Breast cancer is a complex disease. Its aetiology is multifactorial, its period of development can span decades, and its clinical course is highly variable. Evaluation of the role of the immune response in either the development or control of breast cancer is also complex. Nevertheless, there is substantial information that in this disease, the immune response is not a host defence reaction and may even serve to facilitate cancer development. This evidence comes from a variety of sources including clinical–pathological investigations in women that show a correlation between the intensity of lymphocytic infiltration into the tumour mass with poor prognosis, studies in breast cancer patients that demonstrate a similar correlation between delayed hypersensitivity reactivity or in vitro assays of immune reactivity to tumour cell membranes or non-specific antigens and poor prognosis, and analyses of cancer incidence in chronically immunosuppressed, kidney transplant recipients who develop an unexpectedly low incidence of breast cancer. The overall conclusions from these human studies are corroborated by observations in mouse mammary tumour models that also demonstrate immune enhancement of breast cell proliferation in vitro and of breast cancer development in vivo. Potential mechanisms for these effects include production, by inflammatory cell infiltrates, of direct or indirect modulators of breast cell growth, e.g. cytokines, peptide or steroid hormones, enzymes involved in steroid metabolism, as well as of antibodies to growth factors or their receptors. These immune facilitatory mechanisms must be overcome if immune-based therapies are to be applied successfully in breast cancer.

A β-1,3-glucan-binding lectin from the penetration glands of Diplostomum pseudospathaceum cercariae was isolated by affinity chromatography using yeast glucan and curdlan as affinity matrices. Further purification to homogeneity was performed by cation-exchange chromatography. The protein migrated as a double band around 24 kDa in gels after SDS–PAGE. The protein is of strongly basic nature – its pI shown by native IEF was around 10. The mass of the protein determined by MALDI-TOF mass spectrometry was 23·9 kDa. N-terminal sequence as well as some internal sequences showed significant alignments with several cysteine protease sequences found in databases. The protein bound a biotinylated synthetic analogue of the irreversible inhibitor of cysteine proteases, E-64 and, moreover, its proteolytic activity was demonstrated in substrate gels. The enzymatic activity could be inhibited by the cysteine protease inhibitor E-64; therefore, the investigated protein was considered to be a bifunctional molecule possessing both lectin and enzyme activities. Glycanohydrolytic activity was not proved. The detected characters of this molecule lead to a hypothesis on its role in penetration of Diplostomum cercariae into fish hosts – that of binding to the carbohydrates of fish mucus and concurrent cleaving of protein components of the mucus and skin.

A cross-sectional sero-epidemiological study was performed in Magoda, Tanzania, an area where malaria is holoendemic. Blood samples were collected from children (1–4 years) and tested for IgG antibody reactivity against 2 recombinant protein fragments of Plasmodium falciparum Rhoptry-Associated Protein-1 (rRAP-1). The data were related to the prevalence of malarial disease and single P. falciparum or mixed Plasmodium infections. Fever ([ges ]37·5 °C) in combination with parasite densities >5000/μl were used to distinguish between children with asymptomatic malaria infections and those with acute clinical disease. Furthermore, C-reactive protein (CRP) was applied as a surrogate marker of malaria morbidity. The prevalence of Plasmodium infections was 96·0%. Eleven children were defined as clinical malaria cases, all with single P. falciparum infections. The density of P. falciparum was significantly lower in children with mixed Plasmodium infections compared to those with single P. falciparum infections. Children with asymptomatic P. falciparum infections had higher IgG reactivities to rRAP-1, compared to IgG reactivities of children with malarial disease. Children with mixed Plasmodium infections generally showed elevated IgG reactivity to rRAP-1, when compared to children with single P. falciparum infections. The possible relationship between mixed species infections, clinical outcome of the disease and antibody responses to RAP-1 is discussed.

This paper aims to test the influence of route of infection (intravitreal and instillation) on the course of ocular toxoplasmosis in mice, using the Toxoplasma gondii Me-49 strain. All mice inoculated intravitreally or by instillation presented the same pattern of infection. Using either route, parasites were observed in the retinal vessel with the formation of a glial reaction in the inner plexiforme layer and discontinuity of the pigmented epithelium of the retina 7 days after infection. However, when the intravitreal route was used a more intense inflammatory infiltrate was observed in the retina. The results suggest that inoculation route remarkably influences the inflammatory pattern in ocular toxoplasmosis and that the instillation route should be preferentially used in experimental infections in the murine ocular model of infection by T. gondii, specially with small animals where there is extensive needle damage, which is not observed in the instillation route.

The ultrastructure of muscle larvae of Trichinella pseudospiralis was studied by electron microscopy. The overall structure of muscle larvae of T. pseudospiralis resembled that of T. spiralis except for the stichocyte granules. T. pseudospiralis had at least 3 kinds of stichocyte granules distinguishable from each other by their shape, size and inclusions. The granules had some resemblance to α granules or β granules of T. spiralis, but no resemblance to γ granules. In favour of these morphological differences and similarities among T. spiralis and T. pseudospiralis, excretory and secretory (E–S) products (originating from stichocyte granules) of the 2 species differed to some degree. In an analysis by 2-dimensional electrophoresis, some peptide spots migrating at 45 kDa were shared by the 2 species but the other spots were unique to each of the 2 species. Messenger RNA encoding the 43 kDa glycoprotein of stichocyte granules was detected in the muscle larvae of both species but mRNA encoding the 53 kDa glycoprotein was detected only in muscle larvae of T. spiralis.

Throughout the past decade much research has been directed towards identifying the occurrence, epidemiology, and risks associated with waterborne protozoa. While outbreaks are continually documented, sporadic cases of disease associated with exposure to low levels of waterborne protozoa are of increasing concern. Current methodologies may not be sensitive enough to define these low levels of disease. However, risk assessment methods may be utilised to address these low level contamination events. The purpose of this article is to provide an introduction to microbial risk assessment for waterborne protozoa. Risk assessment is a useful tool for evaluating relative risks and can be used for development of policies to decrease risks. Numerous studies have been published on risk assessment methods for pathogenic protozoa including Cryptosporidium and Giardia. One common notion prevails: microbial risk assessment presents interesting complications to the traditional chemical risk assessment paradigm. Single microbial exposures (non-threshold) are capable of causing symptomatic illness unlike traditional chemical exposures, which require a threshold to be reached. Due to the lack of efficient recovery and detection methods for protozoa, we may be underestimating the occurrence, concentration and distribution of these pathogenic micro-organisms. To better utilize the tool of microbial risk assessment for risk management practices, future research should focus in the area of exposure assessment.

Parasites inflict fitness costs on their hosts, but often the exact reduction in fitness is not well understood. We investigated the influence of infection by the gregarine genus Monocystis sp. on growth and female investment (cocoon production) of its earthworm host, Lumbricus terrestris. Earthworms (n=81) were observed in a laboratory setting for 8 months, after which parasite load was determined. The results revealed a significant negative relationship between parasite load and growth, yet no association to cocoon production was found. Although the exact nature, strength, and evolutionary consequence of reduced growth are still unclear, the results are the first indication for a clear, albeit weak effect of Monocystis on host fitness.

We have previously discovered a unique mechanism of epigenetic transcriptional gene silencing in the Entamoeba histolytica trophozoites of strain HM-1:IMSS that resulted in the persistent downregulation of the amoebapore A (ap-a) gene, and that could be successfully applied to silence other virulence genes (cpA5, lgl1). In order to understand how the silencing is maintained throughout generations, we analysed whether modifications occurred at the chromatin level. Chromatin immunoprecipitation assays were done with antibodies specific to the methylated lysine 4 of E. histolytica histone H3. When the genes were in a transcriptionally silent state, the methylation levels of H3K4 in their coding region were significantly reduced. In contrast, the levels of core histone H3 were consistently higher in the silenced genes. Controlled chromatin digestion with micrococcal nuclease was used to assess changes in nucleosome compaction. We found a significant resistance to digestion in the promoter region of the silenced ap-a and cpA5 genes as compared to the parental strain that expresses those genes. Our data lend further support to the idea that histone modifications and heterochromatin formations are at the basis of the transcriptional silencing of genes in E. histolytica.

Small RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

This study reports an outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Following clinical and pathological diagnoses, a total of 112 cattle blood samples were divided into three groups and tested using multiplexed tandem PCR. Group 1 were from aborted heifers in Vietnam; group 2 were from cattle before shipment from group 1 cattle and group 3 were from the same batch of cattle but transported to Taiwan. Theileria orientalis DNA was detected in 72·3% cattle. The prevalences of T. orientalis in groups 1, 2 and 3 were 77·6, 86·9 and 57·5%, respectively, and the difference in prevalence was significant between groups 1 and 3 (P < 0·0001). The infection intensities of genotypes chitose and ikeda of T. orientalis were higher in groups 1 (57 721 and 33 709, respectively) and 3 (5897 and 61 766, respectively) than those in group 2 (2071 and 6331, respectively). Phylogenetic analyses of the major piroplasm surface protein sequences revealed that genotypes chitose and ikeda determined herein were closely related to those previously reported from Australia. This first report of an outbreak of oriental theileriosis in imported cattle emphasizes improved measures for the export and import of cattle infected with T. orientalis.

Plasmodium vinckei chabaudi (which has a 24 h cycle with high synchronicity) has a peak of schizogony in the early morning at about 02.00–04.00. This cycle is orientated to the cycle of the host (mouse or thamnomys). The male gametocytes become ripe for exflagellation in a well-marked wave from 22.00 to 08.00 with peak about 00.00–04.00. This timing presumably corresponds to the hours when the vector mosquitoes are most likely to suck blood, and it is adapted to facilitate transmission. The male gametocytes remain ripe for exflagellation for a mean period of about 6 h and then apparently degenerate.

The feeding behaviour of Glossina morsitans morsitans infected with Trypanosoma vivax, T. congolense or T. brucei was studied and compared with that of uninfected control tsetse. The following parameters were measured. (1) Mean number of feeds taken in 20 consecutive days; (2) mean rate of blood meal intake in μg/sec of the total days fed; (3) mean weight of blood meal intake/day of the total days fed; (4) mean total blood meal engorgement in 20 days; (5) mean number of probes/day of the days fed; (6) mean frequency of the multiple probe feeds and (7) mean number of pre-feeding probes of the multiple probe feeds. Evidence was found to suggest that the rosettes of T. vivax and T. congolense in the proboscis, and colonies of T. brucei parasites in the salivary glands, did not significantly alter the feeding behaviour of the vector.

Transmission of Schistosoma mansoni was monitored by routine snail sampling for Biomphalaria pfeifferi and by supplementary cercariometric measurements in 4 neighbouring study areas in Machakos District, Kenya. After 1 year, extensive, population-based chemotherapy with a single dose of praziquantel was given in 3 areas, but only minimal treatment in the fourth. In the year preceding treatment, seasonal transmission of S. mansoni and other non-human trematodes occurred in all 4 areas, despite some ecological differences and the effects of earlier treatment campaigns in 1 of the study areas. After treatment of all infected subjects in one area in which there had been earlier chemotherapy campaigns, S. mansoni transmission remained very low. It was reduced for at least 2 years after chemotherapy targeted at either all heavily infected subjects or all infected school children, but it was unaffected in an area where treatment was restricted to those few very heavily infected cases at risk of developing, disease. Nowhere was transmission entirely eliminated by chemotherapy and that of non-human trematodes continued unabated. The snail data correspond well with the human, parasitological data. Targeting school children was as effective as more extensive campaigns, but chemotherapy alone never stopped S. mansoni transmission: reinfection was inevitable, at rates determined by ecological factors affecting snail populations.

The influence of genetic factors on acquired resistance to Haemonchus contortus infections in sheep was investigated. Animals whose primary infections were terminated with an anthelmintic failed to develop any immunity against subsequent challenge as judged by worm numbers. Nevertheless, all were better able to retard the development and reduce the fecundity and haematophagic activities of their parasite populations than animals undergoing primary infections. High levels of resistance, as judged by all these parameters, were observed in most animals when the challenge larvae were superimposed on existing worm populations. The patterns of worm establishment and disease indicated that genetic factors operated in determining resistance, since fewer worms became established and less severe clinical and pathophysiological changes were observed in Scottish Blackface than in Finn Dorset sheep with the same haemoglobin type. Similar advantages were displayed by animals with haemoglobin AA and to a lesser extent those with haemoglobin AB over haemoglobin BB types. The importance of breed was further indicated by the occurrence of ‘self-cure’ in the majority of the Scottish Blackfaces but in only one Finn Dorset. There was no evidence that this reaction was associated with haemoglobin type.

In general, female ticks infest the hairy areas of sheep: head and legs; the (comparatively) bare areas: axillary and inguinal; and certain woolled areas: chiefly the short-woolled margins of the hairy and bare areas; they do not attach in the long wool of withers, back, rump and flanks.

More female ticks attach on the head than on the axillary region, and more on the axillary than on the inguinal region, obviously on the principle: first come best served.

Different tick stages tend to attach in different zones of the same regions; two theories are put forward to account for this.

On a hill pasture where the main tick activity occurs in spring only, the average totals of female ticks carried per annum by ewe, hogg and lamb were in the proportions 8:6: 1. Host size is thought to be the chief governing factor.

Under existing British hill pastoral conditions, the upper limits to female tick infestation appear to be of the following order: average spring peak total count per ewe reaches 100–150, with extreme individual limit about 300; and about five times as many ticks are carried per ewe over the entire spring season.

On heavily infested hill pasture, stocked at one sheep to 1–5 acres, typical flocks feed from 100 to 600 female ticks per acre per annum. The densest sheep stocking results in the highest tick density.

For every one female tick, about eight nymphs were fed by sheep in one season.

The better the condition of a sheep, the less bare of wool and apparently (to touch and sight only) the more greasy, and the lower the tick infestation.

In any age group of sheep on one grazing, there is considerable range in weekly infestation weights but individuals tend to keep their relative positions consistently in this range. Sheep differing markedly in their infestation weights show a remarkable lack of difference in the majority of physical and physiological characteristics of body form and body surface. (Fleece humidity was omitted from study.) Notably, there is no indication of significant differences in fleece greasiness. Apparently only two characteristics go with the lighter infestations in Blackfaced sheep. These are coarser fleece hair and less clearance between fleece tips and ground, having doubtful and minor effects respectively.

The most important factor in individual differences of infestation within an age group is very probably individual activity, i.e. the more ground covered in unit time, the more ticks picked up.

Histological, histochemical and ultrastructural methods have been used to study the gut structure and digestive physiology in the parasitic nematode Cyathostomalari E. Banchard, 1849. The nematode is parasitic in the orbito-nasal sinuses of its host and feeds exclusively upon blood.

Three gland cells are present in the oesophagus, one in each sector. The two subventral gland ducts open into the lumen of the oesophagus at the level of the nerve ring, and the dorsal gland duct into the base of the buccal capsule. It is suggested that the latter gland may produce an anticoagulin, histolytic and/or haemolytic secretions, histochemically-inactive in situ. The intestine is syncytial, with no differentiation into secretory components, and its distal surface is provided with a prominent brush border of long microvilli.

A non-specific esterase, thought to originate in the oesophageal gland secretions, causes haemolysis of ingested erythrocytes. The major part of haemoglobin digestion is extracellular, partly by a process of contact digestion on the microvilli, initiated by the esterase, and the protein is then further broken down by an exopeptidase of the ‘leucine aminopeptidase’ type, and the products absorbed into the gastrodermis possibly with the aid of an acid phosphatase present on or in the microvilli. An insoluble iron-containing compound, which is not haematin, results from breakdown of the prosthetic group. A smaller proportion of haemoglobin is taken up by the gastrodermis and digested intracellularly by the action of lysosomal hydrolases. An endopeptidase and an acid phosphatase are present in the gastrodermis, possibly in sacs of GER which are suggested to be primary lysosomes and which fuse with the endocytosed heterophagosomes. The insoluble pigment haematin results from digestion within the heterolysosomes, and this gives rise to the iron-containing gastrodermal pigment granules. The products of extracellular digestion are absorbed by the gastrodermis where they are synthetized into lipid by a lipase, which probably resides in whorls of GER.

This investigation was supported in part by Research Grant AI 06295 of the United States Public Health Service. The final manuscript was prepared during the tenure of a Postdoctoral Fellowship from the British Egg Marketing Board. I wish to extend my gratitude to Dr J. B. Jennings for advice and encouragement throughout this work.

The fine structure of the envelopes surrounding hatched and unhatched oncospheres of Hymenolepis nana has been investigated by transmission and scanning electron microscopy (SEM), together with light microscope histochemical observations of JB-4 embedded material. The oncosphere is surrounded by 3 layers – the capsule, the outer envelope and the inner envelope, the latter giving rise to the embryophore and the ‘oncospheral membrane’. An additional layer – the polar filament layer – lies between the ‘oncospheral membrane’ and the oncosphere. Shell material is deposited on the capsule as a thin layer. It is secreted by the outer envelope, which degenerates once shell formation is complete. The uterus may also contribute to shell formation. The embryophore forms a thin, incomplete and peripheral layer within the inner envelope. In the basal region of this envelope, partial development of an ‘oncospheral membrane’ takes place, but it does not become detached as a separate layer. The polar filaments, which are characteristic of the oncosphere of H. nana, are derived from the epithelial covering of the oncosphere itself, which delaminates to form a separate polar filament layer. The filaments arise from knob-like projections at opposite poles of this layer. The design of the embryonic envelopes in H. nana show a number of modifications from the basic cyclophyllidean pattern, and these can be related to the demands of its ‘direct’ life-cycle.

Experiments were carried out to investigate the possible influence of host-dietary fructose concentration on the relationship between worm burden and the fecundity of Moniliformis dubius. Worm length was found to be dependent both on the fructose concentration of the host's diet and on worm burden. The number of free ovaries/female worm was found to increase linearly with increasing dietary fructose concentration but was independent of worm burden. The length of the ovaries increased with increasing dietary fructose concentration and decreased in response to increases in worm density. The magnitude of the density-dependent reduction in the volume of ovarian tissue/female was decreased in worms from rats fed on diets containing low concentrations of fructose. The numbers of eggs present in the body cavities of the female worms was found to be positively correlated with dietary fructose concentration. At 5 weeks post-infection, mature eggs (containing fully developed acanthor larvae) were found in the body cavities of female worms from rats whose diets contained 3 and 12% fructose, but not in those from rats whose diets contained 1 and 2% fructose (w/w). The experimental results are discussed in the wider context of the influence of host nutrition on parasite population biology.

A population of eels Anguilla anguilla from Lough Derg, R. Shannon, Ireland, harbouring infections of both Acanthocephalus lucii and A. anguillae was studied over three years. Both parasite species had the same intermediate host and eels appeared to be the only definitive host for A. anguillae. Throughout the whole period, A. lucii was the dominant parasite, was over-dispersed throughout the eel population and most frequently occurred as a single species infection. A. anguillae was far less common, its dispersion was close to random at most times and it almost invariably occurred as a mixed species infection. The proportions of the two species remained fairly constant over the period. Despite some indication of site selection in the intestine, the distribution of both species overlapped considerably and there was no evidence of competitive displacement of one species by the other or of resource partitioning in space. The life-histories of both species were similar: they infected eels, bred and were lost from fish at the same time of year and there was no indication of resource partitioning in time. Congeneric species of acanthocephalans can thus co-exist in apparently stable equilibrium in fish as predicted and without any evidence of interactions, but it is still considered that exploitation competition between the species may be occurring in eels.

Piroplasms, including Babesia, Cytauxzoon and Theileria species, frequently infect domestic and wild mammals. At present, there is no information on the occurrence and molecular identity of these tick-borne blood parasites in the meerkat, one of South Africa's most endearing wildlife celebrities. Meerkats live in territorial groups, which may occur on ranchland in close proximity to humans, pets and livestock. Blood collected from 46 healthy meerkats living in the South-African Kalahari desert was screened by microscopy and molecular methods, using PCR and DNA sequencing of 18S rRNA and ITS1 genes. We found that meerkats were infected by 2 species: one species related to Babesia sp. and one species related to Cytauxzoon sp. Ninety one percent of the meerkats were infected by the Cytauxzoon and/or the Babesia species. Co-infection occurred in 46% of meerkats. The pathogenicity and vectors of these two piroplasm species remains to be determined.