In view of the recent discovery (Molecular Cell6, 861–871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology119, 343–348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine.

The initial attachment of Toxoplasma tachyzoites to the target host cell is an important event in the life-cycle of the parasite and a critical stage in infection. Previous studies have shown that polyclonal antibodies directed against the major surface antigen of Toxoplasma gondii (SAG1) inhibit the infection of enterocyte cell lines. Here, we demonstrate that antibodies raised against a central peptide (V41T) of SAG1 and the SAG1 protein itself are able to inhibit the infection of various cell lines by the tachyzoites. Antibodies directed against SAG1 peptides were used to define a site on the SAG1 antigen that interacts with the host cell. The epitope carried by V41T was identified on the tachyzoite surface by immunofluorescence. The peptide sequence seems to be conserved in all the members of the SAG1 Related Sequence family (SRS). Using undifferentiated and differentiated Caco-2 cells, we found that tachyzoites enter preferentially via the basolateral side of the cell. These findings highlight the role of the SRS family members in the mediation of host cell invasion.

The publishers would like to apologise for a typographical error in the June 2001 issue of Parasitology, Volume 122 Part 6.

In the article on pages 641–649 (and on the back cover contents) the final author's name was spelt incorrectly.

The correct spelling of the title and authors of this paper should read:

Determinants for resistance and susceptibility to microfilaraemia in Litomosoides sigmodontis filariasis

W. H. HOFFMANN, A. W. PFAFF, H. SCHULZ-KEY and P. T. SOBOSLAY

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

A Besnoitia species of the teiid lizard Ameiva ameiva (L.), from north Brazil was established in laboratory mice and hamster by the intraperitoneal inoculation of bradyzoites in the tissue cysts. In the lizards all the cyst wall layers were closely apposed. In the mice the layers of the wall were distinguishable, and ultrastructurally the inner cytoplasmic layer contained either a tight network of endoplasmic reticulum or packed mitochondria or both. These components were less frequent or sparse in the inner cytoplasmic layer of cysts in the lizard. The only animals available for experiments in attempts to indicate the definitive host of the parasite were 3 kittens of the domestic cat and a juvenile specimen of the snake Boa constrictor raised in captivity. No evidence of infection could be detected in these animals after feeding them with the tissues of mice harbouring cysts with very large number of bradyzoites.

Data are presented on 2 full epizootic cycles and the start of a third of Ligula intestinalis in roach Rutilus rutilus in a small lake, and the relationships of these cycles to the densities of rudd, Scardinius erythrophthalmus, and Great Crested Grebes, Podiceps cristatus, over 31 years. The parasite was introduced to the lake by P. cristatus in 1973 at a time when the roach population had increased in response to eutrophication to a level at which individual fish growth was stunted and the hithero dominant rudd population had declined in numbers as a consequence of inter-specific competition with roach. Ligula prevalence peaked at 28% in only 2 years: thereafter parasite-induced host mortality caused a decline in the roach population, releasing fish from stunting and allowing the rudd population to recover. The consequent improved growth of roach individuals and their short life-span reduced Ligula transmission rates and prevalence levels declined to approximately 1% although Ligula nevertheless persisted for a further 10 years. Following a massive winter-kill of the fish populations in 1984–1985, fish and Ligula numbers declined to barely detectable levels and the parasite disappeared from samples. Rudd recovered first, then roach and interspecific competition again led to a decline in rudd numbers. This increase in roach numbers led to a decrease in roach growth rates, which coincided with the re-colonization of the lake by Ligula. This second epizootic of Ligula peaked within 2 years in 1991–1992, when up to 78% of roach were infected with a maximum abundance of 2.2 parasites and intensity of 21 parasites. Heavy parasite-induced mortality of roach led to a decline in numbers, an improvement in individual growth rate and a reduction of Ligula transmission rates such that the epizootic died out in 1996. Similar conditions of roach numbers and growth prevailed at the start of a third cycle in 1998. The course of events over the second cycle was so similar to that of the first that it confirms the interpretations of that cycle. Comparison with other localities shows that epizootics of Ligula always coincide with rapid increases in roach numbers, for whatever cause, and stunted growth, which together attract piscivorous birds. At the start of a cycle Ligula is a major determinant of the population dynamics of the roach, but at the end of the cycle the fish population dynamics determine those of the parasite. The cycles are not regulated and the roach–Ligula system is inherently unstable.

Pasteuria penetrans is a Gram-positive endospore-producing bacterium that is a parasite of root-knot nematodes. Attachment of endospores to the cuticle of the nematode is the first stage in the infection process. Western blot analysis with monoclonal and polyclonal antibodies that recognize the 30 kDa heparin-binding domain (HBD) and the 45 kDa gelatin-binding domain (GBD) fragments of human fibronectin (Fn) revealed a series of polypeptides of approximately 40, 45 and 55 kDa present in crude cuticle extracts of Meloidogyne javanica 2nd-stage juveniles. The results suggest that the structure of the nematode fibronectin is different to the fibronectins so far characterized. Pre-treatment of endospores of Pasteuria with either the HBD or the GBD was found to inhibit binding to the nematode cuticle. The larger GBD fragment was the most effective at blocking adhesion. Pre-treatment of the GBD fragment with gelatin prevented the GBD fragment from inhibiting endospore attachment to the nematode cuticle.

A cDNA encoding a Na,K-ATPase alpha subunit homologue, designated SNaK1, was isolated from an adult cDNA library of Schistosoma mansoni. The 3.8 kb DNA contained a 3021 bp open reading frame potentially encoding a 1007 amino acid protein that had an Mr of 111817 and a pI of 5.48. Homology searches for SNaK1 revealed approximately 70% sequence identity with a variety of Na,K-ATPases from evolutionarily diverse organisms. SNaK1 is predicted to contain 10 transmembrane regions typical of this protein family as well as other conserved domains, such as the phosphorylation site and ATP binding domain. Antibodies raised against an amino terminal peptide detected the protein in membrane preparations of eggs, cercariae and adult males and females, suggesting a general role for SNaK1. The mobility of the protein differed in various life-stages suggestive of post-transcriptional or post-translational modification. Immunolocalization of SNaK1 in sections of adult worms using epifluorescence and electron microscopy, revealed antibody labelling in the subtegumental and peripheral layers. Strong staining was discernible in the peripheral muscle band indicating that SNaK1 plays a central role in muscle contraction in adult parasites and may be the primary target of ouabain action. Staining was also detected in the secretory bodies in sections of ducts in this region and over the RER of the presumed gastrodermis. Immunogold labelling was also localized over neuronal vesicles in axons associated with the peripheral muscle layer.

This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3.8 and 6.5 cm long and the females between 4.5 and 8.0 cm. Some individually cultivated females laid eggs which had an average size of 44.4×50.5 μm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4.0 and a temperature of 37 °C, and in air atmosphere with 5% CO2. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1.9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4.2 times, from 22.9 to 95.6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.

Three heat-induced genes of the infective-stage larvae of Trichinella spiralis were successfully identified by the suppression subtractive hybridization (SSH) technique. As indicated by reverse Northern blotting, 19 of 25 clones were scored as differentially transcribed in the heat-shocked infective-stage larvae. The sequencing data showed the presence of 12 different genes. Three were homologous to histone H3, histone H2B and translationally controlled tumour protein (TCTP). A 0.6 kb cDNA of histone H3 was generated by the RACE method and sequenced. It contained an open reading frame of 136 amino acids that demonstrated 94% identity with genes from Drosophila hydei. Semi-quantitative RT-PCR indicated that after heat-shock treatment, the expression levels of histone H3, histone H2B and TCTP increased 4.8, 27 and 5.7-fold, respectively. Northern analysis confirmed the upregulation of histone H3, histone H2B and TCTP transcripts. The upregulation of these genes during stress conditions has not been reported in parasitic organisms. The stress proteins may play an active role to sustain the parasite after exposure to hostile host factors.

A novel DNA-binding peptide of Mr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae of Trichinella pseudospiralis. Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of the Mr of proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.

A simple and robust method for the isolation of gastro-intestinal nematode eggs from faeces is described that uses both salt- and sugar solutions for flotation. Application of this ‘salt–sugar’; isolation method to large numbers of faecal samples of adult dairy cows indicates a 3- or 4-fold reduction in the proportion of e.p.g.-negative cows relative to studies that used other techniques for egg isolation. The procedure detects more eggs than the Wisconsin flotation method in replicate samples and in spiked egg-free faeces. The number of recovered eggs in spiked faecal samples is linear over a range of egg concentrations, and the transparent faecal preparations that result from the protocol can be stored as digital images which can be used as input for an efficient automated egg-counting procedure. The increased rate of processing of faeces combined with the large reduction of the percentage of e.p.g.-negative cows allows more accurate analysis of large numbers of adult or resistant animals for studies of nematode parasitism.

An experiment was undertaken to study the effect of dietary carbohydrates with different digestibility on the populations of Oesophagostomum dentatum in the intestinal tract of pigs. Sixty-four worm-free pigs from a specific pathogen-free farm were randomly divided into 8 equal groups. The animals in 4 groups were assigned to a diet with partially undegradable carbohydrates (diet 1), while the pigs in the 4 remaining groups were given a diet with fermentable carbohydrates (diet 2). Diet 1 was comprised of barley flour, oat husk meal, soybean meal, vitamins and minerals and diet 2 of barley flour, inulin and sugar beet fibre, soybean meal, vitamins and minerals. The pigs in 6 of the groups (n = 48) were inoculated with 6000 infective larvae of O. dentatum. To determine O. dentatum populations at the early stage of infection, 16 pigs were slaughtered 3 weeks p.i., while the remaining 4 groups continued on the diets for a further 9 weeks after which they were slaughtered. In a diet cross-over experiment 6 weeks after inoculation, 8 pigs changed from diet 1 to diet 2 (diet 1>diet 2), and 8 pigs from diet 2 to diet 1 (diet 2>diet 1). The results showed that partially undegradable carbohydrates provided favourable conditions not only for parasite establishment and sustainability, but also for already established O. dentatum infection while, in contrast, the diet composed of highly degradable carbohydrates decreased worm establishment, size and female fecundity. The implications for pastured pigs or pigs fed different complex carbohydrate diets is discussed.

Ten chickens and 2 pigs were fed non-embryonated eggs of Ascaris suum, Trichuris suis and Oesophagostomum dentatum. Each chicken was fed approximately 15000 eggs of each parasite species while approximately 300000 eggs were given to each of the pigs. After passage in chickens 8.3% of O. dentatum eggs were recovered in faeces compared to 61.1% and 41.6% of A. suum and T. suis eggs, respectively. After passage in pigs the percentages were 38.4%, 49.1% and 30.3%, respectively. After embryonation in the laboratory, 1000 eggs of each parasite species having passed through chickens or pigs or having been kept in the laboratory as controls were fed to groups of 6 pigs to check the infectivity. The number of A. suum recovered from pigs was similar in the 3 groups with 34.0, 52.8 and 41.8%, respectively. The recovery of T. suis in the pig passage group was 54.0% which was significantly lower than the recovery in the chicken passage group (81.8%) and the laboratory group (88.0%). The number of O. dentatum recovered was not significantly different among the 3 experimental groups, the percentage recovery being 30.5, 9.2 and 28.5%, respectively. One explanation for the lower infectivity of T. suis in the pig passage group may be that the eggs have been sublethally damaged through their passage. The results demonstrate that chickens and pigs can act as transport hosts for A. suum, T. suis and O. dentatum, and it is highly probable that these domestic animals are able to act also as transport hosts for the human parasite equivalents. This will have important consequences for the environmental and behavioural strategies in human helminth control programmes.