for drug-resistant parasites of both species is significantly longer than the period of drug release from CRCs.