1. We have discovered that Trypanblau and Trypanrot are highly efficient remedies in the treatment of canine piroplasmosis, no drug or mode of treatment having hitherto been found to exert any appreciable effect upon this very fatal disease.

2. The drugs exert a direct and observable effect upon the parasites (a) by causing the pyriform parasites to quickly disappear, and (b) in most cases, by causing the total disappearance of the parasites from microscopic observation in the peripheral blood.

3. The disappearance of the parasites from the blood is usually temporary, the parasites reappearing in small numbers after an interval of 9 to 12 days, but the dogs, as a rule, show no symptoms and gradually progress toward recovery. In our experience there is but slight loss of weight in treated animals, this being in marked contrast to what is usually observed in dogs which recover naturally. In the two dogs which died of a relapse the parasites reappeared after an interval of 4 to 5 days.

4. The experiments were conducted upon 21 dogs of all ages and of a variety of breeds. Many of the dogs were highly susceptible puppies. Twelve dogs were subjected to curative treatment, one dog received preventive treatment, and eight dogs served as controls.

5. Twelve dogs received treatment as follows:

(a) Two dogs were treated with Trypanrot and survived 111 days and 20 days respectively. In the latter case the dog was treated in an advanced stage of the disease, the parasites disappeared, and death appeared to be due to the after-effects of the malady.

Two control dogs died respectively on the 9th and 7th day after they were inoculated.

(b) Ten dogs were treated with Trypanblau and seven made a good recovery. Three of these dogs are still alive, having been respectively under observation for periods of 90, 83 and 65 days. Four of the recovered dogs died respectively of distemper on the 42nd and 43rd day, of severe mange on the 52nd day and of distemper and mange on the 69th day. We failed to cure the disease in 3 out of 10 cases treated: two small wormy ill-nourished puppies died from a relapse (on the 13th and 15th day) after they appeared to be on the road to recovery; one dog, treated only 3 hours prior to death, and when 50% of its corpuscles were infected, succumbed to the disease, but even in this case a visible effect was exerted upon the parasites by the drug.

Of the 7 control dogs, 6 died of piroplasmosis within 7–13 days after inoculation with virulent blood, and the remaining dog died of piroplasmosis on the 36th day after inoculation.

6. After the parasites disappear under the influence of the drug, the temperature usually falls to normal, but slight rises of temperature may at times occur, together with the subsequent reappearance of the parasites.

7. When the parasites reappear after treatment they occur in small numbers and can usually be detected only at the edge of the film. In such cases the parasites tend to occur in higher multiples [(PPPP) or more] within the individual corpuscles. The same appearance has been observed in one of the control dogs, which died of chronic piroplasmosis on the 36th day.

8. In the two puppies 10 and 11, in which death followed upon a relapse, the parasites reappeared after a shorter interval (4 to 5 days) than in any of the other dogs. The blood counts made during the fatal relapse are remarkable as showing a high percentage of dividing forms (D); that rapid invasion of fresh corpuscles was proceeding is also shown by the high percentage of single pyriform parasites (P) which occurred in the corpuscles.

9. One experiment is reported (Dog 13) in which Trypanblau was injected 24 hours after the animal was inoculated, with the result that no parasites appeared in the dog's blood up to the 65th day, and the dog remained perfectly well. The control dog died of piroplasmosis on the 7th day. (This experiment has since been successfully repeated.)

Further experiments of this character will be shortly reported upon.

10. Arsacetin and Soamin exert no curative effect upon canine piroplasmosis.

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.

According to the Red Queen hypothesis for sex, cross-fertilization should be positively associated with the probability of exposure (risk) to virulent parasites. Unfortunately, risk is difficult to measure in the wild, and prevalence of infection is often substituted for risk. Here I suggest that prevalence of infection may not generally suffice as a surrogate for risk, since the Red Queen model can make opposite predictions depending on the distribution of risk in the wild. Specifically, the results of a matching-alleles model suggest that asexual populations should be more infected than sexual populations, when (1) the variance in risk among populations is small, and (2) the mean risk of exposure to parasites is near the point where selection switches to favouring sex over asex. If, however, the variance in risk among populations is large, sexual reproduction should be positively associated with the prevalence of infection. In addition, the coefficient of variation for reproductive mode should increase sharply at the switch point. In light of these results, I re-evaluated data from two studies on the distribution of males in 95 populations of a freshwater snail (Potamopyrgus antipodarum). Populations of these snails are often mixtures of sexual and asexual individuals, and the frequency of males is correlated with the frequency of sexual females in the population. The results show a large, highly skewed variance among populations for prevalence of infection by larval trematodes. The results also show a positive, significant relationship between prevalence of infection and the frequency of males, with a sharp increase in the coefficient of variation at intermediate prevalence. In addition, experimental studies suggest that some of the necessary conditions of the Red Queen hypothesis are also met in this system. Specifically, the most common trematode infecting these snails is (1) adapted to infecting local host populations of the snail, and is (2) more infective to clones that were common in the recent past. It is too early to know if the parasite theory is sufficient to explain the widespread distribution of sex. I suggest that the theory is not sufficient, but that parasites in combination with mutation accumulation in clonal lines may explain the maintenance of sex in species that occasionally produce apomictic mutants.

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.

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.

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.

An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immuno-fluorescence antibody test monoclonal antibody (MAb) IB5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite.

The cutaneous cellular reactions associated with aquisition of resistance by cattle to Hyalomma anatolicum anatolicum feeding were investigated by comparing the nature and sequence of cellular events at H.a. anatolicum feeding sites following primary and tertiary infestations. Cattle acquired resistance to H.a. anatolicum feedings after a single infestation. The resistance was manifested by a significant reduction in the mean weight of engorged females and mean weight of egg mass produced, failure to lay eggs and sometimes death of ticks in situ. The acquisition of resistance corresponded with pronounced epidermal vesiculation and a significant change in the nature of the cellular infiltrate at tick feeding sites indicating the development of cutaneous hypersensitivity. On primary infestation the cellular infiltrate was dominated by neutrophils (43–71%) followed by mononuclear cells (25–35%). Basophil and eosinophil infiltration in small numbers was noticed for the first time at 72 and 144 h, respectively, post-primary infestation. Unlike primary infestation, the dermal infiltrate on tertiary infestation, 24 h after attachment, was characterized by infiltration of basophils (6%) and eosinophils (3%) and a marked degranulation of mast cells and basophils. Basophils showed a consistent increase in absolute as well as relative numbers as the feeding advanced. They were the second most abundant cell types after neutrophils (48–68%) at 72 h (16%), and 144 h (23%). Eosinophils (3–9%) and mononuclear cells also showed a significant increase in their mean numbers as compared with primary infestation. There was a progressive decrease in the number of detectable mast cells following tick infestation and they were significantly lower post-tertiary infestation, compared to primary infestation. These results suggest that the pharmacological mediators released by degranulation of mast cells and basophils are perhaps the major effectors of resistance in cattle to H.a. anatolicum.

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.

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.

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.

Clun forest sheep, worm-free from birth, were given a single dose of 20000 infective larvae of Haemonchus contortus. The total number of eggs/day in the faeces was recorded for 21 infections and data on population size, sex ratio and individual worm development were collected from 76 sheep. The female to male ratio was 1·28±0·07 (s.E.). The relation between increase in worm size and uterine egg content was linear. The number of eggs present in the uteri was found to be an accurate measure of eggs passed. It was shown that the daily faecal egg output is related to total parasite weight and is not a measure of the number of individuals present.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

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.

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.

Maximum levels of protective antibody activity were reached in the circulation of rats given a primary infection or one or two re-infections of Plasmodium berghei at the time the infection was finally eradicated. This association of maximum protective antibody activity with complete clearance of the parasite was also found in recipients of lymphoid cells from immune donor rats and suggests that a high level of protective antibody activity is one essential factor in complete elimination of the parasite.

Excellent technical assistance was given at different times by Mrs J. Page, Mr A. J. Edwards and Miss D. Leader.

Membrane fluidity parameters, lateral diffusion coefficient and mobile fraction were assessed in male and female Schistosoma mansoni teguments immediately after their recovery from infected mice or after 24 or 48 h of culture. Our results show that male and female teguments have different properties and behave differently when in culture. In general, male worms displayed a tendency to increase their lateral diffusion coefficient while females showed a significant reduction. The effects of praziquantel (−) and praziquantel (+) on the fluidity properties of the worm surface were also studied. It was demonstrated that both enantiomers of the drug interact with the tegument inducing a decrease in the average velocity of lipid molecules. However, it is only the active form which reduces the number of molecules that are able to move. In explanation we propose that praziquantel (−) and praziquantel (+) have different ways of inserting into the tegument and that some of the effects of the drug are mediated by altering membrane fluidity.

Mitochondrial DNA (mtDNA) sequence data and amplified fragment length polymorphism (AFLP) patterns were compared for the lungworm Dictyocaulus viviparus, a nematode parasite of cattle. Eight individual D. viviparus samples from each of 8 herds in Sweden and 1 laboratory isolate were analysed, with the aim of describing the diversity and genetic structure in populations using different genetic markers on exactly the same DNA samplesNucleotide sequence data reported in this paper have been submitted to GenBank under the Accession numbers DQ299539-DQ299826.. There was qualitative agreement between the whole-genome AFLP data and the mtDNA sequence data, both indicating relatively strong genetic differentiation among the Swedish farms. However, the AFLP data detected much more genetic variation than did the mtDNA data, even after allowing for the different inheritance patterns of the markers, and indicated that there was much less differentiation among the populations. The mtDNA data therefore seemed to be more informative about the most recent history of the parasite populations, as the general patterns were less obscured by detailed inter-relationships among individual worms. The 4 mtDNA genes sequenced (1542 bp) showed consistent patterns, although there was more genetic variation in the protein-coding genes than in the structural RNA genes. Furthermore, there appeared to be at least 3 distinct genetic groups of D. viviparus infecting Swedish cattle, 1 of which was predominant and showed considerable differentiation between farms, but not necessarily within farms. Second, the 2 smaller genetic groups occurred on farms where the predominant group also occurred, suggesting that these farms have had multiple introductions of D. viviparus.

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.

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.